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WO2024151321A2 - Systèmes et procédés de production de tissu adipeux de culture à l'échelle commerciale - Google Patents

Systèmes et procédés de production de tissu adipeux de culture à l'échelle commerciale Download PDF

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Publication number
WO2024151321A2
WO2024151321A2 PCT/US2023/071704 US2023071704W WO2024151321A2 WO 2024151321 A2 WO2024151321 A2 WO 2024151321A2 US 2023071704 W US2023071704 W US 2023071704W WO 2024151321 A2 WO2024151321 A2 WO 2024151321A2
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cells
protein
adipose
adhered
cultured
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WO2024151321A3 (fr
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David Kaplan
John Se Kit YUEN
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Tufts University
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Tufts University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0653—Adipocytes; Adipose tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1384—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from adipose-derived stem cells [ADSC], from adipose stromal stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/74—Alginate

Definitions

  • the present disclosure generally relates to cultured adipose tissue produced on a macroscale level.
  • the present disclosure further relates to methods for producing cultured adipose tissue on a macroscale level via the harvesting and aggregation of non-adhered adipose cells.
  • the present disclosure also considers the use of flocculation and coagulation techniques in the aggregation of adipose cells.
  • adipose tissue is largely a dense packing (aggregation) of lipid-filled adipocytes held together by a sparse extracellular matrix (ECM). This is opposed to muscle tissue which is comprised of aligned fibers in a multi-hierarchical structure.
  • ECM extracellular matrix
  • 3D culture has been the main approach for generating bulk/macroscale tissues. These tissue engineering strategies involve the in vitro growth of cells over 3D scaffolds.
  • it is challenging to scale up 3D culture due to mass transport limitations with regard to oxygen, nutrients, and waste. It is often quoted in the field that cells cannot remain viable in 3D tissues unless they are within about 200 micrometers of a source of blood or culture media.
  • a method for producing cultured adipose tissue may include the steps of: growing adipogenic precursor cells in a first culture media; differentiating the adipogenic precursor cells to adipose cells in a second culture media; harvesting non-adhered adipose cells; and aggregating the harvested non-adhered adipose cells to provide a cultured adipose tissue.
  • the first culture media and the second culture media may optionally be the same media.
  • a method for producing cultured adipose tissue may include the steps of: growing adipogenic precursor cells on a two-dimensional (2D) substrate; differentiating the adipogenic precursor cells to adipose cells on the 2D substrate; harvesting non-adhered adipose cells; and aggregating the harvested non-adhered adipose cells to provide the cultured adipose tissue.
  • 2D two-dimensional
  • cultured tissues are provided.
  • a cultured adipose tissue has adipose cells embedded in a hydrogel or binder, wherein the cultured adipose tissue has a three-dimensional (3D) shape and a size on the macroscale.
  • a cultured adipose tissue is made by the methods described above.
  • FIG. 1 is a schematic representation of cultured adipose tissue, in accordance with the present disclosure.
  • FIG. 2 is a flow chart of steps that may be involved in producing the cultured adipose tissue, in accordance with the present disclosure.
  • FIG. 3 is a schematic representation of methods of producing the cultured adipose tissue using bioreactors, in accordance with the present disclosure.
  • FIG. 4 is a schematic representation of a continuous process of producing the cultured adipose tissue on a conveyor belt, in accordance with the present disclosure.
  • FIG. 5 is a timeline of 3T3-L1 adipogenic differentiation, according to an embodiment of the present disclosure.
  • this disclosure contemplates all combinations of each of the upper and lower bounds of those ranges, including each of the values within those ranges, both individually and in a range. For example, recitation of a value of from 1 to 10 also contemplates a value of from 1 and 9 or from 3 and 10. When two or more ranges for a particular value are recited, this disclosure contemplates all combinations of the upper and lower bounds of those ranges that are not explicitly recited. For example, recitation of a value of between 1 and 10 or between 2 and 9 also contemplates a value of between 1 and 9 or between 2 and 10.
  • adipogenic precursor cells or “pre-adipocytes” refer to precursor cells capable of differentiating into mature adipose cells.
  • Adipogenic precursor cells or “pre- adipocytes” may be used interchangeably throughout the present disclosure.
  • Non-limiting examples of adipogenic precursor cells include stem cells such as pluripotent stem cells (PSCs), mesenchymal stem cells (MSCs), muscle-derived stem cells (MDSCs), and adipose-derived stem cells (ADSCs) (e.g., porcine, bovine, human, avian (chicken), piscine, etc.).
  • PSCs pluripotent stem cells
  • MSCs mesenchymal stem cells
  • MDSCs muscle-derived stem cells
  • ADSCs adipose-derived stem cells
  • transdifferentiated cells can also be utilized.
  • adipogenic precursor cells may include, but are not limited to, dedifferentiated fat (DFAT) cells (e.g., porcine, bovine, piscine, etc.), preadipocytes (e.g., human, bovine, avian (chicken), murine, piscine, etc.), and fibroblasts (e.g., avian (chicken), bovine, porcine, murine, piscine, etc.).
  • DFAT dedifferentiated fat
  • preadipocytes e.g., human, bovine, avian (chicken), murine, piscine, etc.
  • fibroblasts e.g., avian (chicken), bovine, porcine, murine, piscine, etc.
  • adipose cells are fat cells or adipocytes.
  • Adipose cells are fat cells or adipocytes.
  • Fat cells are used interchangeably throughout the present disclosure.
  • the cultured adipose tissue 10 may include adipose cells 12 (or adipocytes 12) in an extracellular matrix.
  • the cultured adipose tissue 10 may be arranged in a defined three-dimensional (3D) shape and may have a size on the macroscale (i.e., millimeter scale and greater). Although a cube-like structure is shown in FIG. 1 for simplicity, it will be understood that the cultured adipose tissue 10 may have any suitable 3D shape in practice.
  • the cultured adipose tissue 10 may be a food product suitable for consumption.
  • the cultured adipose tissue 10 may be incorporated as an ingredient in a food product suitable for consumption.
  • the cultured adipose tissue 10 is produced using a method that circumvents the mass transport limitations associated with directly culturing bulk or large scale 3D tissues.
  • a mass of adipose cells 12 are cultured from adipogenic precursor cells in culture media.
  • the block 14 may include growing adipogenic precursor cells to confluency (or to a desired coverage/number of cells on a surface or in suspension) in a first culture media, and then differentiating the adipogenic precursor cells into adipose cells 12 in a second culture media.
  • the first culture media may be an adipogenic induction media which supports proliferation of the adipogenic precursor cells
  • the second culture media may be a lipid accumulation media to provide large numbers of lipid-filled adipose cells 12.
  • a single culture medium may be used for both proliferation/growth of the adipogenic precursor cells and for differentiation of the adipogenic precursor cells into adipose cells.
  • the culture time may be tuned to control lipid yield and droplet size. For example, Applicant has found that longer culture times (about a month) yield droplets comparable to in vivo fat (e.g., chicken).
  • the adipose cells 12 may be genetically modified.
  • the adipose cells 12 may be genetically modified to improve their growth and lipid accumulation for more efficient scale up.
  • a skilled artisan will recognize the variety of techniques that allow suitable genetic manipulation, including but not limited to, gene editing technologies such as CRISPR-Cas9 gene editing.
  • the gene modification to generate a given desired property is clearly important.
  • the methods and systems described herein may include gene editing facilities and methods.
  • genetically modified cells are increasingly available, so it is likely that commercial suppliers for genetically modified cells will be available soon.
  • the genetic modification may be a genetic modification to induce adipogenesis.
  • the adipogenic gene modification can be selected from the group consisting of a modification for ectopic expression of: peroxisome proliferator-activated receptor gamma (PPARy), CCAAT/enhancer binding protein alpha and beta (C/EBPa,P), sterol regulatory element binding protein 1 (SREBP-1), fatty acid binding protein 4 (FABP4), zinc finger protein 423 (Zfp423), perilipin 1 (PLIN1), Kruppel-like factor 13 (KLF13), retinoid X receptor a (RXRa), phosphoenol pyruvate carboxykinase 1 (PCK1), early B-cell factor (Ebf) 1-3, runt-related transcription factor 1 (RUNX1), cyclin-dependent kinase 4 (CDK4), combinations thereof, or the like.
  • PPARy peroxisome proliferator-activated receptor gamma
  • the genetic modification can be a genetic modification to downregulate production of adipose triglyceride lipase (TGL), hormone sensitive lipase (HSL), carnitine palmitoyltransferase (CPT), other fat or fatty acid breakdown enzymes, combinations thereof, or the like.
  • TGL adipose triglyceride lipase
  • HSL hormone sensitive lipase
  • CPT carnitine palmitoyltransferase
  • other fat or fatty acid breakdown enzymes combinations thereof, or the like.
  • the genetic modification could cause the production of anti-microbial, anti-fungal, or anti-viral constituents.
  • the genetic modification could cause the production of stabilizing molecules.
  • the stabilizing molecules can prevent protein aggregation (e.g., low molecular weight stabilizers).
  • the genetic modifications are intended for health purposes.
  • the genetic modification can cause expression of vitamins that are not typically present in the cells.
  • the genetic modification can cause expression of resveratrol, the active ingredient in red wine, or catechins, the active ingredient in green tea.
  • the genetic modifications are intended to alter the color of the resulting cultured adipose tissue. Consumer perceptions regarding the color of food can be important. To that end, the genetic modifications can include expression of proteins that provide desirable colors, such as carotenoids, to alter the color of the resulting product to be more appealing to the average consumer. In some cases, the genetic modification can prompt the generation of myoglobin or hemoglobin, which can provide a color that is more typically associated with meat.
  • the genetic modifications can be modifications to hasten proliferation (cell immortalization targets are generally applicable here), improve adipogenesis, improve cell growth (e g., allow growth on a media that has less components or less expensive components), or other modifications that would be understood to improve the overall number and quality of adipose cells for incorporating into the cultured adipose tissue.
  • genetic modification can be used to introduce components that are not produced within the fat cells of the species of interest.
  • cows are a good source of conjugated linoleic acids (CLAs), which are known to be good for human health, but these CLAs are derived from their gut bacteria metabolites, so in vitro cells may be lacking in them.
  • CLAs conjugated linoleic acids
  • the genetic modification may be a gene that is associated with the digestive bacteria of a species of interest.
  • SNPs single nucleotide polymorphisms
  • desired phenotypes e.g., flavor, fattiness, degree of marbling, etc.
  • SNPs identified in the literature may correlate to changes in specific proteins (i.e. may represent a genotype that correlates directly to the phenotype). The changes demonstrated to be favorable for conventional meat products can be incorporated into the cells of the present disclosure.
  • SNPs include, but are not limited to, a G instead of A at locus rs733003230 of the fatty acid desaturase 1 (FADS1) gene and/or a G instead of A at locus LC060926 of the FADS2 gene for increased arachidonic acid in chickens or a G instead of C at position 841 of the fatty acid synthetase gene for increased oleic acid in cattle.
  • FDS1 fatty acid desaturase 1
  • the genetic modifications can be intended to facilitate the systems and methods described herein.
  • the genetic modification can modify the density of surface proteins and/or surface binding sites to facilitate the crosslinking.
  • the surface chemistry can include photoinitiated crosslinking agents.
  • the end product of this system and method is a cultured adipose tissue that is typically going to be combined with some kind of muscle tissue or connective tissue, so it may be beneficial to genetically modify the cells to express muscle binding or connective tissue binding domains, which may make harvesting easier.
  • a cultured adipose tissue that is typically going to be combined with some kind of muscle tissue or connective tissue, so it may be beneficial to genetically modify the cells to express muscle binding or connective tissue binding domains, which may make harvesting easier.
  • the specific genetic modifications that are present may be unknown or may be unknowable.
  • the cells may have no specific genetic modification, but may simply be harvested from a new source with unknown genetic makeup.
  • the systems and methods described herein may encounter cells with unknown genetic makeup.
  • the cells can be subjected to culture media sampling to determine the most efficient culture media for use with those cells.
  • This process can be manual or it can be automated.
  • a lab technician will attempt to grow cells in different culture media and will compare the results to determine a cost/benefit to the different culture media.
  • an artificial intelligence or machine learning algorithm can sample the variable space with respect to culture media composition and can optimize the media to the specific cells.
  • a pre-production process can be performed, where a subset of cells from a population are removed from the population and put through a small-scale version of the methods described herein, to determine suitability of the culture media. In some case, less than 1% of a population of cells, less than 0.5%, less than 0.1%, or less than 0.01% of a population of cells can be selected for sub-sampling to confirm culture media appropriateness prior to a fully scaled up production run.
  • the pre-production process can serve as a final validation that the cells and the media are compatible with one another.
  • the first step/cellular raw materials may be referenced as a cell stock.
  • the acquisition of the cell stock can in general be a process similar to harvesting conventional meat products (i.e., butchery and isolation of cells from the butchered products) or can involve harvesting cells from less traditional places, such as other active cell lines that are being used in cellular agriculture. Additional processing steps for the cells can include immortalization, or clonal isolation to obtain a proliferative + adipogenic + genetically homogenous (for production consistency) cell population/cell line. Cells could also be adapted to single cell suspension prior to use in scaled up production.
  • the culture media used can be provided in a variety of forms, including powdered bases that can be hydrated by pure water and fully formed culture media. In either case, separate and distinct culture media can be included for the proliferation and differentiation steps.
  • the purity of the water is extremely important.
  • the system includes onsite water purification.
  • the method can include hydrating the powder to form a culture media.
  • the culture media can be sterile filtered or sterilized via any means available to those of skill in the art prior to use in the method or at any step of the method.
  • Various contemplated modifications to the culture media include, but are not limited to, bulk mixed powder including all of the non-water components of the culture media, separately maintained ingredients that are mixed on demand to make culture media, growth factors or artificial mimics thereof can be present in the culture media, or a combination thereof.
  • the cells are genetically modified to have intentionally less strict requirements for the culture media.
  • cells can be genetically modified to create their own glutamine (see, for example, WO 2019/014652, which is incorporated herein in its entirety by reference for all purposes), which would eliminate the requirement for glutamine in the culture media. Similar genetic modifications to reduce the need for complex culture media are contemplated.
  • Manipulation of the cells themselves can be done by conventional methods, including seed trains for seeding larger and larger bioreactors. The seed train gradually moves to bioreactors having increasing size until the desired bioreactor size is reached. From there, the cells can be a part of a suspension culture of an adherent culture.
  • cell proliferation is done via cell stock cultured in single cell suspension in various suspension bioreactors.
  • These bioreactors can be a conventional stirred suspension tank, air-lift, vertical wheel, or wave bioreactors.
  • cells can be encapsulated with a material that protects the cells from shear stress. This encapsulating material may itself provide a miniature three-dimensional cell culture environment, which can be advantageous for other reasons discussed elsewhere herein.
  • Other contemplated bioreactor designs include the rotating wall vessel bioreactor described elsewhere herein.
  • the bioreactor used herein can be a dedicated adherent cell bioreactor, such as a fixed bed bioreactor (e.g., the Scale XTM bioreactor, available commercially from Univercells S.A., Charleroi, Belgium, or iCellis® bioreactors, available commercial from Pall Corporation, Port Washington, NY).
  • a fixed bed bioreactor e.g., the Scale XTM bioreactor, available commercially from Univercells S.A., Charleroi, Belgium, or iCellis® bioreactors, available commercial from Pall Corporation, Port Washington, NY.
  • the bioreactor can include stacked culture flask approaches, like "cell factories" or the Hyperstack®, available commercially from Corning Inc., Corning, NY.
  • an adherent approach could be combined with a suspension bioreactor if microcarriers (e.g., pectin microcarriers commercially available from Corning Inc., Corning, NY) can be used as the adherent substrate, but those microcarriers are then circulated in a suspension bioreactor in the conventional fashion.
  • microcarriers e.g., pectin microcarriers commercially available from Corning Inc., Corning, NY
  • the microcarriers can be edible.
  • the microcarriers may end up in the resulting product, if their presence is not undesirable.
  • the same reactor can be used as was used for proliferation, with a change of culture media facilitating the transition.
  • the cells are transferred to a different bioreactor to transition from proliferation to differentiation.
  • cells can be separated from the proliferation media via centrifugation (e.g., disc stack centrifuge, counter flow centrifuge, Gibco Rotea centrifuge, etc.). After removal of the proliferation media, the differentiation media is added to the bioreactor and the cells are cultured in the same fashion as they undergo adipogenesis.
  • centrifugation e.g., disc stack centrifuge, counter flow centrifuge, Gibco Rotea centrifuge, etc.
  • adipocyte buoyancy in suspension culture poses unique problems that are not typically encountered in cell agriculture.
  • a cell floats that is typically a sign that the cell is no longer of any value.
  • the buoyancy can be leveraged as described herein by harvesting cells from a surface or suspended cells and allowing them to replenish from beneath.
  • the culture is ended, and the lipid-laden adipose cells 12 are harvested according to a block 16.
  • the block 16 may include detaching the adipose cells 12 from a substrate and draining the adipose cells of non-cell liquid.
  • Cells can be harvested via the same techniques used to separate cells from culture media between the various stages.
  • “skimming" floating cells from the surface is one particularly promising approach to harvesting cells, given that it is an automatic indicator of buoyancy.
  • “Floating” as used herein may be used interchangeably with “non-adhered” unless discussed otherwise.
  • Other methods of collecting non-adhered cells may comprise collecting non-clustered floating cells, collecting only non-adhered cells, and/or not collecting adhered cells.
  • conventional cell harvesting techniques may be used, such as those disclosed in Dryden et al., "Technical and Economic Considerations of Cell Culture Harvest and Clarification Technologies", Biochem Eng J, Vol. 167, 107892, which is incorporated herein in its entirety by reference. While Applicant believes that some of the cell harvesting techniques described in this application are inventive, there are other inventive concepts which are usable with conventional cell harvesting techniques.
  • the harvested cells Prior to molding the harvested cells in the next portion of certain methods, some postprocessing may be required.
  • the harvested cells might be rinsed to remove/minimize/dilute undesired culture media components. Rinsing can be done by way of counter-flow centrifugation or conventional filtration.
  • cells can be clarified by removing undesirable components, which are not removed by rinsing. This is a process that is regularly used in the pharmaceutical arts, but it is not conventionally used in cellular agriculture. In one specific example, clarification of cells to remove cell debris or excess nucleic acids is expressly contemplated. Other methods such as flocculation via pH or the use of polymers such as chitosan which can induce flocculation are contemplated.
  • harvested adipose cells 12 themselves may be an end product, so the system and method can end there in certain circumstances.
  • the harvested adipose cells 12 can be provided to customers for incorporation into products, including those outlined elsewhere herein.
  • the harvested adipose cells 12 may be aggregated, for instance into a 3D mold (e.g., a 3D printed mold) having a desired 3D shape to generate the 3D adipose tissue 10.
  • a 3D mold e.g., a 3D printed mold
  • the 3D mold can be a permanent and/or reusable mold or it can be single-use.
  • the single-use mold can be made of the same material as the materials identified elsewhere herein as hydrogel materials for use in the cell aggregation step.
  • the entire structure of the single-use mold and the cultured adipose tissue that is molded inside of it can be utilized for inclusion in the various products identified herein. For example, a large chunk of cultured adipose tissue and the 3D mold in which it was made can be chopped into smaller pieces for incorporating into a food product.
  • the material of the 3D mold can be stable or can simply change phase (e.g., similar to the rendering of fat) when exposed to temperatures of at least 40 °C, at least 45 °C, at least 50 °C, at least 55 °C, at least 60 °C, at least 65 °C, at least 70 °C, at least 75 °C, at least 85 °C, at least 90 °C, at least 100 °C, at least 105 °C, at least 125 °C, or at least 150 °C or at temperatures at ranges of from 40 to 150 °C, from 50 to 125 °C, from 60 to 105 °C, or from 70 to 100 °C.
  • the 3D mold material may be stable or generally stable in water, because so many food and cooking environments are aqueous. To the extent that it is not explicitly stated elsewhere, these properties may also apply to the binder/hydrogel material.
  • a thickening agent or thickener may be a suitable binder/hydrogel.
  • any practical shape can be suitable for use, though some may be preferred over others.
  • generally regular spherical shapes may be preferred, while in other cases, irregular shapes may be desired.
  • the 3D shape may be elongated strips.
  • the desired shape is very small, it may be impossible or impractical to make molds of the desired shape. In these cases, it may be useful to make a larger sample that is carved into smaller pieces. For example, if a customer desires a host of small fat "flakes" for incorporating into a food product, it is much easier to make a large piece of cultured adipose tissue and then manipulate that large piece to make smaller flakes. Larger pieces can be cut, sliced, chopped, minced, ground, or other conventional way to manipulate animal-fat-like products.
  • the aggregated material may be formed by way of an extrusion or printing process.
  • the cells and hydrogel material may be co-extruded to form a linear aggregate.
  • the linear aggregate can be cut into pieces to form individual pieces of cultured adipose tissue.
  • the linear aggregate can be solid or hollow, depending on the specific desired application.
  • candy making processes where ropes of extruded or rolled candy is cut into smaller pieces might be one example of a suitable process to mimic for an extrusion process for use with the present invention.
  • Some extruded metal or rubber or plastic processes may be similarly applicable.
  • the cells and binder/hydrogel material can be 3D printed into a desired shape.
  • aggregating as shown in block 18 may involve embedding the harvested adipose cells 12 in a hydrogel or mixing with a binder in a 3D mold.
  • Suitable hydrogels or binders include, but are not limited to, food safe compounds such as alginate, cellulose, gelatin, starch (e.g., tapioca), hyaluronic acid, fibrin, carrageenan, cornstarch, gellan gum, lecithin, maltodextrin, methylcellulose, carboxymethylcellulose, cellulose gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose tragacanth, karaya, acacia, ghatti, beta-glucan, psyllium husk, inulin, chitosan, curdlan, dextran, potato starch, tapioca/arrowroot starch, hyaluronic acid, fibrin, carrageenan, guar gum, inulin, konja
  • the hydrogel or binder is alginate which is a material used as a fat replacer in the food industry.
  • block 18 may include mixing the harvested and drained adipose cells 12 with an alginate solution at a specified volumetric ratio in the 3D mold.
  • a slow gelling alginate solution may be prepared by adding calcium carbonate and glucono delta-lactone (GDL) powders to an alginate solution, and the slow gelling alginate solution may be combined with the harvested and drained adipose tissue at a 1 : 1 volumetric ratio in a 3D printed mold (see Example 3).
  • GDL calcium carbonate and glucono delta-lactone
  • the process is typically a heat-driven process, where water and the binder/hydrogel ingredient are heated, fat cells are mixed in, and the mixture is cooled.
  • agar/agarose is heated to liquify it (e g., 85-100 °C), then cooled (e g., 40-65 °C) and mixed with adipocytes. After mixing, the solution is cooled to form a solid piece of macroscale, structured fat.
  • aggregating as shown in block 18 may involve cross-linking the harvested adipose cells 12 in a 3D mold.
  • the cross-linking may be carried out using a suitable protein-protein cross-linking enzyme such as, but not limited to, transglutaminase, a tyrosinase, a peroxidase, a laccase, a sortase, a subtilisin, and lysyl oxidase.
  • cross-linking the harvested adipose cells includes enzymatically cross-linking the harvested adipose cells using transglutaminase.
  • cross-linking the harvested adipose cells may involve mixing a solution of transglutaminase with the harvested adipose cells at a specified volumetric ratio in a 3D mold (see Example 3).
  • the block 18 may further include adding helper proteins during the cross-linking.
  • the helper proteins (which may simply be referred to as “proteins”) may be selected from casein and gelatin.
  • Chemical crosslinking can also be used when the reactants or catalysts are food safe, such as EDC-NHS reactions between acid and amine groups. Photochemical crosslinking can also be utilized where photosensitizers are food safe.
  • soy protein isolate 6% or higher of soy protein isolate and 5-25 enzyme activity units/gram of protein substrate of transglutaminase is one suitable starting point for these crosslinking conditions.
  • Higher soy protein isolate and/or transglutaminase content can lead to higher strength within the gel. However, the maximum strength is not necessarily desirable, because it would not mimic the mouthfeel of fat.
  • a mixture of pea protein isolate (19.6% w/w), transglutaminase (0.7% w/w), and NaCl (1% w/w) with a pH of 6.5 is incubated at 50 °C to provide a crosslinked protein matrix that is often referred to as a plant based meat.
  • Adipocytes can be mixed into this composition before curing to form a hybrid product of culture adipose tissue and plant based meat.
  • the crosslinking may be dependent on one or more cofactors or metals.
  • exemplary cofactors or metals may include calcium, copper, and heme.
  • the protein is mixed in with the binder/hydrogel and crosslinked (with the transglutaminase, for example). This may be necessary to form a gel.
  • canola oil can be mixed with ⁇ 30% (w/w) of fully hydrogenated canola oil at 65 °C, hot-emulsified with a soy protein suspension (8%, w/w) at a lipid content of 70% (w/w) using a high-shear disperser, and cooled to 37 °C.
  • the concentrated, emulsified fat crystal networks can then incubate with transglutaminase for 1 hr to induce protein crosslinking.
  • cross-linkers may be used for adipose cell aggregation such as, but not limited to, polymers functionalized with aldehyde groups, genipin, phenolic compounds, and combinations thereof.
  • Suitable polymers functionalized with aldehyde groups include, but are not limited to, periodate oxidized pectin, dextran, chitosan, Arabic gum, sucrose, raffinose, stachyose, cyclodextrin, and starch.
  • Suitable phenolic compounds include, but are not limited to, caffeic acid, chlorogenic acid, caftaric acid, quercetin, and rutin derived from plants such as grapes and coffee.
  • aggregation of the cells or the combination of both the aggregation and harvesting of non-adhered adipose cells comprises flocculation.
  • flocculation and grammatical equivalents may refer to the aggregation of unstable and small particles through surface charge neutralization, electrostatic patching and/or bridging after addition of flocculants (or agents that make fine and sub-fine solids or colloids suspended in the solution form large loose flocs through bridging thus achieving solid-liquid separation).
  • Flocculation may be performed by one or more of several techniques, including auto-flocculation, bio-flocculation, chemical flocculation, particle-based flocculation, and electrochemical flocculation.
  • Various flocculation techniques have been reviewed by Matter, et al (2019, Applied Sciences). This is a method with high potential as it might be used to harvest cells from the bioreactor at low cost.
  • Auto-flocculation may be performed at acidic pH (for example, a pH of 4.0). Autoflocculation may be performed at alkaline pH (for example, a pH of 10.4, 11.0, 1.5, 11.6, 12.0, or 12.5). Auto-flocculation may be performed over several days (for example, 16 days).
  • acidic pH for example, a pH of 4.0
  • alkaline pH for example, a pH of 10.4, 11.0, 1.5, 11.6, 12.0, or 12.5
  • Auto-flocculation may be performed over several days (for example, 16 days).
  • Chemical flocculation may be performed with inorganic flocculants such as aluminum sulfate, calcium oxide, iron(III) chloride, and magnesium hydroxide. Chemical flocculation may be performed with organic flocculants such as inulin, starches, chitosan, tannins, alginates, and lysines. Flocculation may preferably be performed with food-safe flocculants such as chitosan, cellulose, lignin and/or other long polymers.
  • inorganic flocculants such as aluminum sulfate, calcium oxide, iron(III) chloride, and magnesium hydroxide. Chemical flocculation may be performed with organic flocculants such as inulin, starches, chitosan, tannins, alginates, and lysines.
  • Flocculation may preferably be performed with food-safe flocculants such as chitosan, cellulose, lignin and/or other long polymers.
  • Particle-based flocculation may be performed using aminoclay-based nanoparticles such as 3-aminopropyltriethoxysilane (APTES) conjugated to magnesium and aminoclays conjugated to aluminum, titanium oxide, and humic acid.
  • Particle-based flocculation may be performed using magnetic particles including ferrous oxide nanoparticles and composites.
  • Coagulation of cells may be an additional or alternative means of cell harvesting and/or aggregation that is similar to flocculation wherein cells aggregate or clump.
  • the adipose cells 12 or the adipose tissue 10 may be supplemented at various stages to tune the sensorial characteristics (e.g., texture, color, and flavor) and/or the nutritional attributes of the cultured adipose tissue 10.
  • supplementation with additives such as, but not limited to, flavorants, colorants, texturizers, vitamins, minerals, amino acids, proteins/peptides, oils, whole foods, and fatty acids is also encompassed by the present disclosure.
  • tunable control of fat nutrition and health may be implemented.
  • the fatty acid composition of the cultured adipose tissue may be tailored via cell feeding strategies, such as by supplementing fatty acids into the culture media during in vitro culture.
  • omega 3 desaturases may be expressed or pathways to produce lipophilic nutrients (e g., beta carotene, vitamin A) may be activated in the adipose cells 12. This may be beneficial to the consumer as certain nutrients are more bioavailable when consumed in food versus a micronutrient supplement.
  • the texture of the cultured fat may be tunable based on variables such as the hydrogel/binder (e.g., alginate) concentration, cross-linker levels, and the inclusion of helper proteins (e.g., casein, gelatin, etc.) during cross-linking.
  • the cultured adipose cells 12 may be supplemented with methylated branched fatty acids to impart a "mutton" flavor in the cultured adipose cells 12.
  • methylated branched fatty acids One specific methylated branched fatty acid is 4-methyloctanoic acid.
  • the relative extracellular matrix production and fat production levels may be optimized pending the texture, taste, and/or nutritional outcomes desired.
  • the starting material can be fish muscle cells, given that fat cells are generally harder to acquire from certain fish species known to be very lean. It has also been shown that certain fish muscle cells can self-immortalize, which is advantageous for large scale production. It is possible to induce lipid uptake in skeletal muscle cells via fatty acid/lipid supplementation, forming fatty muscle cells. Fatty acid/lipid supplementation may also induce transdifferentiation of skeletal muscle cells into adipocytes, or adipocyte-like cells. Genetic approaches may also be used to transdifferentiate skeletal muscle cells, such as via the activation or insertion of PPARy. Without intending to alter the definition provided above, these fatty muscle cells can be used wherever adipocytes or fat cells are referenced herein.
  • FIG. 3 shows scalable processes for the mass production of the cultured adipose tissue 10.
  • the processes may be carried out in a bioreactor 20, such as a stirred suspension tank bioreactor 22 (top) or a hollow fiber bioreactor 24 having hollow fiber membranes 26 (bottom).
  • a bioreactor 20 such as a stirred suspension tank bioreactor 22 (top) or a hollow fiber bioreactor 24 having hollow fiber membranes 26 (bottom).
  • Other types of bioreactors apparent to those skilled in the art may also be used and are within the scope of the present disclosure such as, but not limited to, rotating wall vessel bioreactors (RWVBs) and packed bed bioreactors.
  • Production of the adipose tissue 10 in the bioreactor 20 may involve seeding 28 a first culture media 30 (adipogenic induction media) in the bioreactor 20 with adipogenic precursor cells 32.
  • a first culture media 30 adipogenic induction media
  • the adipogenic precursor cells 32 may then proliferate 34 to confluency (or to a desired coverage/number of cells on a surface or in suspension) in the bioreactor 20.
  • the adipogenic precursor cells 32 may form small aggregates or spheroids 36 as they proliferate (see FIG. 3, top).
  • the spheroids 36 may be dissociated 38 into single adipogenic precursor cells 32 and allowed to proliferate 34 further (see FIG. 3, top).
  • the adipogenic precursor cells 32 may proliferate on the surface of the hollow fiber membranes 26 (see FIG. 4, bottom). In this case, the adipogenic precursor cells 32 may be detached 40 from the hollow fiber membranes 26, and the detached adipogenic precursor cells 32 may be used to reseed the media 30 for further proliferation 34.
  • the cells may accumulate lipids and differentiate 44 into adipose cells 12.
  • the adipose cells 12 may grow separately or in small clusters 46 (see FIG. 3, top).
  • the adipose cells 12 may develop on the surface of the hollow fiber membranes 26.
  • a single culture medium may be used for both proliferation 34 and differentiation 44.
  • the adipose cells 12 may be harvested 48. In the hollow fiber bioreactor 24, the harvesting may involve detaching the adipose cells 12 from the hollow fiber membranes 26.
  • the harvested adipose cells 12 may then be aggregated 50 in a 3D mold to provide the cultured adipose tissue 10.
  • suitable methods for binding and aggregating 50 the adipose cells 12 include cross-linking (e.g., enzymatic cross-linking with transglutaminase), as well as embedding the adipose cells 12 in hydrogels such as alginate.
  • the culturing process of the present disclosure may be compatible with two dimensional (2D) culture strategies.
  • the adipose cells 12 may be cultured in thin layers on a 2D substrate such as culture plates, and then aggregated into the 3D adipose tissue 10 according to the above-described procedures.
  • the adipogenic precursor cells 32 may be grown to confluency (or to a desired coverage/number of cells on a surface or in suspension) and differentiated into the adipose cells 12 on the 2D substrate.
  • Harvesting or collecting the adipose cells 12 from the 2D substrate followed by aggregating the harvested adipose cells 12 may provide the cultured adipose tissue 10.
  • the 2D substrate may by edible and incorporated into the final food product, such that the adipose cells 12 do not need to be detached from the 2D substrate.
  • cells may grow on the 2D substrate and eventually become non-adherent and the adherent and non-adherent cells may be harvested and/or aggregated separately and/or used for distinct purposes as detailed herein.
  • the 2D substrate may be a conveyor belt 52 (see FIG. 4).
  • the continuous production process may involve seeding 54 the adipogenic precursor cells 32 onto the conveyor belt 52 having a culture media thereon.
  • the adipogenic precursor cells 32 may then proliferate 56 to confluency (or to a desired coverage/number of cells on a surface or in suspension) on the conveyor belt 52.
  • Changing the culture media to lipid accumulation media may allow the adipogenic precursor cells 32 to accumulate lipid and differentiate 58 into the adipose cells 12.
  • a single culture medium may be used for both proliferation 56 and differentiation 58.
  • the adipose cells 12 may be harvested 60 by detachment from the conveyor belt 52, and then aggregated 62 according to the above-described procedures to provide the cultured adipose tissue 10.
  • the technology disclosed herein provides a novel and scalable approach to cultured fat generation.
  • the present disclosure leverages large-scale cell proliferation and scale up technology to generate a required amount of in vitro adipose cells, after which the cells are aggregated or packed into a solid 3D construct on the macroscale.
  • the adipose cells are cultured in thin layers (2D culture) or in bioreactors with easy access to the culture media, followed by aggregation into macroscale 3D tissues after sufficient adipocyte maturation.
  • adipocytes or adipocyte clusters recapitulates native fat tissue from a sensory perspective as adipose tissue in vivo is largely a dense aggregation of lipid fdled adipocytes with a sparse extracellular matrix. Furthermore, the compatibility of the adipose tissue production method with 2D culture strategies allows for a continuous production process with a conveyor belt assembly line approach.
  • the method of the present disclosure produces bulk cultured adipose tissue in a way that circumvents the mass transport limitations associated with directly culturing or engineering large 3D tissues. Aggregation at the end of cell culture removes the need for nutrient delivery to the adipose cells via vascularization or an elaborate tissue perforation system. This is because, for food applications, the cultured adipose cells do not need to stay alive once formed into the final edible tissue. This is analogous to meat production in conventional animal agriculture where muscle and fat cells gradually cease to be viable after slaughter. In contrast, for medical applications, cells in 3D tissues may be expected to remain viable to be used for implantation into the body or for testing in an in vitro tissue model. Accordingly, the adipose tissue production method of the present disclosure is less costly than other methods that rely on complex perfusion and mixing systems to distribute nutrients during cell growth. In some embodiments, the food product described herein is produced without vascularization or perfusion.
  • monocultures of adipocytes and preadipocytes may be sufficient for the production of large fat droplets without the need for supporting cell types.
  • Standard cell culture conditions are sufficient for the type of adipocyte culture outlined in this disclosure, and no specific coatings on tissue culture plastics were required to achieve desired adipocyte growth and development.
  • the pre-adipocytes and adipocytes of various livestock species may be grown in serum-free culture media according to the present disclosure, thereby eliminating a major obstacle in in vitro fat culture. These advantages further help reduce production costs.
  • Co-cultures can also be considered for enhanced fat outcomes, such as the use of fibroblasts or muscle cells in the cultures, such as to increase the quality of the fat products or to alter the texture and composition.
  • Applicant has also observed that a large subpopulation of the cultured adipocytes adhere strongly to tissue culture plates and do not float away, avoiding issues of lift-off of adherent adipocytes in vitro due to increasing buoyancy as the adipocytes become fatty.
  • the 2D culture systems disclosed herein self-sort for adherent cell populations, which may provide for adherent and non-adherent cell populations that may be used for separate downstream applications. Applicant has also developed techniques for dealing with or using the non-adherent cell populations, as described elsewhere herein.
  • the systems and methods described herein can include various sensing and control systems to facilitate superior control over the processing/harvesting process.
  • the systems and methods include a measurement component, which observes a property of an individual cell or a group of cells, and an assessment component, which assesses the measurement to make a processing/harvesting decision to select an appropriate time for processing/harvesting the cell or group of cells.
  • processing refers generally to a decision to move a cell or a population of cells to a different processing stage. These processing steps can in some cases be related to developmental differentiation (i.e., the process of becoming an adipose cell).
  • the term harvesting generally refers to a final processing step for adipose cells, after which those cells are aggregated as described herein or otherwise further processed in ways that do not materially alter the cells from their state at harvest.
  • the processing/harvesting decisions can be based on monitoring of individual cells.
  • analytical e.g., optical, etc.
  • interrogation of individual cells can provide meaningful distinctions between adipose cells that are ripe for processing/harvesting versus adipose cells that are not yet ready.
  • individual optical interrogation of cells revealing underlying lipid accumulation can be performed, followed by comparing that interrogation to a benchmark threshold for cells known to have a desired lipid accumulation (or other measurable property).
  • These techniques can include active techniques, where the property or properties of the cells are directly measured, and more passive techniques, where the property or properties of the cells are more indirectly observed.
  • the processing/harvesting decisions can be based on monitoring of populations of cells.
  • analytical e.g., optical, electrical, etc.
  • populations of cells can have optical measurements taken (e.g., absorption, scattering measurements, etc.) or electrical measurements taken (e.g., impedance, capacitance, etc ), which can represent important meaningful properties of the population of cells.
  • optical measurements taken e.g., absorption, scattering measurements, etc.
  • electrical measurements taken e.g., impedance, capacitance, etc
  • These techniques can include active techniques, where the property or properties of the population of cells are measured directly, and more passive techniques, where the property or properties of the population of cells are more indirectly observed. Lipid accumulation may be a suitable determinant for monitoring.
  • the processing/harvesting decisions can be based on cell growth.
  • growth can be determined in terms of growth of individual cells (i.e., cell size and/or mass and/or density). In some cases, growth can be determined in terms of growth of the number of cells (i.e., cell count).
  • the processing/harvesting decisions can be based on a degree of cell differentiation.
  • the system and method can include steps relating to differentiation of certain cell types into adipose cells and/or precursors of adipose cells.
  • the monitoring can be user-originated, such that a user can specifically request that the system or method observe a given cell or a given population of cells at a specific time.
  • the monitoring can be automated, such that the monitoring occurs at a predetermined time or that a computer program selected a time based on a given set of criteria. Skilled artisans in the automation arts will recognize a host of options for automating the monitoring of the individual cells or populations of cells.
  • the monitoring involves a destructive sampling process, where one or more cells are removed from the system or method in order to make a representative measurement.
  • sampling techniques can be used, such as physical removal of cells or populations of cells.
  • a user can arbitrarily select the cells for sampling or the user can be directed in some fashion to select specific cells and/or a specific location.
  • the system can include labels that direct the user to a proper sampling location. In some cases, these labels can be digital labels or they can be printed labels.
  • automated sampling can involve routinely retrieving cells from a specific location in the system/method and simply acquiring whatever sample happens to be occupying that location at the given time. This is a good bulk sampling technique to be used in cases where the overall number of cells required for sampling is adequately small when compared with the overall cell population size.
  • automated sampling can involve complex decision trees and/or machine-learning-derived algorithms for selecting cells or populations of cells for interrogation. Examples of suitable selection algorithms include, but are not limited to, random sampling techniques, weighted sampling techniques, and the like.
  • the monitoring involves a non-destructive sampling process, where the cells are not disturbed to a degree that their growth, differentiation, and/or proliferation remain generally unaltered.
  • the user can manually move an analytical device into a location for interrogating a cell or population of cells.
  • a user can arbitrarily select the cells for interrogation or the user can be directed in some fashion to select specific cells and/or a specific location.
  • the systems and methods can utilize labels to direct a user to a proper location. Such labels can be digital or analog.
  • the monitoring is performed on adhered cells.
  • the monitoring systems are configured to interrogate a location where the substrate is positioned during processing. Because the systems and methods are typically deployed in a reproducible fashion, the substrates will typically be located in predictable locations.
  • the monitoring can be performed on non-adhered cells. Those aspects of the present disclosure are described in greater detailed elsewhere herein where "floating" cells are discussed.
  • the monitoring is a specific monitoring of lipid droplet size within a cell or within a population of cells.
  • lipid droplet size is an important factor in determining when an adipose cell is ready for harvesting. This lipid droplet size is optically visible and can be interrogated by known optical methods. It should be appreciated that the lipid droplet size monitoring can also be used to select for undesirable cells, in a similar fashion to the way that it selects for desirable cells.
  • the monitoring can include monitoring the culture media. This analyzing of the culture media can be used for making harvesting decisions or can inform control of other aspects of the method.
  • the culture media can be monitored to determine a concentration of ammonia.
  • concentration of ammonia can be used as a feedback for manual or automated control of the system.
  • the ammonia can be extracted and processed into an end product.
  • ammonia can be extracted and processed into fertilizer using methods known in the art.
  • the culture media can be monitored to determine a concentration of lactic acid in the culture media.
  • concentration of lactic acid can be used as a feedback for manual or automated control of the system.
  • waste products that are not useful as recycled source materials in the system and method described herein can be monitored and removed from the system. If those waste products are capable of being transformed into a useful product, then the system can include those facilities.
  • the lactic acid can be extracted and processed into an end product.
  • lactic acid can be extracted and processed into poly(lactic acid) using methods known in the art.
  • harvesting decisions can be phenotypic.
  • the secretome of the cells can be used to make a harvesting decision.
  • the level of adipokine surrounding the cells can be used as a measure of readiness for harvest.
  • the level of adiponectin could be utilized for harvesting decisions.
  • the systems and methods described herein are particularly useful for processing cells that detach from the substrate during the process of differentiating into fat cells (herein referred to as “floating” or “non-adhered” cells).
  • the systems and methods of this disclosure provide approaches to collecting (or “harvesting” or “aggregating”) and utilizing these floating cells, which have traditionally been discarded as waste. By making productive use of these previously-wasted products, much higher efficiency is achieved by virtue of not wasting this large population of cells.
  • the surface of a growth medium reservoir can be monitored to determine a concentration of floating cells.
  • a liquid-air interface is monitored. If the concentration of floating cells exceeds a predetermined threshold, then the systems and methods can harvest the floating cells. These harvested floating cells can be processed in the fashion described elsewhere herein.
  • an interface between two liquids having different densities can be monitored to determine a concentration of cells at the interface.
  • a liquid-liquid interface is monitored.
  • multiple interfaces between multiple liquids having different densities can be monitored to determined concentrations of cells at the multiple interfaces.
  • multiple liquid-liquid interfaces are monitored.
  • these cells can be divided into quality grades based on one or more properties.
  • cells having a lower density can be assigned a higher grade.
  • cells that remain adhered to a substrate for a shorter length of time are assigned a higher quality grade.
  • cells that remain adhered to a substrate for a longer length of time are assigned the higher quality grade. It should be appreciated that some applications may have a preference for cells that rapidly loser their adherence versus cells that slowly lose their adherence and vice versa. Without wishing to be bound by any particular theory, the systems and methods described herein provide a platform for assigning quality grades to a given cell.
  • the floating cells can be delivered to a chromatographic system and/or a microfluidics system and/or a cell sorting system and/or a cell picker, as will be understood by those having ordinary skill in the cell manipulation arts.
  • a cell skimmer is one particularly useful device for removing cells from the surface.
  • the microfluidics system can be tailored specifically for handling floating cells described herein.
  • One example of tailoring the microfluidics system for floating cells is to adjust the density of the carrier fluid prior to entry into the microfluidics system, such that the cells are neutral buoyance and are no longer "floating" in the medium.
  • a specific stationary phase and mobile phase may be selected for a desired result. For instance, if size selection is an important criterion, then a stationary phase that has porosity that provides differentiated retention properties based on size could be useful. As another example, if surface chemistry of the adipose cells is an important criterion, then a stationary and/or mobile phase could be selected based on those desirable surface chemistries. In one specific example, certain cells may be more amenable to embedding into a hydrogel if they have a specific surface chemistry, so a chromatography system could be used to isolate cells that are more or less amenable to embedding into the hydrogel.
  • certain cells may be more amenable to direct cross-linking to other cells if they have a specific surface chemistry, so a chromatography system could be used to isolate cells that are more or less amenable to direct cross-linking with one another.
  • the cell sorting system can be used to differentiate and categorize the cells based on measurable properties, as would be appreciated by a skilled artisan. Applicant does not intend to provide specific inventive contribution with respect to the cell sorting itself, but rather integrates and utilizes the understood concept of cell sorting into inventive concepts that form a part of the broader disclosure described herein.
  • the cells are sorted through multiple different centrifuges.
  • the cells that make it through multiple centrifuges will have different properties from those that do not and those different categories of cells can be divided into grades, in a similar fashion as USDA grading.
  • the cells could be graded as superior or inferior relative to one another.
  • the cell sorting can involve the use of a density gradient.
  • the different densities can allow harvesting of cells having different buoyancies.
  • the different buoyancies can also be used to grade the cells.
  • some cells that do not meet certain grading threshold or certain quality standards i.e., cells that do not have enough fat in them) can be returned to the system at the relevant stage in the process.
  • sedimented cells can be isolated and further processed as biomass.
  • the sedimented cells can be recycled for raw materials and reintroduced into the system and/or method at various points in the system/process.
  • sedimented cells can be harvested and have components for culture media extracted from them.
  • the sedimented cells are processed for other uses.
  • sedimented cells might be suitable fdler material in animal feedstock.
  • sedimented cells might be suitable for use as a starting material for a further culture.
  • the systems and methods described herein may provide useful sorting and/or isolation of relevant cell populations, such as the sedimented cells described herein.
  • These sedimented cells are artificially selected by virtue of the processing decisions made. These artificial selections can imbue the cell populations with desirable properties (e.g., density can be desirable for certain applications that are not trying to make adipose tissue) and further cultures can grow and expand those populations of cells to have larger populations that are enriched in those desirable properties.
  • the sedimented cells themselves may be usable as a single cell protein for growth of bacteria or fungus.
  • the cell itself serves as the protein source.
  • sedimented cells are typically very high in nucleic acid content. Therefore, in some cases, nucleic acid content is reduced prior to harvesting non-adhered adipose cells. In some cases, the sedimented cells can have nucleic acid content reduced before further use. However, the cells may still be useful without reducing the nucleic acid content, for example, as livestock feed. In some cases, the sedimented cells, with or without reducing the nucleic acid content, can be mixed into one or more of the food products described herein for the purpose of adding more of a "meat" flavor. Reducing nucleic acid content may comprise “rinsing” cells as understood in the art and discussed herein elsewhere.
  • any of the proposed genetic modifications discussed above are also applicable as additives, so long as the science does not render it impossible.
  • the carotenoids for adding color can simply be added to the cells as supplements.
  • those fatty acids can be added to the cells via supplementing. The same is true for myoglobin and hemoglobin.
  • dyes may be added to the cells and/or binder/hydrogel in order to provide a more aesthetically pleasing appearance.
  • the cultured adipose tissue may be particularly useful for introducing lipid-soluble nutrient into subjects.
  • a lipid-soluble nutrient that is challenging for uptake in humans can be supplemented into the cells here and the resulting cultured adipose tissue may enable greater uptake of those lipid-soluble nutrients than if they were administered outside of the cultured adipose tissue.
  • additives that add no proven nutritional or therapeutic value are also contemplated.
  • additives can be purely aesthetic.
  • one additive could be gold nanoparticles, which could be added at any stage of the process and taken up into the cells, thereby producing "gold fat”.
  • various materials from the nutraceutical industry can be incorporated into the cells at various stages of the process, including but not limited to silver compositions which are believed by some to have antimicrobial properties.
  • the systems and methods described herein can provide advantageous ability to administer growth factors to the cells, in the interest of providing them with more optimal receipt and uptake of the growth factors.
  • Certain growth factors may advantageously be administered to cells for the purpose of aiding and/or enhancing their development in the systems and methods disclosed herein.
  • some of the growth factors can be incorporated into the hydrogel or binder in which harvested adipose cells 12 are aggregated.
  • additives can be incorporated into the hydrogel or binder in which harvested adipose cells 12 are aggregated.
  • additives can include, but are not limited to, flavorants, colorants, texturizers, vitamins, minerals, amino acids, proteins/peptides, and fatty acids. These additives can be added to the hydrogel or binder in amounts that are tuned to the desired outcome.
  • Growth factors that are suitable for use as additives include, but are not limited to, fibroblast growth factor 2, transforming growth factor beta 3, insulin-like growth factor, or the like. Growth factors may be fish-specific growth factors.
  • cultured cells are generally deficient in oleic acid, linoleic acid, or arachidonic acid. These can all be additives in the present disclosure, thereby obviating the deficiency.
  • Alternative or additional additives include, but are not limited to, flavorants, colorants, texturizers, vitamins, minerals, amino acids, proteins/peptides, whole foods, oils, and fatty acids.
  • Particular fatty acids such as linoleic acid, arachidonic acid, alpha-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid, may be preferable but are not exclusive.
  • additives may be selected from the group comprising soybean oil, coconut oil, palm oil, cocoa butter, shea butter, mango butter, olive oil, canola oil, sunflower oil, flaxseed oil, avocado oil, peanut oil, sesame oil, corn oil, vegetable oil, margarine, shortening, and vegetable ghee.
  • Additives comprising fatty acids or oils may be hydrogenated at least partially (which includes wholly hydrogenated) or may be present in their oleogel, bigel, or emulgel form.
  • Whole foods may be selected from the group comprising, but not limited to, tofu, tempeh, instantan, jackfruit, quinoa, chia seeds, bananas, oats, avocados, flax seeds, rice, tapioca/arrowroot, and potatoes.
  • One particularly useful additive for the present disclosure is a fatty acid. Without wishing to be bound by any particular theory, it is believed that cell lines that are generally lacking in the production of a desirable fatty acid can be supplemented in the development process to include more of the desirable fatty acid. Again, without wishing to be bound by any particular theory, it is believed that in vitro fat cells have historically been lacking in certain fatty acids.
  • omega 3 fatty acids are also typically lacking in omega 3 fatty acids as these are also dietary. These would be alpha-linolenic acid (18:3), eicosapentaenoic acid (EP A, 20:5), and docosahexaenoic acid (DHA, 22:6). Omega 3s are generally less abundant in terrestrial (land) animals so a lack of them is not that detrimental. However, they can make up a significant proportion of fish lipids, so it may be important to have for fish adipocytes.
  • the supplementing concentrations can be chosen to provide desirable end concentrations.
  • soybean oil can be added to one or more of the stages of the system and method. Soybean oil has been shown to increase lipid drop size in in vitro adipose tissue. By adding soybean oil, a desired droplet size can be achieved more quickly, thereby enhancing efficiency of the overall process.
  • Alternative or additional exemplary oils may include coconut oil, palm oil, cocoa butter, shea butter, mango butter, olive oil, canola oil, sunflower oil, flaxseed oil, avocado oil, peanut oil, sesame oil, com oil, vegetable oil, margarine, shortening, and vegetable ghee. Oils may be partially or wholly hydrogenated and may be constituted in their oleogel, bigel, or emulgel form.
  • the additive might be present in the form of an uptake-enhancing additive for the explicit purpose of enhancing uptake of other additives.
  • an uptake-enhancing additive for the explicit purpose of enhancing uptake of other additives.
  • the uptake-enhancing additive can be an enzyme that modifies a different additive to make that different additive more suitable for uptake (e.g., a lipase with large lipids).
  • the uptake-enhancing additive can be a permeability-enhancing agent, which enhances the permeability of the cell membrane of the adipose cells.
  • One specific avenue for introducing growth factors and/or agents and/or additive into the inventive compositions is by way of adding the growth factors and/or agents and/or additives into the first culture media or the second culture media at the time that those culture media are made and/or when those culture media are introduced into their respective tanks.
  • the culture media can be tailored for proliferation and differentiation, as well for other steps of the process.
  • the various steps can use more than one culture media.
  • one culture media could be used for a first part of a proliferation process and the cells can be transferred to a different culture media for a later part of the proliferation process.
  • the culture media is typically comprised of a basal medium containing basic nutrients (sugars/carbohydrates, lipids/fatty acids, amino acids, vitamins, minerals, salts, water) combined with a growth factor or complex component containing proteins, peptides, hormones, or other bioactive compounds responsible for directing cell behavior, (e.g., to promote proliferation/cell survival, or to promote adipogenesis, or to suppress other cell pathways such as osteogenesis)
  • Basal media will likely be best optimized for the specific adipogenic progenitor cell that is used but may be similar to or based off of existing formulations such as Dulbecco’s modified eagle medium (DMEM), Ham’s F12, Ham’s MCDB 131, Iscove’s modified Dulbecco’s medium (IMDM).
  • Growth factors for adipogenic progenitor proliferation media formulations may include non-exhaustively: serum albumins, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), fetuin, bone morphogenic proteins (BMPs, e.g., BMP4), Wnt proteins, leukemia inhibitor factor (LIF), hydrocortisone, testosterone, progesterone, estrogen, hypoxia inducible factors (HZFs), stem cell growth factor- (SCGF-0), tumor necrosis factor alpha (TNFa), interleukin- 1 beta (IL- 10) and other interleukins (ILs), transforming growth factor beta (TGF0), insulin-like growth factor 1 (IGF-1), insulin, transferrin, myoglobin, vitronectin, truncated vitronectin peptides, laminin, laminin peptides (e g., 511-E8), fibronectin,
  • Growth factors specifically for adipogenic progenitor cell differentiation include non- exhaustively: Phospholipids, cholesterols (e.g., very low density lipoprotein, VLDL), fatty acids (e.g., oleic acid, linoleic acid), triacylglycerides (e.g., soybean oil, olive oil, rapeseed oil, fish oil), cyclodextrins (to solubilize non-polar ingredients), PPARy agonists (e.g., rosiglitazone, pioglitazone, pristanic acid, phytanic acid, lutein, beta-carotene, all-trans retinoic acid, 9-cis retinoic acid), ascorbic acid, isobutylmethylxanthine (IBMX), dexamethasone, hydrocortisone, insulin, insulin-like growth factor 1 (IGF-1). In some cases, components that promote differentiation may overlap with ingredients in the basal medium formulation
  • the effects of more complex growth factors may be replicated more cost effectively using peptides or small molecules.
  • Growth factors, in particular large protein growth factors may also be modified to impart features such as improved thermal stability over time.
  • certain growth factors or media components can be excluded to reduce costs, should the cells be engineered to not require such components/be engineered to generate the components themselves from components or basic/simple nutrients in the basal media.
  • cells can be engineered to produce glutamine synthetase, which would reduce or eliminate the need for glutamine in the culture media formulation.
  • fish-specific versions of the growth factors mentioned herein may be used to for improved/enhanced function (relative to the typically used mammalian versions) when culturing adipogenic progenitor cells from fish or seafood.
  • lactic acid may be extracted from the culture media (or first culture media or second culture media) and processed into polylactic acid. This beneficially repurposes a potential waste product.
  • the computational facilities of the system can include various artificial intelligence and machine learning algorithms to optimize production.
  • machine learning techniques that use a production variable as a target for optimization could work toward a more efficient culture media selection.
  • the optimization process can be automated with appropriate robotics.
  • the present disclosure provides a host of end-use applications for the materials provided by the systems and methods described herein. These end-use applications can be applicable both to the desirable products produced by the systems and methods described herein (i.e., adipose tissue in many/most cases) and the undesirable or waste products produced by the systems and methods described herein (i.e., sedimented cells or non-adipose tissue in many/most cases), unless the context clearly dictates otherwise.
  • the materials produced by the systems and methods described herein can generally be incorporated into edible products.
  • the materials produced by the systems and methods described herein can generally be incorporated into consumer products not limited to cosmetics, skin creams, lotions, soaps, detergents, emulsifiers, lubricants, fuels, and/or animal feeds.
  • the materials produced by the systems and methods described herein can be included in cosmetics, such as make-up, skin creams, lotions, and the like.
  • the materials produced by the systems and methods described herein can be included in soaps, detergents, or other generally emulsifying compositions.
  • the materials produced by the systems and methods described herein can be included in industrial compositions, such as lubricants used in a variety of industries, liquids useful in extraction industries like the oil and gas industry, and the like.
  • the materials produced by the systems and methods described herein can be included in sustainability systems for environmental purposes, such as animal feeds, aquaculture compositions, and the like.
  • the materials produced by the systems and methods described herein can be included in end-use compositions that include some proportion of material that is produced from the systems and methods described herein and some proportion from naturally occurring sources.
  • the cultured adipose tissue described herein can be combined with lean meat from a natural source to make ground meat having an artificially enhanced fat content.
  • the materials produced can have components extracted and that can be the end product.
  • the cultured adipose tissue can be rendered to remove fatty acids and those fatty acids can be the end product.
  • a system for executing the methods described herein includes one or more raw material sources.
  • the raw material sources can feed raw materials into a first bioreactor.
  • the first bioreactor can provide a first output to optionally proceed to a second bioreactor and can provide waste via a waste output to be collected for reuse and recycling.
  • the second bioreactor is not present and the first output is moved on to other portions of the system for further processing.
  • the system can include any number of bioreactors. Those bioreactors can be arranged to operate in series, in parallel, or in any combination of the two.
  • the system includes various extraction, filtration, and monitoring systems, as described elsewhere herein.
  • the raw material sources can be generally conventional material storage and supply technologies as would be appropriate for cell agriculture applications.
  • One important requirement of the raw material sources is that the materials need to be sterile when they are eventually introduced into the system for executing the methods described above.
  • Another optional feature of the raw material sources is temperature control. Particularly for the cellular material, the temperature control is important.
  • the conduits and other means of transport for the various material flows also require sterility and optionally include temperature controls.
  • the raw material sources can have quality control facilities.
  • the raw material sources can have cellular testing facilities (e g., PCR, morphology assessment, genetic testing, etc.).
  • the raw material sources can have appropriate facilities to confirm sterility, such as sampling and testing facilities as would be understood by those having ordinary skill in the art.
  • the raw material sources can be adapted to sterilize the raw materials, using techniques known in the art.
  • At least one portion of the raw material sources is adapted to provide a supply of cells for use in methods described herein.
  • the raw material sources can take any physical form that meets the requirements of the applications described, including but not limited to, storage tanks, hoppers, reservoirs, silos, and other means of storing raw materials on-site for use in the methods described herein.
  • Receiving raw materials into the raw materials sources can be handled in generally conventional ways, with the understanding that the ordering and delivery of cells is an emerging field.
  • the raw material sources are fed by an on-site supply of raw materials.
  • the raw material sources are supplied with locally sourced cells.
  • the system can include the necessary processing facilities to isolate cells of interest from livestock or other local sources of cells.
  • the raw material sources for cells include cells that are embedded in a matrix, such as the natural tissue matrix from which they originated.
  • the cells used herein can be provided to the system in the form of agricultural silage mixtures.
  • the cells themselves may be extracted from agricultural waste products.
  • the raw material sources are supplied or refilled via pipelines or other conduits for materials. In some cases, the raw material sources are themselves pipelines or other conduits for materials.
  • the system include an entire sub-facility dedicated to producing cells for use in the methods described herein.
  • This sub-facility can include all of the various processing systems that are required for processing agricultural and livestock products to isolate cells for use as described herein.
  • the systems may be paired with conventional farming and food distributions systems, such as butcheries or meat packing facilities. It should be appreciated that the initial stages of the growth of cellular agriculture are unlikely to supplant traditional meat sources, so the systems described herein may benefit from being integrated into a conventional food supply chain. In these cases, products that are typically viewed as waste could provide useful cells for use in the methods described herein.
  • the system can be divided into raw material production facilities, where cells and other reagents are produced, and end product production facilities, where the desired adipose tissue is produced.
  • the raw material sources can include sources for the various reagents used in the methods, including the culture media. As with the cellular material, the reagents used in the methods can also be locally sourced.
  • At least one portion of the raw material sources is adapted to provide culture media for use in the methods described herein.
  • These raw material sources can include powdered storage media that is combined with purified water to make culture media or they can include the completed culture media itself.
  • the system is adapted to receive commercial-scale quantities of material, so the system includes receiving docks and pipe infrastructure to receive trailer trucks and tanker trucks of raw materials. Similarly, rail, air, and sea transport features are contemplated. As outlined above, pipelines are also a viable option for receiving raw material.
  • the raw materials can be recycled materials, including recycled agricultural materials and other recycled materials that can be useful in the processes described herein. In some cases, the raw materials are recycled from the system or method described herein.
  • the raw materials are delivered from the raw material sources into the first bioreactor.
  • the means for delivery are conventional, with sterility maintained.
  • Material can be transferred between bioreactors using conventional delivery means, with sterility maintained.
  • Waste streams can be moved in conventional ways. However, the systems and methods described herein make improved use of waste. For example, most of the waste streams in this system and method can be recycled within the system.
  • Cellular material can be isolated from the culture media in ways understood to those having ordinary skill in the art, including centrifugation and filtration.
  • a device for separating cells from culture media is a counter flow centrifuge.
  • the cells can be isolated from culture media by using micro-carrier, such as those made out of pectin.
  • the cells can be harvested using techniques made popular in different industries, such as the pharmaceutical industry and brewing industry, where cellular processes are used widely.
  • the cells can be harvested as floating cells.
  • certain specific harvesting devices can be used, which may not be applicable in other environments.
  • a skimmer could be used to skim the surface of a bioreactor to remove surface cells.
  • a screen or mesh could be located beneath the surface of the culture media and then raised out of the culture media to harvest the floating cells while the culture media is drained and left behind.
  • the cells can be harvested as sedimented cells.
  • certain specific harvesting devices can be used, which may not be applicable in other environments.
  • suction filtration of a bottom portion of the bioreactor may be one suitable way to harvest sedimented cells.
  • adhered cells can be harvested from a substrate using conventional methods. It should be appreciated that harvesting the adhered cells are not specifically the focus of most of this disclosure, though many of the features described herein are applicable to adhered cells. For the avoidance of doubt, the features of the present disclosure are also contemplated for use with harvested adhered cells.
  • Applicant has developed a technique for harvesting cells from the surface, which is different from existing techniques.
  • a layer of fat cells or otherwise
  • the layer is pipetted off the top by placing the tip of the pipette into the fat layer and introducing suction.
  • this conventional process causes problems with certain samples and the fat layer cannot be isolated.
  • the inventors surprisingly discovered that removing fat cells by maintaining suction on a pipette as the tip is drawn close to the fat layer, contacting the fat layer with the tip of the pipette, then lifting the pipette away from the surface, thereby removing a small section of the layer.
  • This process is repeated for different locations on the layer, until most or all of the layer has been removed.
  • This process can be manual or automated. If automated, a robotic arm with sensors can bring the pipette tip to the surface with adequate control to selectively remove the fat layer.
  • the culture media within a given bioreactor is replaced with every usage.
  • the culture media is retained for some length of time before being replaced.
  • the culture media is slowly regenerated over time by supplementing the portions of the media that are removed in the process.
  • the system can include culture media fdters, which filter culture media for reintroduction into the bioreactors.
  • the system can include an analytical device, such as optical spectrophotometer, a gas chromatography system, a mass spectrometry system, other analytical devices known in the art to useful for assessing quality control of liquid compositions such as culture media, or a combination thereof. This analytical device can be used to assess new or used culture media to determine if it is appropriate for further use.
  • the analytical device is used to search for contaminants, such as undesirable bacterial growth or harsh chemical solvents. If contamination is identified, then the culture media can be routed to waste or to a refining facility for removal of the contaminants.
  • the analytical device is used to confirm that certain desirable components are present.
  • the analytical device can confirm the presence or amount of one of the growth factors discussed above. If the analytical device identifies one or more desirable components are missing from a new or used culture media, then the culture media can be supplemented with the missing desirable components.
  • the analytical device inspects for both contaminants and desirable components and directs the system to make the necessary remedial corrections, if needed.
  • the facility is equipped with the necessary scanning and tracking devices that would be required to keep track of the sourcing of the various components that go into the methods described herein.
  • one of the powerful advantages that may be provided with cellular agriculture is the reduction in transportation costs that are required to transport meat from places where the animals are harvests to the consumer.
  • the system can be located nearer to the consumer, so the bulk of the shipping cost and environmental impact is related to raw material shipping.
  • the system includes the necessary computing facilities to generate and/or modify blockchains in ways that are understood by those in the art to be useful for tracking the authenticity or sourcing of food products. Again, without wishing to be bound by any particular theory, it is believed that there may be advantage to providing consumers with evidence of how little shipping was required to produce a given food product.
  • blockchain sourcing authentication techniques or other methods known in the art to provide similar capabilities
  • the culture adipose tissue produced by the methods described herein can have corresponding entries into a blockchain regarding the cultured adipose tissue's provenance.
  • the system and method described herein has the full capability to acquire and record any data that is necessary for complying with food regulatory authorities.
  • Logs of processing parameters, test results, and other information that is relevant to regulators can be created and saved. The logs can be made manually or automatically.
  • the digital records can be utilized to improve the system and method.
  • the improvements can be based on measured properties of the products, but in other cases, the improvements can be based on user feedback. There is inherently a lag between the facility producing a product and consumers enjoying it, so if there is a wave of product that has a particularly positive or negative customer response, then it would be possible to look up specific information from the run that led to that customer response for the purpose of intentionally repeating or not repeating it.
  • the data can include both information about the cell source material and the other formulation information from the process. If a certain cell line, process, and set of formulations produces a superior product, then the formula can be retrieved for reproduction.
  • Data can be collected from multiple facilities, thereby providing a global data set, from which broader conclusions can be drawn. For example, if all plants around the world show an inefficiency in the use of a given reagent, then this flags the problem as likely being related to the portions of the system or method that are deployed in all facilities.
  • the present disclosure provides a food product prepared directly from the culture adipose tissue that is made by the method described herein.
  • This product stands in contrast to the other food products described herein, where the cultured adipose tissue is integrated with other components to make a food product that includes the cultured adipose tissue.
  • the disclosed food product is a shaped and cooked piece of the cultured adipose tissue described herein. Without wishing to be bound by any particular theory, it is believed that the cultured adipose tissue disclosed herein can be treated as a food product with similar properties to certain animal products.
  • slices of the cultured adipose tissue can be fried to form cultured adipose chips, which may resemble pork rinds in certain embodiments.
  • slices of the culture adipose tissue can be seared to form a seared fatty tissue morsel.
  • the cultured adipose tissue is cooked or fried.
  • the cultured adipose tissue is shaped to a particular three- dimensional configuration as desired.
  • the food products described herein both those that integrate the culture adipose tissue and those that are composed entirely of cultured adipose tissue, can be adapted in the ways described above with respect to altering the nutritional or flavor contents, either by way of genetic manipulation or process manipulation. In this fashion, a previously unhealthy product can be adapted to provide a healthier alternative.
  • a cultured adipose tissue chip that is flavored to taste like pork rinds could be adapted to have a different fatty acid blend, thereby producing fewer negative health consequences to a consumer than a comparison conventional pork rind.
  • comparisons regarding the health consequences of food may require larger studies and statistical averages rather than direct measurements of individual cases.
  • Example 1 Timeline of 3T3-L1 adipogenic differentiation
  • FIG. 5 shows a timeline for differentiation of 3T3-L1 adipogenic cells. Days 0 (dO), 2 (t/2), 15 and 30 (d3O) are indicated on the timeline. Confluent pre-adipocytes were grown in adipogenic induction media for the first two days and then switched to lipid accumulation media until harvest for cultured fat tissue formation on day 15 (see Example 2). Additional samples were grown in lipid accumulation media for 30 days to analyze lipid accumulation over longer-term culture.
  • Lipidomics and immunostaining were carried out on day 13. Additional samples were grown in lipid accumulation media for 1 month to analyze lipid accumulation over longer-term culture.
  • Example 2 Harvest of lipid-laden adipocytes and formation of 3D cultured fat constructs
  • lipid-filled adipocytes were detached using a cell scraper. The adipocytes were then drained of non-cell liquid using a 0.22 micrometer vacuum filter. After detaching and draining, the in vitro adipocytes were then combined with transglutaminase or alginate and formed into discrete macroscale tissues in a 3D printed mold. Finally, 3D cultured fat constructs were mechanically tested for compressive strength, fluorescently stained for lipid and analyzed for volatile compounds.
  • Example 3 Methods for generating 3D cultured fat using alginate or transglutaminase
  • Transglutaminase aggregation Cultured fat was produced by mixing a 15% solution of transglutaminase with drained adipose tissue at a 2:8 volumetric ratio in a 3D printed mold.
  • Example 4 Generating Macroscale Cultured Fat via the Aggregation of In Vitro Grown Adipocytes
  • Native (in vivo) adipose tissue is largely a dense packing (aggregation) of lipid-filled adipocytes, held together by a sparse extracellular matrix (ECM) network. This is opposed to muscle tissue, which is comprised of aligned fibers in a multi -hi erar chi cal structure. Since native adipose is an aggregation of adipocyte globules with less structural features than in muscle it is possible to recapitulate it on an organoleptic basis by aggregating separately grown adipocytes or adipocyte clusters.
  • ECM extracellular matrix
  • adipocytes separately, or in small clusters, is desirable because it is currently infeasible to directly grow large tissues on the macroscale (millimeter scale and up) using contemporary tissue engineering techniques.
  • By growing individual or small clusters of cells we are able to produce-at-scale a general mass of adipose cells, followed by the formation of actual adipose tissue through various methods of binding and aggregating the cells into a solid 3D construct.
  • Methods of binding and aggregating the fat cells include enzymatic cross-linking with transglutaminase, as well as embedding in hydrogels such as alginate (a material which is already used as a fat replacer in the food industry).
  • An adipose aggregation approach circumvents the mass transport issue of macroscale 3D culture/tissue engineering by deliberately culturing individual adipocytes or small adipocyte clusters via 2D culture or various scalable bioreactors. Only after the separated adipocytes have grown and accumulated sufficient lipid does cell culture end, followed by the manual aggregation of the cultured cells into denser 3D tissues without the need for vascularization and perfusion. This is uniquely possible with cultured fat because for food purposes, for example, once the tissue is formed and harvested the cultured cells do not need to stay alive. This process is analogous to meat production in conventional animal agriculture where muscle and fat cells gradually cease to be viable after slaughter. For conventional tissue engineering (e.g., medical applications etc.) cells in 3D tissues are expected to remain viable to be used for implantation into the body, or for use and testing in an in vitro tissue model.
  • tissue engineering e.g., medical applications etc.
  • Example 5 Avoiding lift-off issues faced with adherent adipocytes
  • Applicant has also observed that a large subpopulation of the cultured adipocytes adhere strongly to tissue culture plates and do not float away, avoiding issues of lift-off of adherent adipocytes in vitro due to increasing buoyancy as the adipocytes become fatty.
  • the 2D culture systems self-sort for adherent cell populations.
  • techniques have been developed for dealing with the non-adherent cell populations.
  • An assessment of such benchmark threshold or property/properties allows for the processing/harvesting decision to select an appropriate time for processing/harvesting cells (or cell). This decision can be made based on cell growth ((i.e., cell size and/or mass and/or density, number of cells or cell count).
  • lipid droplet size is an important factor in determining when an adipose cell is ready for harvesting. This lipid droplet size is optically visible and can be interrogated by known optical methods. As such, monitoring of lipid droplet size within a cell or within a population of cells could be used for harvesting decisions.
  • the cell secretome can also be analyzed. Specifically, the level of adipokine or adiponectin surrounding the cells can be used as a measure of readiness for harvest.
  • monitoring can be performed on non-adhered (or “floating”) cells.
  • floating cells In the case where multiple different density gradients are deployed and/or where multiple different populations of floating cells are collected, these cells can be divided into quality grades based on one or more properties as discussed above.
  • Floating cells can be delivered to a number of useful devices including chromatographic systems, microfluidics systems, cell sorting systems, cell pickers, and cell skimmers, to name a few examples.
  • the microfluidics system can be tailored specifically for handling floating cells described herein.
  • One example of tailoring the microfluidics system for floating cells is to adjust the density of the carrier fluid prior to entry into the microfluidics system, such that the cells are neutral buoyance and are no longer "floating" in the medium.
  • the harvested cells might be rinsed to remove/minimize/dilute undesired culture media components. Rinsing can be done by way of counter-flow centrifugation or conventional filtration. As another example, cells can be clarified by removing undesirable components, which are not removed by rinsing. This is a process that is regularly used in the pharmaceutical arts, but it is not conventionally used in cellular agriculture. In one specific example, clarification of cells to remove cell debris or excess nucleic acids is expressly contemplated. Other methods such as flocculation via pH or the use of polymers such as chitosan which can induce flocculation are contemplated. Other methods of collecting non-adhered cells may comprise collecting non-clustered floating cells, collecting only non-adhered cells, and/or not collecting adhered cells.
  • a specific stationary phase and mobile phase may be selected for a desired result. For instance, if size selection is an important criterion, then a stationary phase that has porosity that provides differentiated retention properties based on size could be useful. As another example, if surface chemistry of the adipose cells is an important criterion, then a stationary and/or mobile phase could be selected based on those desirable surface chemistries. In one specific example, certain cells may be more amenable to embedding into a hydrogel if they have a specific surface chemistry, so a chromatography system could be used to isolate cells that are more or less amenable to embedding into the hydrogel.
  • certain cells may be more amenable to direct cross-linking to other cells if they have a specific surface chemistry, so a chromatography system could be used to isolate cells that are more or less amenable to direct cross-linking with one another.
  • the cell sorting system can be used to differentiate and categorize the cells based on measurable properties, as would be appreciated by a skilled artisan. Applicant does not intend to provide specific inventive contribution with respect to the cell sorting itself, but rather integrates and utilizes the understood concept of cell sorting into inventive concepts that form a part of the broader disclosure described herein.
  • the cells are sorted through multiple different centrifuges.
  • the cells that make it through multiple centrifuges will have different properties from those that do not and those different categories of cells can be divided into grades, in a similar fashion as USDA grading.
  • the cells could be graded as superior or inferior relative to one another.
  • the systems and methods of this disclosure provide approaches to collecting and utilizing these floating cells, which have traditionally been discarded as waste. By making productive use of these previously wasted products, much higher efficiency is achieved by virtue of not wasting this large population of cells.
  • sedimented cells may be inversely applicable to sedimented cells.
  • the bottom of a reaction tank could be monitored for sedimented cells.
  • Sedimented cells may be preferred for certain applications and those sedimented cells can be collected and further processed for those applications.
  • adipose tissue which is generally buoyant in aqueous environments such as growth cultures, the sedimented cells are typically waste products and can be processed as such.
  • Sedimented cells can be isolated and further processed as biomass or recycled for raw materials and reintroduced into the system and/or method at various points in the system/process. For example, sedimented cells can be harvested and have components for culture media extracted from them. In certain cases sedimented cells are harvested separately from non-adhered cells. Other uses may include filler material in animal feedstock, starting material for a further culture, and/or a single cell protein for growth of bacteria or fungus.
  • sedimented cells are typically very high in nucleic acid content.
  • the sedimented cells can have nucleic acid content reduced before further use.
  • the cells can still be useful without reducing the nucleic acid content, for example, as livestock feed.
  • the sedimented cells, with or without reducing the nucleic acid content can be mixed into one or more of the food products described herein for the purpose of adding more of a "meat" flavor.
  • Example 7 Advantages in culturing adipocytes
  • the techniques described herein can provide advantageous ability to administer growth factors to the cells, in the interest of providing them with more optimal receipt and uptake of the growth factors.
  • Growth factors and other additives can be incorporated into the hydrogel or binder in which harvested adipose cells 12 are aggregated. These additives can include flavorants, colorants, texturizers, vitamins, minerals, amino acids, proteins/peptides, and fatty acids and can be added to the hydrogel or binder in amounts that are tuned to the desired outcome.
  • Growth factors that are suitable for use as additives can include fibroblast growth factor 2, transforming growth factor beta 3, and insulin-like growth factor. It is believed that cultured cells are generally deficient in oleic acid, linoleic acid, and/or arachidonic acid. Thus, these can all be additives, thereby obviating the deficiency.
  • One particularly useful additive for the present disclosure is a fatty acid. It is believed that cell lines that are generally lacking in the production of a desirable fatty acid can be supplemented in the development process to include more of the desirable fatty acid and that in vitro fat cells have historically been lacking in certain fatty acids.
  • the most abundantly missing fatty acid is linoleic acid (18:2), as mammals take this from the diet rather than synthesizing it themselves.
  • the lack of 20:4 can be both good and bad, as it's been reported to be important for flavor but also a precursor molecule to pro-inflammatory compounds (eicosanoids).
  • Jn vitro mammalian cells are also typically lacking in omega 3 fatty acids as these are also dietary. These would be alphalinolenic acid (18:3), eicosapentaenoic acid (EP A, 20:5), and docosahexaenoic acid (DHA, 22:6). Omega 3s are generally less abundant in terrestrial (land) animals so a lack of them is not that detrimental. However, they can make up a significant proportion of fish lipids, so it may be important to have for fish adipocytes. In a general sense, when multiple different fatty acids are supplemented, the supplementing concentrations can be chosen to provide desirable end concentrations.
  • soybean oil can be added. Soybean oil has been shown to increase lipid drop size in in vitro adipose tissue. By adding soybean oil, a desired droplet size can be achieved more quickly, thereby enhancing efficiency of the overall process.
  • Certain additives may be present in the form of an uptake-enhancing additive for the explicit purpose of enhancing uptake of other additives.
  • the uptake of triglycerides was enhanced by administering an agent that induced the release of a relevant lipase, which allowed the breakdown of the triglycerides for entry into the cells.
  • Co-administered agents may be required to be administered with a given additive in the interest of improving uptake of the additive.
  • the uptake-enhancing additive can be an enzyme that modifies a different additive to make that different additive more suitable for uptake (e.g., a lipase with large lipids).
  • the uptakeenhancing additive can be a permeability-enhancing agent, which enhances the permeability of the cell membrane of the adipose cells.
  • One specific avenue for introducing growth factors and/or agents and/or additive into the inventive compositions is by way of adding the growth factors and/or agents and/or additives into the first culture media or the second culture media at the time that those culture media are made and/or when those culture media are introduced into their respective tanks.
  • the culture media can be tailored for proliferation and differentiation, as well for other steps of the process.
  • the various steps can use more than one culture media.
  • one culture media could be used for a first part of a proliferation process and the cells can be transferred to a different culture media for a later part of the proliferation process.
  • the culture media is typically comprised of a basal medium containing basic nutrients (sugars/carbohydrates, lipids/fatty acids, amino acids, vitamins, minerals, salts, water) combined with a growth factor or complex component containing proteins, peptides, hormones, or other bioactive compounds responsible for directing cell behavior, (e.g., to promote proliferation/cell survival, or to promote adipogenesis, or to suppress other cell pathways such as osteogenesis).
  • basic nutrients sucgars/carbohydrates, lipids/fatty acids, amino acids, vitamins, minerals, salts, water
  • a growth factor or complex component containing proteins, peptides, hormones, or other bioactive compounds responsible for directing cell behavior, (e.g., to promote proliferation/cell survival, or to promote adipogenesis, or to suppress other cell pathways such as osteogenesis).
  • Basal media will likely be best optimized for the specific adipogenic progenitor cell that is used but may be similar to or based off of existing formulations such as Dulbecco’s modified eagle medium (DMEM), Ham’s F12, Ham’s MCDB 131, Iscove’s modified Dulbecco’s medium (IMDM).
  • DMEM Dulbecco modified eagle medium
  • IMDM Iscove’s modified Dulbecco’s medium
  • Growth factors for adipogenic progenitor proliferation media formulations may include non-exhaustively: serum albumins, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), fetuin, bone morphogenic proteins (BMPs, e.g., BMP4), Wnt proteins, leukemia inhibitor factor (LIF), hydrocortisone, testosterone, progesterone, estrogen, hypoxia inducible factors (HIFs), stem cell growth factor-P (SCGF-P), tumor necrosis factor alpha (TNFa), interleukin- 1 beta (IL- 1 ) and other interleukins (ILs), transforming growth factor beta (TGFP), insulin-like growth factor 1 (IGF-1), insulin, transferrin, myoglobin, vitronectin, truncated vitronectin peptides, laminin, laminin peptides (e.g., 511-E8), fibronectin,
  • Growth factors specifically for adipogenic progenitor cell differentiation include non- exhaustively: Phospholipids, cholesterols (e.g., very low density lipoprotein, VLDL), fatty acids (e.g., oleic acid, linoleic acid), triacylglycerides (e.g., soybean oil, olive oil, rapeseed oil, fish oil), cyclodextrins (to solubilize non-polar ingredients), PPARy agonists (e.g., rosiglitazone, pioglitazone, pristanic acid, phytanic acid, lutein, beta-carotene, all-trans retinoic acid, 9-cis retinoic acid), ascorbic acid, isobutylmethylxanthine (IBMX), dexamethasone, hydrocortisone, insulin, insulin-like growth factor 1 (IGF-1). In some cases, components that promote differentiation may overlap with ingredients in the basal medium formulation
  • Example 8 A system for culturing adipocytes
  • a system for culturing adipocytes includes one or more raw material sources feeding into a first bioreactor.
  • the first bioreactor can provide a first output to optionally proceed to a second bioreactor and can provide waste via a waste output to be collected for reuse and recycling.
  • a second bioreactor may not be present and the first output is moved on to other portions of the system for further processing.
  • the system can include any number of bioreactors. Those bioreactors can be arranged to operate in series, in parallel, or in any combination of the two.
  • the system includes various extraction, filtration, and monitoring systems.
  • the raw material sources can be generally conventional material storage and supply technologies as would be appropriate for cell agriculture applications.
  • the raw material sources need to be sterile when they are eventually introduced into the system for executing the methods described above.
  • Another optional feature of the raw material sources is temperature control. Particularly for the cellular material, the temperature control is important.
  • the conduits and other means of transport for the various material flows also require sterility and optionally include temperature controls.
  • the raw material sources can have quality control facilities, cellular testing facilities (e.g., PCR, morphology assessment, genetic testing, etc.), and/or appropriate facilities to confirm sterility, such as sampling and testing facilities.
  • the raw material sources can be adapted to sterilize the raw materials. At least one portion of the raw material sources is adapted to provide a supply of cells for use in the methods described herein.
  • the raw material sources can take any physical form that meets the requirements of the applications described, including but not limited to, storage tanks, hoppers, reservoirs, silos, and other means of storing raw materials onsite for use in the methods described herein. Receiving raw materials into the raw materials sources can be handled in generally conventional ways, with the understanding that the ordering and delivery of cells is an emerging field.
  • the system is adapted to receive commercial-scale delivery of cells as raw material for the methods described herein.
  • sterility is of paramount importance.
  • the raw material sources may be fed by an on-site supply of raw materials and/or with locally sourced cells.
  • the system can include the necessary processing facilities to isolate cells of interest from livestock or other local sources of cells.
  • the raw material sources for cells may include cells that are embedded in a matrix, such as the natural tissue matrix from which they originated and may be provided to the system in the form of agricultural silage mixtures.
  • the cells themselves may be extracted from agricultural waste products.
  • the system may include an entire sub-facility dedicated to producing cells for use in the methods described herein.
  • This sub-facility can include all of the various processing systems that are required for processing agricultural and livestock products to isolate cells
  • the systems may be paired with conventional farming and food distributions systems, such as butcheries or meat packing facilities. It should be appreciated that the initial stages of the growth of cellular agriculture are unlikely to supplant traditional meat sources, so the systems described herein may benefit from being integrated into a conventional food supply chain. In these cases, products that are typically viewed as waste could provide useful cells for use in the methods described herein.
  • the system can be divided into raw material production facilities, where cells and other reagents are produced, and end product production facilities, where the desired adipose tissue is produced. At least one portion of the raw material sources is adapted to provide culture media for use in the methods described herein. These raw material sources can include powdered storage media that is combined with purified water to make culture media or they can include the completed culture media itself.
  • the system is adapted to receive commercial-scale quantities of material, so the system includes receiving docks and pipe infrastructure to receive trailer trucks and tanker trucks of raw materials. Similarly, rail, air, and sea transport features are contemplated. As outlined above, pipelines are also a viable option for receiving raw material.
  • the raw materials can be recycled materials, including recycled agricultural materials and other recycled materials that can be useful in the processes described herein. In some cases, the raw materials are recycled from the system or method described herein.
  • the raw materials are delivered from the raw material sources into the first bioreactor.
  • the means for delivery into the first bioreactor and for the transfer between bioreactors are conventional, with sterility maintained.
  • Waste streams can be moved in conventional ways. However, the systems and methods described herein make improved use of waste. For example, most of the waste streams in this system and method can be recycled within the system.
  • Cellular material can be isolated from the culture media in ways understood to those having ordinary skill in the art, including centrifugation and filtration.
  • a device for separating cells from culture media is a counter flow centrifuge.
  • the cells can be isolated from culture media by using micro-carrier, such as those made out of pectin.
  • the cells can be harvested using techniques made popular in different industries, such as the pharmaceutical industry and brewing industry, where cellular processes are used widely. As discussed in Example 7, the cells can be harvested as floating cells. In these cases, certain specific harvesting devices can be used, which may not be applicable in other environments. For example, a skimmer could be used to skim the surface of a bioreactor to remove surface cells.
  • a screen or mesh could be located beneath the surface of the culture media and then raised out of the culture media to harvest the floating cells while the culture media is drained and left behind.
  • the cells can be harvested as sedimented cells.
  • certain specific harvesting devices can be used, which may not be applicable in other environments. For example, suction filtration of a bottom portion of the bioreactor may be one suitable way to harvest sedimented cells.
  • the culture media within a given bioreactor is replaced with every usage. In some cases, the culture media is retained for some length of time before being replaced. In some cases, the culture media is slowly regenerated over time by supplementing the portions of the media that are removed in the process.
  • the system can include culture media filters, which filter culture media for reintroduction into the bioreactors.
  • the system can include an analytical device, such as optical spectrophotometer, a gas chromatography system, a mass spectrometry system, other analytical devices known in the art to useful for assessing quality control of liquid compositions such as culture media, or a combination thereof. This analytical device can be used to assess new or used culture media to determine if it is appropriate for further use.
  • the analytical device may be used to search for contaminants, such as undesirable bacterial growth or harsh chemical solvents. If contamination is identified, then the culture media can be routed to waste or to a refining facility for removal of the contaminants.
  • the analytical device may be used to confirm that certain desirable components are present. For example, the analytical device can confirm the presence or amount of one of the growth factors discussed above. If the analytical device identifies one or more desirable components are missing from a new or used culture media, then the culture media can be supplemented with the missing desirable components. In some cases, the analytical device inspects for both contaminants and desirable components and directs the system to make the necessary remedial corrections, if needed.
  • the facility may be equipped with the necessary scanning and tracking devices that would be required to keep track of the sourcing of the various components that go into the methods described herein.
  • one of the powerful advantages that may be provided with cellular agriculture is the reduction in transportation costs that are required to transport meat from places where the animals are harvests to the consumer.
  • the system can be located nearer to the consumer, so the bulk of the shipping cost and environmental impact is related to raw material shipping.
  • the system includes the necessary computing facilities to generate and/or modify blockchains in ways that are understood by those in the art to be useful for tracking the authenticity or sourcing of food products. Again, without wishing to be bound by any particular theory, it is believed that there may be advantage to providing consumers with evidence of how little shipping was required to produce a given food product.
  • blockchain sourcing authentication techniques or other methods known in the art to provide similar capabilities
  • the culture adipose tissue produced by the methods described herein can have corresponding entries into a blockchain regarding the cultured adipose tissue's provenance.
  • the system and method described herein has the full capability to acquire and record any data that is necessary for complying with food regulatory authorities.
  • Logs of processing parameters, test results, and other information that is relevant to regulators can be created and saved.
  • the logs can be made manually or automatically.
  • the digital records can be utilized to improve the system and method. In some cases, the improvements can be based on measured properties of the products, but in other cases, the improvements can be based on user feedback.
  • the data can include both information about the cell source material and the other formulation information from the process. If a certain cell line, process, and set of formulations produces a superior product, then the formula can be retrieved for reproduction. Data can be collected from multiple facilities, thereby providing a global data set, from which broader conclusions can be drawn. For example, if all plants around the world show an inefficiency in the use of a given reagent, then this flags the problem as likely being related to the portions of the system or method that are deployed in all facilities.
  • Example 9 A technique for harvesting cells from the surface
  • Applicant has developed a technique for harvesting cells from the surface, which is different from existing techniques.
  • a layer of fat cells or otherwise
  • the layer is pipetted off the top by placing the tip of the pipette into the fat layer and introducing suction.
  • this conventional process causes problems with certain samples and the fat layer cannot be isolated.
  • the inventors surprisingly discovered that removing fat cells by maintaining suction on a pipette as the tip is drawn close to the fat layer, contacting the fat layer with the tip of the pipette, then lifting the pipette away from the surface, thereby removing a small section of the layer.
  • This process is repeated for different locations on the layer, until most or all of the layer has been removed.
  • This process can be manual or automated. If automated, a robotic arm with sensors can bring the pipette tip to the surface with adequate control to selectively remove the fat layer.
  • Example 10 Aggregation of adipose cells by flocculation
  • Applicant proposes the aggregation of non-adhered adipose cells by flocculation.
  • Flocculation often refers to the aggregation of unstable and small particles through surface charge neutralization, electrostatic patching and/or bridging after addition of flocculants (or agents that make fine and sub-fine solids or colloids suspended in the solution form large loose flocs through bridging thus achieving solid-liquid separation).
  • Flocculation may be performed by one or more of several techniques, including auto-flocculation, bio-flocculation, chemical flocculation, particle-based flocculation, and electrochemical flocculation.
  • Various flocculation techniques have been reviewed by Matter, etal (2019, Applied Sciences).
  • Auto-flocculation may be performed at acidic pH (for example, a pH of 4.0). Autoflocculation may be performed at alkaline pH (for example, a pH of 10.4, 11.0, 1.5, 11.6, 12.0, or 12.5). Auto-flocculation may be performed over several days (for example, 16 days).
  • Chemical flocculation may be performed with inorganic flocculants such as aluminum sulfate, calcium oxide, iron(III) chloride, and magnesium hydroxide. Chemical flocculation may be performed with organic flocculants such as inulin, starches, chitosan, tannins, alginates, and lysines. Food-safe flocculants (such as chitosan and other long polymers) may be preferred.
  • inorganic flocculants such as aluminum sulfate, calcium oxide, iron(III) chloride, and magnesium hydroxide.
  • Chemical flocculation may be performed with organic flocculants such as inulin, starches, chitosan, tannins, alginates, and lysines. Food-safe flocculants (such as chitosan and other long polymers) may be preferred.
  • Particle-based flocculation may be performed using aminoclay-based nanoparticles such as 3-aminopropyltriethoxysilane (APTES) conjugated to magnesium and aminoclays conjugated to aluminum, titanium oxide, and humic acid.
  • Particle-based flocculation may be performed using magnetic particles including ferrous oxide nanoparticles and composites.
  • Adipose cells aggregated through means discussed in the present example may be further processed or used in the various methods, food products, systems (etc.) described in the present application.
  • Example 11 Food product prepared by adipose tissue made by the methods herein
  • a food product may be prepared directly from the culture adipose tissue that is made by the method described herein. This product stands in contrast to the other food products described herein, where the cultured adipose tissue is integrated with other components to make a food product that includes the cultured adipose tissue.
  • the disclosed food product is a shaped and cooked piece of the cultured adipose tissue described herein. It is believed that the cultured adipose tissue disclosed herein can be treated as a food product with similar properties to certain animal products.
  • slices of the cultured adipose tissue can be fried to form cultured adipose chips, which may resemble pork rinds in certain embodiments.
  • slices of the culture adipose tissue can be seared to form a seared fatty tissue morsel.
  • a method for producing cultured adipose tissue comprising: growing adipogenic precursor cells in a first culture media; differentiating the adipogenic precursor cells to adipose cells in a second culture media; harvesting non-adhered adipose cells; and aggregating the harvested non-adhered adipose cells to provide the cultured adipose tissue.
  • a method for producing cultured adipose tissue comprising: growing adipogenic precursor cells in a culture media; differentiating the adipogenic precursor cells to adipose cells in the culture media; harvesting non-adhered adipose cells; and aggregating the harvested non-adhered adipose cells to provide the cultured adipose tissue.
  • bioreactor is selected from the group consisting of a stirred suspension tank bioreactor, airlift bioreactor, bubble column bioreactor, fluidized bed bioreactor, packed bed bioreactor, vertical wheel bioreactor, and wave bag bioreactor .
  • a method for producing cultured adipose tissue comprising: growing adipogenic precursor cells on a two-dimensional (2D) substrate; differentiating the adipogenic precursor cells to adipose cells on the 2D substrate; harvesting non-adhered adipose cells; and aggregating the harvested non-adhered adipose cells to provide the cultured adipose tissue.
  • 2D two-dimensional
  • a method for producing cultured adipose tissue comprising: culturing adipose cells from adipogenic precursor cells in culture media; harvesting non-adhered adipose cells after a predetermined or desired amount of adipose cells are produced; and aggregating the harvested non-adhered adipose cells to provide the cultured adipose tissue.
  • adipogenic precursor cells are selected from the group consisting of mesenchymal stem cells, dedifferentiated fat cells, fibroblasts, and fibroadipogenic progenitor cells.
  • hydrogel or binder is selected from the group consisting of alginate, cellulose, gelatin, starch, tara gum, agar, agarose, carrageenan, cornstarch, gellan gum, lecithin, maltodextrin, methylcellulose, carboxymethylcellulose, cellulose gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose tragacanth, karaya, acacia, ghatti, beta-glucan, psyllium husk, inulin, chitosan, curdlan, dextran, potato starch, tapioca/arrowroot starch, hyaluronic acid, fibrin, carrageenan, guar gum, inulin, konjac, oat bran, pectin, locust bean gum, xanthan gum, soy protein, wheat protein, pea protein, chickpea protein, almond protein, rice protein, hemp protein, quinoa protein, sunflower
  • cross-linking the harvested non-adhered adipose cells comprises cross-linking the harvested non-adhered adipose cells using an enzyme selected from the group consisting of a transglutaminase, a tyrosinase, a peroxidase, a laccase, a sortase, a subtilisin, and lysyl oxidase.
  • cross-linking the harvested non-adhered adipose cells comprises enzymatically cross-linking the harvested non-adhered adipose cells using transglutaminase.
  • cross-linking the harvested non-adhered adipose cells with transglutaminase comprises mixing a solution of transglutaminase with the harvested non-adhered adipose cells at a predetermined volumetric ratio.
  • cross-linking the harvested non-adhered adipose cells comprises cross-linking the harvested non-adhered adipose cells using a cross-linker selected from the group consisting of polymers functionalized with aldehyde groups, genipin, and phenolic compounds.
  • flocculation comprises a flocculation technique selected from the group comprising auto-flocculation, bio-flocculation, chemical flocculation, particle-based flocculation, and electrochemical flocculation
  • monitoring comprises optical or electrical monitoring.
  • monitoring comprises measuring lipid droplet size within a cell or within a population of cells.
  • the at least one additive is a fatty acid selected from the group comprising linoleic acid, arachidonic acid, alpha-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid.
  • a cultured adipose tissue comprising adipose cells embedded in a hydrogel or binder, wherein the cultured adipose tissue has a three-dimensional (3D) shape and a size on the macroscale.
  • a cultured adipose tissue comprising adipose cells cross-linked together, wherein the cultured adipose tissue has a three-dimensional (3D) shape and a size on the macroscale.
  • centrifugation comprises counter flow centrifugation.
  • a method for producing cultured adipose tissue comprising: growing adipogenic precursor cells in a first culture media; differentiating the adipogenic precursor cells to adipose cells in a second culture media; harvesting non-adhered adipose cells; and aggregating the harvested non-adhered adipose cells to provide the cultured adipose tissue, optionally wherein the first culture media and the second culture media are the same media.
  • growing the adipogenic precursor cells in the first culture media comprises seeding the adipogenic precursor cells into a bioreactor containing the first culture media and allowing the adipogenic precursor cells to proliferate in the bioreactor.
  • differentiating the adipogenic precursor cells to adipose cells comprises changing the first culture media to the second culture media in the bioreactor.
  • bioreactor is selected from the group consisting of a stirred suspension tank bioreactor, rotating wall vessel bioreactor, hollow fiber bioreactor, airlift bioreactor, bubble column bioreactor, fluidized bed bioreactor, packed bed bioreactor, vertical wheel bioreactor, and wave bag bioreactor.
  • a method for producing cultured adipose tissue comprising: growing adipogenic precursor cells on a two-dimensional (2D) substrate; differentiating the adipogenic precursor cells to adipose cells on the 2D substrate; harvesting non-adhered adipose cells; and aggregating the harvested non-adhered adipose cells to provide the cultured adipose tissue.
  • 2D two-dimensional
  • aggregating the harvested non-adhered adipose cells comprises mixing the harvested non-adhered adipose cells with a hydrogel or binder in a 3D mold.
  • the hydrogel or binder is selected from the group consisting of alginate, cellulose, gelatin, starch, tara gum, agar, agarose, carrageenan, cornstarch, gellan gum, lecithin, maltodextrin, methylcellulose, carboxymethylcellulose, cellulose gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose tragacanth, karaya, acacia, ghatti, beta-glucan, psyllium husk, inulin, chitosan, curdlan, dextran, potato starch, tapioca/arrowroot starch, hyaluronic acid, fibrin, carrageenan, guar gum, inulin, konjac,
  • aggregating the harvested non-adhered adipose cells comprises mixing the harvested non-adhered adipose cells with alginate.
  • mixing the harvested non-adhered adipose cells with alginate comprises: adding calcium carbonate and glucono delta-lactone to a solution of the alginate; and combining the harvested non-adhered adipose cells with the solution of the alginate in the 3D mold.
  • cross-linking the harvested non-adhered adipose cells comprises cross-linking the harvested non-adhered adipose cells using an enzyme selected from the group consisting of a transglutaminase, a tyrosinase, a peroxidase, a laccase, a sortase, a subtilisin, and lysyl oxidase.
  • cross-linking the harvested non-adhered adipose cells comprises cross-linking the harvested non-adhered adipose cells using a cross-linker selected from the group consisting of polymers functionalized with aldehyde groups, genipin, and phenolic compounds, optionally wherein the polymers functionalized with aldehyde groups are selected from a group consisting of periodate oxidized pectin, dextran, chitosan, Arabic gum, sucrose, raffinose, stachyose, cyclodextrin, and starch and optionally wherein the phenolic compound is selected from the group consisting of caffeic acid, chlorogenic acid, caftaric acid, quercetin, and rutin.
  • a cross-linker selected from the group consisting of polymers functionalized with aldehyde groups, genipin, and phenolic compounds, optionally wherein the polymers functionalized with aldehyde groups are selected from a group
  • the method of claim 1 or claim 5, wherein aggregating the harvested non-adhered adipose cells comprises adding a protein during the aggregation.
  • the protein is selected from the group consisting of casein, gelatin, pea protein, soy protein, wheat protein, corn protein, chickpea protein, almond protein, rice protein, hemp protein, quinoa protein, sunflower protein, potato protein, algae protein, yeast protein, bacterial protein, mycoprotein, hydrolysates of any of the aforementioned proteins, and combinations thereof.
  • the method of claim 1 or claim 5, wherein the harvesting and/or the aggregating steps comprise flocculation or coagulation of the non-adhered adipose cells.
  • flocculation comprises a flocculation technique selected from the group comprising auto-flocculation, bio-flocculation, chemical flocculation, particle-based flocculation, and electrochemical flocculation
  • flocculation or coagulation is performed with a food-safe flocculant or coagulant, optionally wherein the food-safe flocculant or coagulant is selected from the group consisting of chitosans, celluloses, lignins, inulins, starches, tannins, alginates, lysines, long polymers, and combinations thereof.
  • the method of claim 1 or claim 5, wherein the adipose cells express omega 3 desaturases.
  • the at least one additive is selected from the group consisting of linoleic acid, arachidonic acid, alpha-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, soybean oil, coconut oil, palm oil, cocoa butter, shea butter, mango butter, olive oil, canola oil, sunflower oil, flaxseed oil, avocado oil, peanut oil, sesame oil, com oil, vegetable oil, margarine, shortening, vegetable ghee, tofu, tempeh, herean,jackfruit, quinoa, chia seeds, bananas, oats, avocados, flax seeds, rice, tapioca/arrowroot, and potatoes.
  • the at least one additive is selected from the group consisting of linoleic acid, arachidonic acid, alpha-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, soybean oil, coconut oil, palm oil
  • harvesting the non-adhered adipose cells comprises at least one of skimming non-adhered cells, collecting non-clustered floating cells, or collecting only non-adhered adipose cells.
  • a cultured adipose tissue comprising adipose cells embedded in a hydrogel or binder, wherein the cultured adipose tissue has a three-dimensional (3D) shape and a size on the macroscale.
  • the cultured adipose tissue of claim 36 wherein the hydrogel or binder is selected from the group consisting of alginate, cellulose, gelatin, starch, tara gum, agar, agarose, carrageenan, cornstarch, gellan gum, lecithin, maltodextrin, methylcellulose, carboxymethylcellulose, cellulose gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose tragacanth, karaya, acacia, ghatti, beta-glucan, psyllium husk, inulin, chitosan, curdlan, dextran, potato starch, tapioca/arrowroot starch, hyaluronic acid, fibrin, carrageenan, guar gum, inulin, konjac, oat bran, pectin, locust bean gum, xanthan gum, soy protein, wheat protein, pea protein, chickpea protein, almond protein, rice protein, hemp protein, soy
  • a cultured adipose tissue comprising adipose cells cross-linked together, wherein the cultured adipose tissue has a three-dimensional (3D) shape and a size on the macroscale.
  • the cultured adipose tissue of claim 38 wherein the adipose cells are cross-linked with a cross-linker selected from the group consisting of polymers functionalized with aldehyde groups, genipin, and phenolic compounds.
  • a cross-linker selected from the group consisting of polymers functionalized with aldehyde groups, genipin, and phenolic compounds.
  • the system of claim 44 comprising at least two vessels, wherein the at least two vessels are arranged to operate in series, in parallel, or in any combination of the two.
  • the system of any one of claims 44-45, wherein the adipose precursor cells are extracted from agricultural waste products.
  • the present disclosure generally relates to cultured adipose tissue produced on a macroscale level.
  • the present disclosure further relates to methods for producing cultured adipose tissue on a macroscale level via the harvesting and aggregation of non-adhered adipose cells.
  • the present disclosure also considers the use of flocculation and coagulation techniques in the aggregation of adipose cells.
  • adipose tissue is largely a dense packing (aggregation) of lipid-filled adipocytes held together by a sparse extracellular matrix (ECM). This is opposed to muscle tissue which is comprised of aligned fibers in a multi-hierarchical structure.
  • ECM extracellular matrix
  • 3D culture has been the main approach for generating bulk/macroscale tissues. These tissue engineering strategies involve the in vitro growth of cells over 3D scaffolds.
  • it is challenging to scale up 3D culture due to mass transport limitations with regard to oxygen, nutrients, and waste. It is often quoted in the field that cells cannot remain viable in 3D tissues unless they are within about 200 micrometers of a source of blood or culture media.
  • a method for producing cultured adipose tissue may include the steps of: growing adipogenic precursor cells in a first culture media; differentiating the adipogenic precursor cells to adipose cells in a second culture media; harvesting non-adhered adipose cells; and aggregating the harvested non-adhered adipose cells to provide a cultured adipose tissue.
  • the first culture media and the second culture media may optionally be the same media.
  • a method for producing cultured adipose tissue may include the steps of: growing adipogenic precursor cells on a two-dimensional (2D) substrate; differentiating the adipogenic precursor cells to adipose cells on the 2D substrate; harvesting non-adhered adipose cells; and aggregating the harvested non-adhered adipose cells to provide the cultured adipose tissue.
  • 2D two-dimensional
  • cultured tissues are provided.
  • a cultured adipose tissue has adipose cells embedded in a hydrogel or binder, wherein the cultured adipose tissue has a three-dimensional (3D) shape and a size on the macroscale.
  • a cultured adipose tissue is made by the methods described above.
  • FIG. 1 is a schematic representation of cultured adipose tissue, in accordance with the present disclosure.
  • FIG. 2 is a flow chart of steps that may be involved in producing the cultured adipose tissue, in accordance with the present disclosure.
  • FIG. 3 is a schematic representation of methods of producing the cultured adipose tissue using bioreactors, in accordance with the present disclosure.
  • FIG. 4 is a schematic representation of a continuous process of producing the cultured adipose tissue on a conveyor belt, in accordance with the present disclosure.
  • FIG. 5 is a timeline of 3T3-L1 adipogenic differentiation, according to an embodiment of the present disclosure.
  • this disclosure contemplates all combinations of each of the upper and lower bounds of those ranges, including each of the values within those ranges, both individually and in a range. For example, recitation of a value of from 1 to 10 also contemplates a value of from 1 and 9 or from 3 and 10. When two or more ranges for a particular value are recited, this disclosure contemplates all combinations of the upper and lower bounds of those ranges that are not explicitly recited. For example, recitation of a value of between 1 and 10 or between 2 and 9 also contemplates a value of between 1 and 9 or between 2 and 10.
  • adipogenic precursor cells or “pre-adipocytes” refer to precursor cells capable of differentiating into mature adipose cells.
  • Adipogenic precursor cells or “pre- adipocytes” may be used interchangeably throughout the present disclosure.
  • Non-limiting examples of adipogenic precursor cells include stem cells such as pluripotent stem cells (PSCs), mesenchymal stem cells (MSCs), muscle-derived stem cells (MDSCs), and adipose-derived stem cells (ADSCs) (e.g., porcine, bovine, human, avian (chicken), piscine, etc.).
  • PSCs pluripotent stem cells
  • MSCs mesenchymal stem cells
  • MDSCs muscle-derived stem cells
  • ADSCs adipose-derived stem cells
  • transdifferentiated cells can also be utilized.
  • adipogenic precursor cells may include, but are not limited to, dedifferentiated fat (DFAT) cells (e.g., porcine, bovine, piscine, etc.), preadipocytes (e.g., human, bovine, avian (chicken), murine, piscine, etc.), and fibroblasts (e.g., avian (chicken), bovine, porcine, murine, piscine, etc.).
  • DFAT dedifferentiated fat
  • preadipocytes e.g., human, bovine, avian (chicken), murine, piscine, etc.
  • fibroblasts e.g., avian (chicken), bovine, porcine, murine, piscine, etc.
  • adipose cells are fat cells or adipocytes.
  • Adipose cells are fat cells or adipocytes.
  • Fat cells are used interchangeably throughout the present disclosure.
  • the cultured adipose tissue 10 may include adipose cells 12 (or adipocytes 12) in an extracellular matrix.
  • the cultured adipose tissue 10 may be arranged in a defined three-dimensional (3D) shape and may have a size on the macroscale (i.e., millimeter scale and greater). Although a cube-like structure is shown in FIG. 1 for simplicity, it will be understood that the cultured adipose tissue 10 may have any suitable 3D shape in practice.
  • the cultured adipose tissue 10 may be a food product suitable for consumption.
  • the cultured adipose tissue 10 may be incorporated as an ingredient in a food product suitable for consumption.
  • the cultured adipose tissue 10 is produced using a method that circumvents the mass transport limitations associated with directly culturing bulk or large scale 3D tissues.
  • a mass of adipose cells 12 are cultured from adipogenic precursor cells in culture media.
  • the block 14 may include growing adipogenic precursor cells to confluency (or to a desired coverage/number of cells on a surface or in suspension) in a first culture media, and then differentiating the adipogenic precursor cells into adipose cells 12 in a second culture media.
  • the first culture media may be an adipogenic induction media which supports proliferation of the adipogenic precursor cells
  • the second culture media may be a lipid accumulation media to provide large numbers of lipid-filled adipose cells 12.
  • a single culture medium may be used for both proliferation/growth of the adipogenic precursor cells and for differentiation of the adipogenic precursor cells into adipose cells.
  • the culture time may be tuned to control lipid yield and droplet size. For example, Applicant has found that longer culture times (about a month) yield droplets comparable to in vivo fat (e.g., chicken).
  • the adipose cells 12 may be genetically modified.
  • the adipose cells 12 may be genetically modified to improve their growth and lipid accumulation for more efficient scale up.
  • a skilled artisan will recognize the variety of techniques that allow suitable genetic manipulation, including but not limited to, gene editing technologies such as CRISPR-Cas9 gene editing.
  • the gene modification to generate a given desired property is clearly important.
  • the methods and systems described herein may include gene editing facilities and methods.
  • genetically modified cells are increasingly available, so it is likely that commercial suppliers for genetically modified cells will be available soon.
  • the genetic modification may be a genetic modification to induce adipogenesis.
  • the adipogenic gene modification can be selected from the group consisting of a modification for ectopic expression of: peroxisome proliferator-activated receptor gamma (PPARy), CCAAT/enhancer binding protein alpha and beta (C/EBPa,P), sterol regulatory element binding protein 1 (SREBP-1), fatty acid binding protein 4 (FABP4), zinc finger protein 423 (Zfp423), perilipin 1 (PLIN1), Kruppel-like factor 13 (KLF13), retinoid X receptor a (RXRa), phosphoenol pyruvate carboxykinase 1 (PCK1), early B-cell factor (Ebf) 1-3, runt-related transcription factor 1 (RUNX1), cyclin-dependent kinase 4 (CDK4), combinations thereof, or the like.
  • PPARy peroxisome proliferator-activated receptor gamma
  • the genetic modification can be a genetic modification to downregulate production of adipose triglyceride lipase (TGL), hormone sensitive lipase (HSL), carnitine palmitoyltransferase (CPT), other fat or fatty acid breakdown enzymes, combinations thereof, or the like.
  • TGL adipose triglyceride lipase
  • HSL hormone sensitive lipase
  • CPT carnitine palmitoyltransferase
  • other fat or fatty acid breakdown enzymes combinations thereof, or the like.
  • the genetic modification could cause the production of anti-microbial, anti-fungal, or anti-viral constituents.
  • the genetic modification could cause the production of stabilizing molecules.
  • the stabilizing molecules can prevent protein aggregation (e.g., low molecular weight stabilizers).
  • the genetic modifications are intended for health purposes.
  • the genetic modification can cause expression of vitamins that are not typically present in the cells.
  • the genetic modification can cause expression of resveratrol, the active ingredient in red wine, or catechins, the active ingredient in green tea.
  • the genetic modifications are intended to alter the color of the resulting cultured adipose tissue. Consumer perceptions regarding the color of food can be important. To that end, the genetic modifications can include expression of proteins that provide desirable colors, such as carotenoids, to alter the color of the resulting product to be more appealing to the average consumer. In some cases, the genetic modification can prompt the generation of myoglobin or hemoglobin, which can provide a color that is more typically associated with meat.
  • the genetic modifications can be modifications to hasten proliferation (cell immortalization targets are generally applicable here), improve adipogenesis, improve cell growth (e g., allow growth on a media that has less components or less expensive components), or other modifications that would be understood to improve the overall number and quality of adipose cells for incorporating into the cultured adipose tissue.
  • genetic modification can be used to introduce components that are not produced within the fat cells of the species of interest.
  • cows are a good source of conjugated linoleic acids (CLAs), which are known to be good for human health, but these CLAs are derived from their gut bacteria metabolites, so in vitro cells may be lacking in them.
  • CLAs conjugated linoleic acids
  • the genetic modification may be a gene that is associated with the digestive bacteria of a species of interest.
  • SNPs single nucleotide polymorphisms
  • desired phenotypes e.g., flavor, fattiness, degree of marbling, etc.
  • SNPs identified in the literature may correlate to changes in specific proteins (i.e. may represent a genotype that correlates directly to the phenotype). The changes demonstrated to be favorable for conventional meat products can be incorporated into the cells of the present disclosure.
  • SNPs include, but are not limited to, a G instead of A at locus rs733003230 of the fatty acid desaturase 1 (FADS1) gene and/or a G instead of A at locus LC060926 of the FADS2 gene for increased arachidonic acid in chickens or a G instead of C at position 841 of the fatty acid synthetase gene for increased oleic acid in cattle.
  • FDS1 fatty acid desaturase 1
  • the genetic modifications can be intended to facilitate the systems and methods described herein.
  • the genetic modification can modify the density of surface proteins and/or surface binding sites to facilitate the crosslinking.
  • the surface chemistry can include photoinitiated crosslinking agents.
  • the end product of this system and method is a cultured adipose tissue that is typically going to be combined with some kind of muscle tissue or connective tissue, so it may be beneficial to genetically modify the cells to express muscle binding or connective tissue binding domains, which may make harvesting easier.
  • a cultured adipose tissue that is typically going to be combined with some kind of muscle tissue or connective tissue, so it may be beneficial to genetically modify the cells to express muscle binding or connective tissue binding domains, which may make harvesting easier.
  • the specific genetic modifications that are present may be unknown or may be unknowable.
  • the cells may have no specific genetic modification, but may simply be harvested from a new source with unknown genetic makeup.
  • the systems and methods described herein may encounter cells with unknown genetic makeup.
  • the cells can be subjected to culture media sampling to determine the most efficient culture media for use with those cells.
  • This process can be manual or it can be automated.
  • a lab technician will attempt to grow cells in different culture media and will compare the results to determine a cost/benefit to the different culture media.
  • an artificial intelligence or machine learning algorithm can sample the variable space with respect to culture media composition and can optimize the media to the specific cells.
  • a pre-production process can be performed, where a subset of cells from a population are removed from the population and put through a small-scale version of the methods described herein, to determine suitability of the culture media. In some case, less than 1% of a population of cells, less than 0.5%, less than 0.1%, or less than 0.01% of a population of cells can be selected for sub-sampling to confirm culture media appropriateness prior to a fully scaled up production run.
  • the pre-production process can serve as a final validation that the cells and the media are compatible with one another.
  • the first step/cellular raw materials may be referenced as a cell stock.
  • the acquisition of the cell stock can in general be a process similar to harvesting conventional meat products (i.e., butchery and isolation of cells from the butchered products) or can involve harvesting cells from less traditional places, such as other active cell lines that are being used in cellular agriculture. Additional processing steps for the cells can include immortalization, or clonal isolation to obtain a proliferative + adipogenic + genetically homogenous (for production consistency) cell population/cell line. Cells could also be adapted to single cell suspension prior to use in scaled up production.
  • the culture media used can be provided in a variety of forms, including powdered bases that can be hydrated by pure water and fully formed culture media. In either case, separate and distinct culture media can be included for the proliferation and differentiation steps.
  • the purity of the water is extremely important.
  • the system includes onsite water purification.
  • the method can include hydrating the powder to form a culture media.
  • the culture media can be sterile filtered or sterilized via any means available to those of skill in the art prior to use in the method or at any step of the method.
  • Various contemplated modifications to the culture media include, but are not limited to, bulk mixed powder including all of the non-water components of the culture media, separately maintained ingredients that are mixed on demand to make culture media, growth factors or artificial mimics thereof can be present in the culture media, or a combination thereof.
  • the cells are genetically modified to have intentionally less strict requirements for the culture media.
  • cells can be genetically modified to create their own glutamine (see, for example, WO 2019/014652, which is incorporated herein in its entirety by reference for all purposes), which would eliminate the requirement for glutamine in the culture media. Similar genetic modifications to reduce the need for complex culture media are contemplated.
  • Manipulation of the cells themselves can be done by conventional methods, including seed trains for seeding larger and larger bioreactors. The seed train gradually moves to bioreactors having increasing size until the desired bioreactor size is reached. From there, the cells can be a part of a suspension culture of an adherent culture.
  • cell proliferation is done via cell stock cultured in single cell suspension in various suspension bioreactors.
  • These bioreactors can be a conventional stirred suspension tank, air-lift, vertical wheel, or wave bioreactors.
  • cells can be encapsulated with a material that protects the cells from shear stress. This encapsulating material may itself provide a miniature three-dimensional cell culture environment, which can be advantageous for other reasons discussed elsewhere herein.
  • Other contemplated bioreactor designs include the rotating wall vessel bioreactor described elsewhere herein.
  • the bioreactor used herein can be a dedicated adherent cell bioreactor, such as a fixed bed bioreactor (e.g., the Scale XTM bioreactor, available commercially from Univercells S.A., Charleroi, Belgium, or iCellis® bioreactors, available commercial from Pall Corporation, Port Washington, NY).
  • a fixed bed bioreactor e.g., the Scale XTM bioreactor, available commercially from Univercells S.A., Charleroi, Belgium, or iCellis® bioreactors, available commercial from Pall Corporation, Port Washington, NY.
  • the bioreactor can include stacked culture flask approaches, like "cell factories" or the Hyperstack®, available commercially from Corning Inc., Corning, NY.
  • an adherent approach could be combined with a suspension bioreactor if microcarriers (e.g., pectin microcarriers commercially available from Corning Inc., Corning, NY) can be used as the adherent substrate, but those microcarriers are then circulated in a suspension bioreactor in the conventional fashion.
  • microcarriers e.g., pectin microcarriers commercially available from Corning Inc., Corning, NY
  • the microcarriers can be edible.
  • the microcarriers may end up in the resulting product, if their presence is not undesirable.
  • the same reactor can be used as was used for proliferation, with a change of culture media facilitating the transition.
  • the cells are transferred to a different bioreactor to transition from proliferation to differentiation.
  • cells can be separated from the proliferation media via centrifugation (e.g., disc stack centrifuge, counter flow centrifuge, Gibco Rotea centrifuge, etc.). After removal of the proliferation media, the differentiation media is added to the bioreactor and the cells are cultured in the same fashion as they undergo adipogenesis.
  • centrifugation e.g., disc stack centrifuge, counter flow centrifuge, Gibco Rotea centrifuge, etc.
  • adipocyte buoyancy in suspension culture poses unique problems that are not typically encountered in cell agriculture.
  • a cell floats that is typically a sign that the cell is no longer of any value.
  • the buoyancy can be leveraged as described herein by harvesting cells from a surface or suspended cells and allowing them to replenish from beneath.
  • the culture is ended, and the lipid-laden adipose cells 12 are harvested according to a block 16.
  • the block 16 may include detaching the adipose cells 12 from a substrate and draining the adipose cells of non-cell liquid.
  • Cells can be harvested via the same techniques used to separate cells from culture media between the various stages.
  • “skimming" floating cells from the surface is one particularly promising approach to harvesting cells, given that it is an automatic indicator of buoyancy.
  • “Floating” as used herein may be used interchangeably with “non-adhered” unless discussed otherwise.
  • Other methods of collecting non-adhered cells may comprise collecting non-clustered floating cells, collecting only non-adhered cells, and/or not collecting adhered cells.
  • conventional cell harvesting techniques may be used, such as those disclosed in Dryden et al., "Technical and Economic Considerations of Cell Culture Harvest and Clarification Technologies", Biochem Eng J, Vol. 167, 107892, which is incorporated herein in its entirety by reference. While Applicant believes that some of the cell harvesting techniques described in this application are inventive, there are other inventive concepts which are usable with conventional cell harvesting techniques.
  • the harvested cells Prior to molding the harvested cells in the next portion of certain methods, some postprocessing may be required.
  • the harvested cells might be rinsed to remove/minimize/dilute undesired culture media components. Rinsing can be done by way of counter-flow centrifugation or conventional filtration.
  • cells can be clarified by removing undesirable components, which are not removed by rinsing. This is a process that is regularly used in the pharmaceutical arts, but it is not conventionally used in cellular agriculture. In one specific example, clarification of cells to remove cell debris or excess nucleic acids is expressly contemplated. Other methods such as flocculation via pH or the use of polymers such as chitosan which can induce flocculation are contemplated.
  • harvested adipose cells 12 themselves may be an end product, so the system and method can end there in certain circumstances.
  • the harvested adipose cells 12 can be provided to customers for incorporation into products, including those outlined elsewhere herein.
  • the harvested adipose cells 12 may be aggregated, for instance into a 3D mold (e.g., a 3D printed mold) having a desired 3D shape to generate the 3D adipose tissue 10.
  • a 3D mold e.g., a 3D printed mold
  • the 3D mold can be a permanent and/or reusable mold or it can be single-use.
  • the single-use mold can be made of the same material as the materials identified elsewhere herein as hydrogel materials for use in the cell aggregation step.
  • the entire structure of the single-use mold and the cultured adipose tissue that is molded inside of it can be utilized for inclusion in the various products identified herein. For example, a large chunk of cultured adipose tissue and the 3D mold in which it was made can be chopped into smaller pieces for incorporating into a food product.
  • the material of the 3D mold can be stable or can simply change phase (e.g., similar to the rendering of fat) when exposed to temperatures of at least 40 °C, at least 45 °C, at least 50 °C, at least 55 °C, at least 60 °C, at least 65 °C, at least 70 °C, at least 75 °C, at least 85 °C, at least 90 °C, at least 100 °C, at least 105 °C, at least 125 °C, or at least 150 °C or at temperatures at ranges of from 40 to 150 °C, from 50 to 125 °C, from 60 to 105 °C, or from 70 to 100 °C.
  • the 3D mold material may be stable or generally stable in water, because so many food and cooking environments are aqueous. To the extent that it is not explicitly stated elsewhere, these properties may also apply to the binder/hydrogel material.
  • a thickening agent or thickener may be a suitable binder/hydrogel.
  • any practical shape can be suitable for use, though some may be preferred over others.
  • generally regular spherical shapes may be preferred, while in other cases, irregular shapes may be desired.
  • the 3D shape may be elongated strips.
  • the desired shape is very small, it may be impossible or impractical to make molds of the desired shape. In these cases, it may be useful to make a larger sample that is carved into smaller pieces. For example, if a customer desires a host of small fat "flakes" for incorporating into a food product, it is much easier to make a large piece of cultured adipose tissue and then manipulate that large piece to make smaller flakes. Larger pieces can be cut, sliced, chopped, minced, ground, or other conventional way to manipulate animal-fat-like products.
  • the aggregated material may be formed by way of an extrusion or printing process.
  • the cells and hydrogel material may be co-extruded to form a linear aggregate.
  • the linear aggregate can be cut into pieces to form individual pieces of cultured adipose tissue.
  • the linear aggregate can be solid or hollow, depending on the specific desired application.
  • candy making processes where ropes of extruded or rolled candy is cut into smaller pieces might be one example of a suitable process to mimic for an extrusion process for use with the present invention.
  • Some extruded metal or rubber or plastic processes may be similarly applicable.
  • the cells and binder/hydrogel material can be 3D printed into a desired shape.
  • aggregating as shown in block 18 may involve embedding the harvested adipose cells 12 in a hydrogel or mixing with a binder in a 3D mold.
  • Suitable hydrogels or binders include, but are not limited to, food safe compounds such as alginate, cellulose, gelatin, starch (e.g., tapioca), hyaluronic acid, fibrin, carrageenan, cornstarch, gellan gum, lecithin, maltodextrin, methylcellulose, carboxymethylcellulose, cellulose gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose tragacanth, karaya, acacia, ghatti, beta-glucan, psyllium husk, inulin, chitosan, curdlan, dextran, potato starch, tapioca/arrowroot starch, hyaluronic acid, fibrin, carrageenan, guar gum, inulin, konja
  • the hydrogel or binder is alginate which is a material used as a fat replacer in the food industry.
  • block 18 may include mixing the harvested and drained adipose cells 12 with an alginate solution at a specified volumetric ratio in the 3D mold.
  • a slow gelling alginate solution may be prepared by adding calcium carbonate and glucono delta-lactone (GDL) powders to an alginate solution, and the slow gelling alginate solution may be combined with the harvested and drained adipose tissue at a 1 : 1 volumetric ratio in a 3D printed mold (see Example 3).
  • GDL calcium carbonate and glucono delta-lactone
  • the process is typically a heat-driven process, where water and the binder/hydrogel ingredient are heated, fat cells are mixed in, and the mixture is cooled.
  • agar/agarose is heated to liquify it (e g., 85-100 °C), then cooled (e g., 40-65 °C) and mixed with adipocytes. After mixing, the solution is cooled to form a solid piece of macroscale, structured fat.
  • aggregating as shown in block 18 may involve cross-linking the harvested adipose cells 12 in a 3D mold.
  • the cross-linking may be carried out using a suitable protein-protein cross-linking enzyme such as, but not limited to, transglutaminase, a tyrosinase, a peroxidase, a laccase, a sortase, a subtilisin, and lysyl oxidase.
  • cross-linking the harvested adipose cells includes enzymatically cross-linking the harvested adipose cells using transglutaminase.
  • cross-linking the harvested adipose cells may involve mixing a solution of transglutaminase with the harvested adipose cells at a specified volumetric ratio in a 3D mold (see Example 3).
  • the block 18 may further include adding helper proteins during the cross-linking.
  • the helper proteins (which may simply be referred to as “proteins”) may be selected from casein and gelatin.
  • Chemical crosslinking can also be used when the reactants or catalysts are food safe, such as EDC-NHS reactions between acid and amine groups. Photochemical crosslinking can also be utilized where photosensitizers are food safe.
  • soy protein isolate 6% or higher of soy protein isolate and 5-25 enzyme activity units/gram of protein substrate of transglutaminase is one suitable starting point for these crosslinking conditions.
  • Higher soy protein isolate and/or transglutaminase content can lead to higher strength within the gel. However, the maximum strength is not necessarily desirable, because it would not mimic the mouthfeel of fat.
  • a mixture of pea protein isolate (19.6% w/w), transglutaminase (0.7% w/w), and NaCl (1% w/w) with a pH of 6.5 is incubated at 50 °C to provide a crosslinked protein matrix that is often referred to as a plant based meat.
  • Adipocytes can be mixed into this composition before curing to form a hybrid product of culture adipose tissue and plant based meat.
  • the crosslinking may be dependent on one or more cofactors or metals.
  • exemplary cofactors or metals may include calcium, copper, and heme.
  • the protein is mixed in with the binder/hydrogel and crosslinked (with the transglutaminase, for example). This may be necessary to form a gel.
  • canola oil can be mixed with ⁇ 30% (w/w) of fully hydrogenated canola oil at 65 °C, hot-emulsified with a soy protein suspension (8%, w/w) at a lipid content of 70% (w/w) using a high-shear disperser, and cooled to 37 °C.
  • the concentrated, emulsified fat crystal networks can then incubate with transglutaminase for 1 hr to induce protein crosslinking.
  • cross-linkers may be used for adipose cell aggregation such as, but not limited to, polymers functionalized with aldehyde groups, genipin, phenolic compounds, and combinations thereof.
  • Suitable polymers functionalized with aldehyde groups include, but are not limited to, periodate oxidized pectin, dextran, chitosan, Arabic gum, sucrose, raffinose, stachyose, cyclodextrin, and starch.
  • Suitable phenolic compounds include, but are not limited to, caffeic acid, chlorogenic acid, caftaric acid, quercetin, and rutin derived from plants such as grapes and coffee.
  • aggregation of the cells or the combination of both the aggregation and harvesting of non-adhered adipose cells comprises flocculation.
  • flocculation and grammatical equivalents may refer to the aggregation of unstable and small particles through surface charge neutralization, electrostatic patching and/or bridging after addition of flocculants (or agents that make fine and sub-fine solids or colloids suspended in the solution form large loose flocs through bridging thus achieving solid-liquid separation).
  • Flocculation may be performed by one or more of several techniques, including auto-flocculation, bio-flocculation, chemical flocculation, particle-based flocculation, and electrochemical flocculation.
  • Various flocculation techniques have been reviewed by Matter, et al (2019, Applied Sciences). This is a method with high potential as it might be used to harvest cells from the bioreactor at low cost.
  • Auto-flocculation may be performed at acidic pH (for example, a pH of 4.0). Autoflocculation may be performed at alkaline pH (for example, a pH of 10.4, 11.0, 1.5, 11.6, 12.0, or 12.5). Auto-flocculation may be performed over several days (for example, 16 days).
  • acidic pH for example, a pH of 4.0
  • alkaline pH for example, a pH of 10.4, 11.0, 1.5, 11.6, 12.0, or 12.5
  • Auto-flocculation may be performed over several days (for example, 16 days).
  • Chemical flocculation may be performed with inorganic flocculants such as aluminum sulfate, calcium oxide, iron(III) chloride, and magnesium hydroxide. Chemical flocculation may be performed with organic flocculants such as inulin, starches, chitosan, tannins, alginates, and lysines. Flocculation may preferably be performed with food-safe flocculants such as chitosan, cellulose, lignin and/or other long polymers.
  • inorganic flocculants such as aluminum sulfate, calcium oxide, iron(III) chloride, and magnesium hydroxide. Chemical flocculation may be performed with organic flocculants such as inulin, starches, chitosan, tannins, alginates, and lysines.
  • Flocculation may preferably be performed with food-safe flocculants such as chitosan, cellulose, lignin and/or other long polymers.
  • Particle-based flocculation may be performed using aminoclay-based nanoparticles such as 3-aminopropyltriethoxysilane (APTES) conjugated to magnesium and aminoclays conjugated to aluminum, titanium oxide, and humic acid.
  • Particle-based flocculation may be performed using magnetic particles including ferrous oxide nanoparticles and composites.
  • Coagulation of cells may be an additional or alternative means of cell harvesting and/or aggregation that is similar to flocculation wherein cells aggregate or clump.
  • the adipose cells 12 or the adipose tissue 10 may be supplemented at various stages to tune the sensorial characteristics (e.g., texture, color, and flavor) and/or the nutritional attributes of the cultured adipose tissue 10.
  • supplementation with additives such as, but not limited to, flavorants, colorants, texturizers, vitamins, minerals, amino acids, proteins/peptides, oils, whole foods, and fatty acids is also encompassed by the present disclosure.
  • tunable control of fat nutrition and health may be implemented.
  • the fatty acid composition of the cultured adipose tissue may be tailored via cell feeding strategies, such as by supplementing fatty acids into the culture media during in vitro culture.
  • omega 3 desaturases may be expressed or pathways to produce lipophilic nutrients (e g., beta carotene, vitamin A) may be activated in the adipose cells 12. This may be beneficial to the consumer as certain nutrients are more bioavailable when consumed in food versus a micronutrient supplement.
  • the texture of the cultured fat may be tunable based on variables such as the hydrogel/binder (e.g., alginate) concentration, cross-linker levels, and the inclusion of helper proteins (e.g., casein, gelatin, etc.) during cross-linking.
  • the cultured adipose cells 12 may be supplemented with methylated branched fatty acids to impart a "mutton" flavor in the cultured adipose cells 12.
  • methylated branched fatty acids One specific methylated branched fatty acid is 4-methyloctanoic acid.
  • the relative extracellular matrix production and fat production levels may be optimized pending the texture, taste, and/or nutritional outcomes desired.
  • the starting material can be fish muscle cells, given that fat cells are generally harder to acquire from certain fish species known to be very lean. It has also been shown that certain fish muscle cells can self-immortalize, which is advantageous for large scale production. It is possible to induce lipid uptake in skeletal muscle cells via fatty acid/lipid supplementation, forming fatty muscle cells. Fatty acid/lipid supplementation may also induce transdifferentiation of skeletal muscle cells into adipocytes, or adipocyte-like cells. Genetic approaches may also be used to transdifferentiate skeletal muscle cells, such as via the activation or insertion of PPARy. Without intending to alter the definition provided above, these fatty muscle cells can be used wherever adipocytes or fat cells are referenced herein.
  • FIG. 3 shows scalable processes for the mass production of the cultured adipose tissue 10.
  • the processes may be carried out in a bioreactor 20, such as a stirred suspension tank bioreactor 22 (top) or a hollow fiber bioreactor 24 having hollow fiber membranes 26 (bottom).
  • a bioreactor 20 such as a stirred suspension tank bioreactor 22 (top) or a hollow fiber bioreactor 24 having hollow fiber membranes 26 (bottom).
  • Other types of bioreactors apparent to those skilled in the art may also be used and are within the scope of the present disclosure such as, but not limited to, rotating wall vessel bioreactors (RWVBs) and packed bed bioreactors.
  • Production of the adipose tissue 10 in the bioreactor 20 may involve seeding 28 a first culture media 30 (adipogenic induction media) in the bioreactor 20 with adipogenic precursor cells 32.
  • a first culture media 30 adipogenic induction media
  • the adipogenic precursor cells 32 may then proliferate 34 to confluency (or to a desired coverage/number of cells on a surface or in suspension) in the bioreactor 20.
  • the adipogenic precursor cells 32 may form small aggregates or spheroids 36 as they proliferate (see FIG. 3, top).
  • the spheroids 36 may be dissociated 38 into single adipogenic precursor cells 32 and allowed to proliferate 34 further (see FIG. 3, top).
  • the adipogenic precursor cells 32 may proliferate on the surface of the hollow fiber membranes 26 (see FIG. 4, bottom). In this case, the adipogenic precursor cells 32 may be detached 40 from the hollow fiber membranes 26, and the detached adipogenic precursor cells 32 may be used to reseed the media 30 for further proliferation 34.
  • the cells may accumulate lipids and differentiate 44 into adipose cells 12.
  • the adipose cells 12 may grow separately or in small clusters 46 (see FIG. 3, top).
  • the adipose cells 12 may develop on the surface of the hollow fiber membranes 26.
  • a single culture medium may be used for both proliferation 34 and differentiation 44.
  • the adipose cells 12 may be harvested 48. In the hollow fiber bioreactor 24, the harvesting may involve detaching the adipose cells 12 from the hollow fiber membranes 26.
  • the harvested adipose cells 12 may then be aggregated 50 in a 3D mold to provide the cultured adipose tissue 10.
  • suitable methods for binding and aggregating 50 the adipose cells 12 include cross-linking (e.g., enzymatic cross-linking with transglutaminase), as well as embedding the adipose cells 12 in hydrogels such as alginate.
  • the culturing process of the present disclosure may be compatible with two dimensional (2D) culture strategies.
  • the adipose cells 12 may be cultured in thin layers on a 2D substrate such as culture plates, and then aggregated into the 3D adipose tissue 10 according to the above-described procedures.
  • the adipogenic precursor cells 32 may be grown to confluency (or to a desired coverage/number of cells on a surface or in suspension) and differentiated into the adipose cells 12 on the 2D substrate.
  • Harvesting or collecting the adipose cells 12 from the 2D substrate followed by aggregating the harvested adipose cells 12 may provide the cultured adipose tissue 10.
  • the 2D substrate may by edible and incorporated into the final food product, such that the adipose cells 12 do not need to be detached from the 2D substrate.
  • cells may grow on the 2D substrate and eventually become non-adherent and the adherent and non-adherent cells may be harvested and/or aggregated separately and/or used for distinct purposes as detailed herein.
  • the 2D substrate may be a conveyor belt 52 (see FIG. 4).
  • the continuous production process may involve seeding 54 the adipogenic precursor cells 32 onto the conveyor belt 52 having a culture media thereon.
  • the adipogenic precursor cells 32 may then proliferate 56 to confluency (or to a desired coverage/number of cells on a surface or in suspension) on the conveyor belt 52.
  • Changing the culture media to lipid accumulation media may allow the adipogenic precursor cells 32 to accumulate lipid and differentiate 58 into the adipose cells 12.
  • a single culture medium may be used for both proliferation 56 and differentiation 58.
  • the adipose cells 12 may be harvested 60 by detachment from the conveyor belt 52, and then aggregated 62 according to the above-described procedures to provide the cultured adipose tissue 10.
  • the technology disclosed herein provides a novel and scalable approach to cultured fat generation.
  • the present disclosure leverages large-scale cell proliferation and scale up technology to generate a required amount of in vitro adipose cells, after which the cells are aggregated or packed into a solid 3D construct on the macroscale.
  • the adipose cells are cultured in thin layers (2D culture) or in bioreactors with easy access to the culture media, followed by aggregation into macroscale 3D tissues after sufficient adipocyte maturation.
  • adipocytes or adipocyte clusters recapitulates native fat tissue from a sensory perspective as adipose tissue in vivo is largely a dense aggregation of lipid fdled adipocytes with a sparse extracellular matrix. Furthermore, the compatibility of the adipose tissue production method with 2D culture strategies allows for a continuous production process with a conveyor belt assembly line approach.
  • the method of the present disclosure produces bulk cultured adipose tissue in a way that circumvents the mass transport limitations associated with directly culturing or engineering large 3D tissues. Aggregation at the end of cell culture removes the need for nutrient delivery to the adipose cells via vascularization or an elaborate tissue perforation system. This is because, for food applications, the cultured adipose cells do not need to stay alive once formed into the final edible tissue. This is analogous to meat production in conventional animal agriculture where muscle and fat cells gradually cease to be viable after slaughter. In contrast, for medical applications, cells in 3D tissues may be expected to remain viable to be used for implantation into the body or for testing in an in vitro tissue model. Accordingly, the adipose tissue production method of the present disclosure is less costly than other methods that rely on complex perfusion and mixing systems to distribute nutrients during cell growth. In some embodiments, the food product described herein is produced without vascularization or perfusion.
  • monocultures of adipocytes and preadipocytes may be sufficient for the production of large fat droplets without the need for supporting cell types.
  • Standard cell culture conditions are sufficient for the type of adipocyte culture outlined in this disclosure, and no specific coatings on tissue culture plastics were required to achieve desired adipocyte growth and development.
  • the pre-adipocytes and adipocytes of various livestock species may be grown in serum-free culture media according to the present disclosure, thereby eliminating a major obstacle in in vitro fat culture. These advantages further help reduce production costs.
  • Co-cultures can also be considered for enhanced fat outcomes, such as the use of fibroblasts or muscle cells in the cultures, such as to increase the quality of the fat products or to alter the texture and composition.
  • Applicant has also observed that a large subpopulation of the cultured adipocytes adhere strongly to tissue culture plates and do not float away, avoiding issues of lift-off of adherent adipocytes in vitro due to increasing buoyancy as the adipocytes become fatty.
  • the 2D culture systems disclosed herein self-sort for adherent cell populations, which may provide for adherent and non-adherent cell populations that may be used for separate downstream applications. Applicant has also developed techniques for dealing with or using the non-adherent cell populations, as described elsewhere herein.
  • the systems and methods described herein can include various sensing and control systems to facilitate superior control over the processing/harvesting process.
  • the systems and methods include a measurement component, which observes a property of an individual cell or a group of cells, and an assessment component, which assesses the measurement to make a processing/harvesting decision to select an appropriate time for processing/harvesting the cell or group of cells.
  • processing refers generally to a decision to move a cell or a population of cells to a different processing stage. These processing steps can in some cases be related to developmental differentiation (i.e., the process of becoming an adipose cell).
  • the term harvesting generally refers to a final processing step for adipose cells, after which those cells are aggregated as described herein or otherwise further processed in ways that do not materially alter the cells from their state at harvest.
  • the processing/harvesting decisions can be based on monitoring of individual cells.
  • analytical e.g., optical, etc.
  • interrogation of individual cells can provide meaningful distinctions between adipose cells that are ripe for processing/harvesting versus adipose cells that are not yet ready.
  • individual optical interrogation of cells revealing underlying lipid accumulation can be performed, followed by comparing that interrogation to a benchmark threshold for cells known to have a desired lipid accumulation (or other measurable property).
  • These techniques can include active techniques, where the property or properties of the cells are directly measured, and more passive techniques, where the property or properties of the cells are more indirectly observed.
  • the processing/harvesting decisions can be based on monitoring of populations of cells.
  • analytical e.g., optical, electrical, etc.
  • populations of cells can have optical measurements taken (e.g., absorption, scattering measurements, etc.) or electrical measurements taken (e.g., impedance, capacitance, etc ), which can represent important meaningful properties of the population of cells.
  • optical measurements taken e.g., absorption, scattering measurements, etc.
  • electrical measurements taken e.g., impedance, capacitance, etc
  • These techniques can include active techniques, where the property or properties of the population of cells are measured directly, and more passive techniques, where the property or properties of the population of cells are more indirectly observed. Lipid accumulation may be a suitable determinant for monitoring.
  • the processing/harvesting decisions can be based on cell growth.
  • growth can be determined in terms of growth of individual cells (i.e., cell size and/or mass and/or density). In some cases, growth can be determined in terms of growth of the number of cells (i.e., cell count).
  • the processing/harvesting decisions can be based on a degree of cell differentiation.
  • the system and method can include steps relating to differentiation of certain cell types into adipose cells and/or precursors of adipose cells.
  • the monitoring can be user-originated, such that a user can specifically request that the system or method observe a given cell or a given population of cells at a specific time.
  • the monitoring can be automated, such that the monitoring occurs at a predetermined time or that a computer program selected a time based on a given set of criteria. Skilled artisans in the automation arts will recognize a host of options for automating the monitoring of the individual cells or populations of cells.
  • the monitoring involves a destructive sampling process, where one or more cells are removed from the system or method in order to make a representative measurement.
  • sampling techniques can be used, such as physical removal of cells or populations of cells.
  • a user can arbitrarily select the cells for sampling or the user can be directed in some fashion to select specific cells and/or a specific location.
  • the system can include labels that direct the user to a proper sampling location. In some cases, these labels can be digital labels or they can be printed labels.
  • automated sampling can involve routinely retrieving cells from a specific location in the system/method and simply acquiring whatever sample happens to be occupying that location at the given time. This is a good bulk sampling technique to be used in cases where the overall number of cells required for sampling is adequately small when compared with the overall cell population size.
  • automated sampling can involve complex decision trees and/or machine-learning-derived algorithms for selecting cells or populations of cells for interrogation. Examples of suitable selection algorithms include, but are not limited to, random sampling techniques, weighted sampling techniques, and the like.
  • the monitoring involves a non-destructive sampling process, where the cells are not disturbed to a degree that their growth, differentiation, and/or proliferation remain generally unaltered.
  • the user can manually move an analytical device into a location for interrogating a cell or population of cells.
  • a user can arbitrarily select the cells for interrogation or the user can be directed in some fashion to select specific cells and/or a specific location.
  • the systems and methods can utilize labels to direct a user to a proper location. Such labels can be digital or analog.
  • the monitoring is performed on adhered cells.
  • the monitoring systems are configured to interrogate a location where the substrate is positioned during processing. Because the systems and methods are typically deployed in a reproducible fashion, the substrates will typically be located in predictable locations.
  • the monitoring can be performed on non-adhered cells. Those aspects of the present disclosure are described in greater detailed elsewhere herein where "floating" cells are discussed.
  • the monitoring is a specific monitoring of lipid droplet size within a cell or within a population of cells.
  • lipid droplet size is an important factor in determining when an adipose cell is ready for harvesting. This lipid droplet size is optically visible and can be interrogated by known optical methods. It should be appreciated that the lipid droplet size monitoring can also be used to select for undesirable cells, in a similar fashion to the way that it selects for desirable cells.
  • the monitoring can include monitoring the culture media. This analyzing of the culture media can be used for making harvesting decisions or can inform control of other aspects of the method.
  • the culture media can be monitored to determine a concentration of ammonia.
  • concentration of ammonia can be used as a feedback for manual or automated control of the system.
  • the ammonia can be extracted and processed into an end product.
  • ammonia can be extracted and processed into fertilizer using methods known in the art.
  • the culture media can be monitored to determine a concentration of lactic acid in the culture media.
  • concentration of lactic acid can be used as a feedback for manual or automated control of the system.
  • waste products that are not useful as recycled source materials in the system and method described herein can be monitored and removed from the system. If those waste products are capable of being transformed into a useful product, then the system can include those facilities.
  • the lactic acid can be extracted and processed into an end product.
  • lactic acid can be extracted and processed into poly(lactic acid) using methods known in the art.
  • harvesting decisions can be phenotypic.
  • the secretome of the cells can be used to make a harvesting decision.
  • the level of adipokine surrounding the cells can be used as a measure of readiness for harvest.
  • the level of adiponectin could be utilized for harvesting decisions.
  • the systems and methods described herein are particularly useful for processing cells that detach from the substrate during the process of differentiating into fat cells (herein referred to as “floating” or “non-adhered” cells).
  • the systems and methods of this disclosure provide approaches to collecting (or “harvesting” or “aggregating”) and utilizing these floating cells, which have traditionally been discarded as waste. By making productive use of these previously-wasted products, much higher efficiency is achieved by virtue of not wasting this large population of cells.
  • the surface of a growth medium reservoir can be monitored to determine a concentration of floating cells.
  • a liquid-air interface is monitored. If the concentration of floating cells exceeds a predetermined threshold, then the systems and methods can harvest the floating cells. These harvested floating cells can be processed in the fashion described elsewhere herein.
  • an interface between two liquids having different densities can be monitored to determine a concentration of cells at the interface.
  • a liquid-liquid interface is monitored.
  • multiple interfaces between multiple liquids having different densities can be monitored to determined concentrations of cells at the multiple interfaces.
  • multiple liquid-liquid interfaces are monitored.
  • these cells can be divided into quality grades based on one or more properties.
  • cells having a lower density can be assigned a higher grade.
  • cells that remain adhered to a substrate for a shorter length of time are assigned a higher quality grade.
  • cells that remain adhered to a substrate for a longer length of time are assigned the higher quality grade. It should be appreciated that some applications may have a preference for cells that rapidly loser their adherence versus cells that slowly lose their adherence and vice versa. Without wishing to be bound by any particular theory, the systems and methods described herein provide a platform for assigning quality grades to a given cell.
  • the floating cells can be delivered to a chromatographic system and/or a microfluidics system and/or a cell sorting system and/or a cell picker, as will be understood by those having ordinary skill in the cell manipulation arts.
  • a cell skimmer is one particularly useful device for removing cells from the surface.
  • the microfluidics system can be tailored specifically for handling floating cells described herein.
  • One example of tailoring the microfluidics system for floating cells is to adjust the density of the carrier fluid prior to entry into the microfluidics system, such that the cells are neutral buoyance and are no longer "floating" in the medium.
  • a specific stationary phase and mobile phase may be selected for a desired result. For instance, if size selection is an important criterion, then a stationary phase that has porosity that provides differentiated retention properties based on size could be useful. As another example, if surface chemistry of the adipose cells is an important criterion, then a stationary and/or mobile phase could be selected based on those desirable surface chemistries. In one specific example, certain cells may be more amenable to embedding into a hydrogel if they have a specific surface chemistry, so a chromatography system could be used to isolate cells that are more or less amenable to embedding into the hydrogel.
  • certain cells may be more amenable to direct cross-linking to other cells if they have a specific surface chemistry, so a chromatography system could be used to isolate cells that are more or less amenable to direct cross-linking with one another.
  • the cell sorting system can be used to differentiate and categorize the cells based on measurable properties, as would be appreciated by a skilled artisan. Applicant does not intend to provide specific inventive contribution with respect to the cell sorting itself, but rather integrates and utilizes the understood concept of cell sorting into inventive concepts that form a part of the broader disclosure described herein.
  • the cells are sorted through multiple different centrifuges.
  • the cells that make it through multiple centrifuges will have different properties from those that do not and those different categories of cells can be divided into grades, in a similar fashion as USDA grading.
  • the cells could be graded as superior or inferior relative to one another.
  • the cell sorting can involve the use of a density gradient.
  • the different densities can allow harvesting of cells having different buoyancies.
  • the different buoyancies can also be used to grade the cells.
  • some cells that do not meet certain grading threshold or certain quality standards i.e., cells that do not have enough fat in them) can be returned to the system at the relevant stage in the process.
  • sedimented cells can be isolated and further processed as biomass.
  • the sedimented cells can be recycled for raw materials and reintroduced into the system and/or method at various points in the system/process.
  • sedimented cells can be harvested and have components for culture media extracted from them.
  • the sedimented cells are processed for other uses.
  • sedimented cells might be suitable fdler material in animal feedstock.
  • sedimented cells might be suitable for use as a starting material for a further culture.
  • the systems and methods described herein may provide useful sorting and/or isolation of relevant cell populations, such as the sedimented cells described herein.
  • These sedimented cells are artificially selected by virtue of the processing decisions made. These artificial selections can imbue the cell populations with desirable properties (e.g., density can be desirable for certain applications that are not trying to make adipose tissue) and further cultures can grow and expand those populations of cells to have larger populations that are enriched in those desirable properties.
  • the sedimented cells themselves may be usable as a single cell protein for growth of bacteria or fungus.
  • the cell itself serves as the protein source.
  • sedimented cells are typically very high in nucleic acid content. Therefore, in some cases, nucleic acid content is reduced prior to harvesting non-adhered adipose cells. In some cases, the sedimented cells can have nucleic acid content reduced before further use. However, the cells may still be useful without reducing the nucleic acid content, for example, as livestock feed. In some cases, the sedimented cells, with or without reducing the nucleic acid content, can be mixed into one or more of the food products described herein for the purpose of adding more of a "meat" flavor. Reducing nucleic acid content may comprise “rinsing” cells as understood in the art and discussed herein elsewhere.
  • any of the proposed genetic modifications discussed above are also applicable as additives, so long as the science does not render it impossible.
  • the carotenoids for adding color can simply be added to the cells as supplements.
  • those fatty acids can be added to the cells via supplementing. The same is true for myoglobin and hemoglobin.
  • dyes may be added to the cells and/or binder/hydrogel in order to provide a more aesthetically pleasing appearance.
  • the cultured adipose tissue may be particularly useful for introducing lipid-soluble nutrient into subjects.
  • a lipid-soluble nutrient that is challenging for uptake in humans can be supplemented into the cells here and the resulting cultured adipose tissue may enable greater uptake of those lipid-soluble nutrients than if they were administered outside of the cultured adipose tissue.
  • additives that add no proven nutritional or therapeutic value are also contemplated.
  • additives can be purely aesthetic.
  • one additive could be gold nanoparticles, which could be added at any stage of the process and taken up into the cells, thereby producing "gold fat”.
  • various materials from the nutraceutical industry can be incorporated into the cells at various stages of the process, including but not limited to silver compositions which are believed by some to have antimicrobial properties.
  • the systems and methods described herein can provide advantageous ability to administer growth factors to the cells, in the interest of providing them with more optimal receipt and uptake of the growth factors.
  • Certain growth factors may advantageously be administered to cells for the purpose of aiding and/or enhancing their development in the systems and methods disclosed herein.
  • some of the growth factors can be incorporated into the hydrogel or binder in which harvested adipose cells 12 are aggregated.
  • additives can be incorporated into the hydrogel or binder in which harvested adipose cells 12 are aggregated.
  • additives can include, but are not limited to, flavorants, colorants, texturizers, vitamins, minerals, amino acids, proteins/peptides, and fatty acids. These additives can be added to the hydrogel or binder in amounts that are tuned to the desired outcome.
  • Growth factors that are suitable for use as additives include, but are not limited to, fibroblast growth factor 2, transforming growth factor beta 3, insulin-like growth factor, or the like. Growth factors may be fish-specific growth factors.
  • cultured cells are generally deficient in oleic acid, linoleic acid, or arachidonic acid. These can all be additives in the present disclosure, thereby obviating the deficiency.
  • Alternative or additional additives include, but are not limited to, flavorants, colorants, texturizers, vitamins, minerals, amino acids, proteins/peptides, whole foods, oils, and fatty acids.
  • Particular fatty acids such as linoleic acid, arachidonic acid, alpha-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid, may be preferable but are not exclusive.
  • additives may be selected from the group comprising soybean oil, coconut oil, palm oil, cocoa butter, shea butter, mango butter, olive oil, canola oil, sunflower oil, flaxseed oil, avocado oil, peanut oil, sesame oil, corn oil, vegetable oil, margarine, shortening, and vegetable ghee.
  • Additives comprising fatty acids or oils may be hydrogenated at least partially (which includes wholly hydrogenated) or may be present in their oleogel, bigel, or emulgel form.
  • Whole foods may be selected from the group comprising, but not limited to, tofu, tempeh, instantan, jackfruit, quinoa, chia seeds, bananas, oats, avocados, flax seeds, rice, tapioca/arrowroot, and potatoes.
  • One particularly useful additive for the present disclosure is a fatty acid. Without wishing to be bound by any particular theory, it is believed that cell lines that are generally lacking in the production of a desirable fatty acid can be supplemented in the development process to include more of the desirable fatty acid. Again, without wishing to be bound by any particular theory, it is believed that in vitro fat cells have historically been lacking in certain fatty acids.
  • omega 3 fatty acids are also typically lacking in omega 3 fatty acids as these are also dietary. These would be alpha-linolenic acid (18:3), eicosapentaenoic acid (EP A, 20:5), and docosahexaenoic acid (DHA, 22:6). Omega 3s are generally less abundant in terrestrial (land) animals so a lack of them is not that detrimental. However, they can make up a significant proportion of fish lipids, so it may be important to have for fish adipocytes.
  • the supplementing concentrations can be chosen to provide desirable end concentrations.
  • soybean oil can be added to one or more of the stages of the system and method. Soybean oil has been shown to increase lipid drop size in in vitro adipose tissue. By adding soybean oil, a desired droplet size can be achieved more quickly, thereby enhancing efficiency of the overall process.
  • Alternative or additional exemplary oils may include coconut oil, palm oil, cocoa butter, shea butter, mango butter, olive oil, canola oil, sunflower oil, flaxseed oil, avocado oil, peanut oil, sesame oil, com oil, vegetable oil, margarine, shortening, and vegetable ghee. Oils may be partially or wholly hydrogenated and may be constituted in their oleogel, bigel, or emulgel form.
  • the additive might be present in the form of an uptake-enhancing additive for the explicit purpose of enhancing uptake of other additives.
  • an uptake-enhancing additive for the explicit purpose of enhancing uptake of other additives.
  • the uptake-enhancing additive can be an enzyme that modifies a different additive to make that different additive more suitable for uptake (e.g., a lipase with large lipids).
  • the uptake-enhancing additive can be a permeability-enhancing agent, which enhances the permeability of the cell membrane of the adipose cells.
  • One specific avenue for introducing growth factors and/or agents and/or additive into the inventive compositions is by way of adding the growth factors and/or agents and/or additives into the first culture media or the second culture media at the time that those culture media are made and/or when those culture media are introduced into their respective tanks.
  • the culture media can be tailored for proliferation and differentiation, as well for other steps of the process.
  • the various steps can use more than one culture media.
  • one culture media could be used for a first part of a proliferation process and the cells can be transferred to a different culture media for a later part of the proliferation process.
  • the culture media is typically comprised of a basal medium containing basic nutrients (sugars/carbohydrates, lipids/fatty acids, amino acids, vitamins, minerals, salts, water) combined with a growth factor or complex component containing proteins, peptides, hormones, or other bioactive compounds responsible for directing cell behavior, (e.g., to promote proliferation/cell survival, or to promote adipogenesis, or to suppress other cell pathways such as osteogenesis)
  • Basal media will likely be best optimized for the specific adipogenic progenitor cell that is used but may be similar to or based off of existing formulations such as Dulbecco’s modified eagle medium (DMEM), Ham’s F12, Ham’s MCDB 131, Iscove’s modified Dulbecco’s medium (IMDM).
  • Growth factors for adipogenic progenitor proliferation media formulations may include non-exhaustively: serum albumins, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), fetuin, bone morphogenic proteins (BMPs, e.g., BMP4), Wnt proteins, leukemia inhibitor factor (LIF), hydrocortisone, testosterone, progesterone, estrogen, hypoxia inducible factors (HZFs), stem cell growth factor- (SCGF-0), tumor necrosis factor alpha (TNFa), interleukin- 1 beta (IL- 10) and other interleukins (ILs), transforming growth factor beta (TGF0), insulin-like growth factor 1 (IGF-1), insulin, transferrin, myoglobin, vitronectin, truncated vitronectin peptides, laminin, laminin peptides (e g., 511-E8), fibronectin,
  • Growth factors specifically for adipogenic progenitor cell differentiation include non- exhaustively: Phospholipids, cholesterols (e.g., very low density lipoprotein, VLDL), fatty acids (e.g., oleic acid, linoleic acid), triacylglycerides (e.g., soybean oil, olive oil, rapeseed oil, fish oil), cyclodextrins (to solubilize non-polar ingredients), PPARy agonists (e.g., rosiglitazone, pioglitazone, pristanic acid, phytanic acid, lutein, beta-carotene, all-trans retinoic acid, 9-cis retinoic acid), ascorbic acid, isobutylmethylxanthine (IBMX), dexamethasone, hydrocortisone, insulin, insulin-like growth factor 1 (IGF-1). In some cases, components that promote differentiation may overlap with ingredients in the basal medium formulation
  • the effects of more complex growth factors may be replicated more cost effectively using peptides or small molecules.
  • Growth factors, in particular large protein growth factors may also be modified to impart features such as improved thermal stability over time.
  • certain growth factors or media components can be excluded to reduce costs, should the cells be engineered to not require such components/be engineered to generate the components themselves from components or basic/simple nutrients in the basal media.
  • cells can be engineered to produce glutamine synthetase, which would reduce or eliminate the need for glutamine in the culture media formulation.
  • fish-specific versions of the growth factors mentioned herein may be used to for improved/enhanced function (relative to the typically used mammalian versions) when culturing adipogenic progenitor cells from fish or seafood.
  • lactic acid may be extracted from the culture media (or first culture media or second culture media) and processed into polylactic acid. This beneficially repurposes a potential waste product.
  • the computational facilities of the system can include various artificial intelligence and machine learning algorithms to optimize production.
  • machine learning techniques that use a production variable as a target for optimization could work toward a more efficient culture media selection.
  • the optimization process can be automated with appropriate robotics.
  • the present disclosure provides a host of end-use applications for the materials provided by the systems and methods described herein. These end-use applications can be applicable both to the desirable products produced by the systems and methods described herein (i.e., adipose tissue in many/most cases) and the undesirable or waste products produced by the systems and methods described herein (i.e., sedimented cells or non-adipose tissue in many/most cases), unless the context clearly dictates otherwise.
  • the materials produced by the systems and methods described herein can generally be incorporated into edible products.
  • the materials produced by the systems and methods described herein can generally be incorporated into consumer products not limited to cosmetics, skin creams, lotions, soaps, detergents, emulsifiers, lubricants, fuels, and/or animal feeds.
  • the materials produced by the systems and methods described herein can be included in cosmetics, such as make-up, skin creams, lotions, and the like.
  • the materials produced by the systems and methods described herein can be included in soaps, detergents, or other generally emulsifying compositions.
  • the materials produced by the systems and methods described herein can be included in industrial compositions, such as lubricants used in a variety of industries, liquids useful in extraction industries like the oil and gas industry, and the like.
  • the materials produced by the systems and methods described herein can be included in sustainability systems for environmental purposes, such as animal feeds, aquaculture compositions, and the like.
  • the materials produced by the systems and methods described herein can be included in end-use compositions that include some proportion of material that is produced from the systems and methods described herein and some proportion from naturally occurring sources.
  • the cultured adipose tissue described herein can be combined with lean meat from a natural source to make ground meat having an artificially enhanced fat content.
  • the materials produced can have components extracted and that can be the end product.
  • the cultured adipose tissue can be rendered to remove fatty acids and those fatty acids can be the end product.
  • a system for executing the methods described herein includes one or more raw material sources.
  • the raw material sources can feed raw materials into a first bioreactor.
  • the first bioreactor can provide a first output to optionally proceed to a second bioreactor and can provide waste via a waste output to be collected for reuse and recycling.
  • the second bioreactor is not present and the first output is moved on to other portions of the system for further processing.
  • the system can include any number of bioreactors. Those bioreactors can be arranged to operate in series, in parallel, or in any combination of the two.
  • the system includes various extraction, filtration, and monitoring systems, as described elsewhere herein.
  • the raw material sources can be generally conventional material storage and supply technologies as would be appropriate for cell agriculture applications.
  • One important requirement of the raw material sources is that the materials need to be sterile when they are eventually introduced into the system for executing the methods described above.
  • Another optional feature of the raw material sources is temperature control. Particularly for the cellular material, the temperature control is important.
  • the conduits and other means of transport for the various material flows also require sterility and optionally include temperature controls.
  • the raw material sources can have quality control facilities.
  • the raw material sources can have cellular testing facilities (e g., PCR, morphology assessment, genetic testing, etc.).
  • the raw material sources can have appropriate facilities to confirm sterility, such as sampling and testing facilities as would be understood by those having ordinary skill in the art.
  • the raw material sources can be adapted to sterilize the raw materials, using techniques known in the art.
  • At least one portion of the raw material sources is adapted to provide a supply of cells for use in methods described herein.
  • the raw material sources can take any physical form that meets the requirements of the applications described, including but not limited to, storage tanks, hoppers, reservoirs, silos, and other means of storing raw materials on-site for use in the methods described herein.
  • Receiving raw materials into the raw materials sources can be handled in generally conventional ways, with the understanding that the ordering and delivery of cells is an emerging field.
  • the raw material sources are fed by an on-site supply of raw materials.
  • the raw material sources are supplied with locally sourced cells.
  • the system can include the necessary processing facilities to isolate cells of interest from livestock or other local sources of cells.
  • the raw material sources for cells include cells that are embedded in a matrix, such as the natural tissue matrix from which they originated.
  • the cells used herein can be provided to the system in the form of agricultural silage mixtures.
  • the cells themselves may be extracted from agricultural waste products.
  • the raw material sources are supplied or refilled via pipelines or other conduits for materials. In some cases, the raw material sources are themselves pipelines or other conduits for materials.
  • the system include an entire sub-facility dedicated to producing cells for use in the methods described herein.
  • This sub-facility can include all of the various processing systems that are required for processing agricultural and livestock products to isolate cells for use as described herein.
  • the systems may be paired with conventional farming and food distributions systems, such as butcheries or meat packing facilities. It should be appreciated that the initial stages of the growth of cellular agriculture are unlikely to supplant traditional meat sources, so the systems described herein may benefit from being integrated into a conventional food supply chain. In these cases, products that are typically viewed as waste could provide useful cells for use in the methods described herein.
  • the system can be divided into raw material production facilities, where cells and other reagents are produced, and end product production facilities, where the desired adipose tissue is produced.
  • the raw material sources can include sources for the various reagents used in the methods, including the culture media. As with the cellular material, the reagents used in the methods can also be locally sourced.
  • At least one portion of the raw material sources is adapted to provide culture media for use in the methods described herein.
  • These raw material sources can include powdered storage media that is combined with purified water to make culture media or they can include the completed culture media itself.
  • the system is adapted to receive commercial-scale quantities of material, so the system includes receiving docks and pipe infrastructure to receive trailer trucks and tanker trucks of raw materials. Similarly, rail, air, and sea transport features are contemplated. As outlined above, pipelines are also a viable option for receiving raw material.
  • the raw materials can be recycled materials, including recycled agricultural materials and other recycled materials that can be useful in the processes described herein. In some cases, the raw materials are recycled from the system or method described herein.
  • the raw materials are delivered from the raw material sources into the first bioreactor.
  • the means for delivery are conventional, with sterility maintained.
  • Material can be transferred between bioreactors using conventional delivery means, with sterility maintained.
  • Waste streams can be moved in conventional ways. However, the systems and methods described herein make improved use of waste. For example, most of the waste streams in this system and method can be recycled within the system.
  • Cellular material can be isolated from the culture media in ways understood to those having ordinary skill in the art, including centrifugation and filtration.
  • a device for separating cells from culture media is a counter flow centrifuge.
  • the cells can be isolated from culture media by using micro-carrier, such as those made out of pectin.
  • the cells can be harvested using techniques made popular in different industries, such as the pharmaceutical industry and brewing industry, where cellular processes are used widely.
  • the cells can be harvested as floating cells.
  • certain specific harvesting devices can be used, which may not be applicable in other environments.
  • a skimmer could be used to skim the surface of a bioreactor to remove surface cells.
  • a screen or mesh could be located beneath the surface of the culture media and then raised out of the culture media to harvest the floating cells while the culture media is drained and left behind.
  • the cells can be harvested as sedimented cells.
  • certain specific harvesting devices can be used, which may not be applicable in other environments.
  • suction filtration of a bottom portion of the bioreactor may be one suitable way to harvest sedimented cells.
  • adhered cells can be harvested from a substrate using conventional methods. It should be appreciated that harvesting the adhered cells are not specifically the focus of most of this disclosure, though many of the features described herein are applicable to adhered cells. For the avoidance of doubt, the features of the present disclosure are also contemplated for use with harvested adhered cells.
  • Applicant has developed a technique for harvesting cells from the surface, which is different from existing techniques.
  • a layer of fat cells or otherwise
  • the layer is pipetted off the top by placing the tip of the pipette into the fat layer and introducing suction.
  • this conventional process causes problems with certain samples and the fat layer cannot be isolated.
  • the inventors surprisingly discovered that removing fat cells by maintaining suction on a pipette as the tip is drawn close to the fat layer, contacting the fat layer with the tip of the pipette, then lifting the pipette away from the surface, thereby removing a small section of the layer.
  • This process is repeated for different locations on the layer, until most or all of the layer has been removed.
  • This process can be manual or automated. If automated, a robotic arm with sensors can bring the pipette tip to the surface with adequate control to selectively remove the fat layer.
  • the culture media within a given bioreactor is replaced with every usage.
  • the culture media is retained for some length of time before being replaced.
  • the culture media is slowly regenerated over time by supplementing the portions of the media that are removed in the process.
  • the system can include culture media fdters, which filter culture media for reintroduction into the bioreactors.
  • the system can include an analytical device, such as optical spectrophotometer, a gas chromatography system, a mass spectrometry system, other analytical devices known in the art to useful for assessing quality control of liquid compositions such as culture media, or a combination thereof. This analytical device can be used to assess new or used culture media to determine if it is appropriate for further use.
  • the analytical device is used to search for contaminants, such as undesirable bacterial growth or harsh chemical solvents. If contamination is identified, then the culture media can be routed to waste or to a refining facility for removal of the contaminants.
  • the analytical device is used to confirm that certain desirable components are present.
  • the analytical device can confirm the presence or amount of one of the growth factors discussed above. If the analytical device identifies one or more desirable components are missing from a new or used culture media, then the culture media can be supplemented with the missing desirable components.
  • the analytical device inspects for both contaminants and desirable components and directs the system to make the necessary remedial corrections, if needed.
  • the facility is equipped with the necessary scanning and tracking devices that would be required to keep track of the sourcing of the various components that go into the methods described herein.
  • one of the powerful advantages that may be provided with cellular agriculture is the reduction in transportation costs that are required to transport meat from places where the animals are harvests to the consumer.
  • the system can be located nearer to the consumer, so the bulk of the shipping cost and environmental impact is related to raw material shipping.
  • the system includes the necessary computing facilities to generate and/or modify blockchains in ways that are understood by those in the art to be useful for tracking the authenticity or sourcing of food products. Again, without wishing to be bound by any particular theory, it is believed that there may be advantage to providing consumers with evidence of how little shipping was required to produce a given food product.
  • blockchain sourcing authentication techniques or other methods known in the art to provide similar capabilities
  • Ill tissue produced by the methods described herein can have corresponding entries into a blockchain regarding the cultured adipose tissue's provenance.
  • the system and method described herein has the full capability to acquire and record any data that is necessary for complying with food regulatory authorities.
  • Logs of processing parameters, test results, and other information that is relevant to regulators can be created and saved. The logs can be made manually or automatically.
  • the digital records can be utilized to improve the system and method.
  • the improvements can be based on measured properties of the products, but in other cases, the improvements can be based on user feedback. There is inherently a lag between the facility producing a product and consumers enjoying it, so if there is a wave of product that has a particularly positive or negative customer response, then it would be possible to look up specific information from the run that led to that customer response for the purpose of intentionally repeating or not repeating it.
  • the data can include both information about the cell source material and the other formulation information from the process. If a certain cell line, process, and set of formulations produces a superior product, then the formula can be retrieved for reproduction.
  • Data can be collected from multiple facilities, thereby providing a global data set, from which broader conclusions can be drawn. For example, if all plants around the world show an inefficiency in the use of a given reagent, then this flags the problem as likely being related to the portions of the system or method that are deployed in all facilities.
  • the present disclosure provides a food product prepared directly from the culture adipose tissue that is made by the method described herein.
  • This product stands in contrast to the other food products described herein, where the cultured adipose tissue is integrated with other components to make a food product that includes the cultured adipose tissue.
  • the disclosed food product is a shaped and cooked piece of the cultured adipose tissue described herein. Without wishing to be bound by any particular theory, it is believed that the cultured adipose tissue disclosed herein can be treated as a food product with similar properties to certain animal products.
  • slices of the cultured adipose tissue can be fried to form cultured adipose chips, which may resemble pork rinds in certain embodiments.
  • slices of the culture adipose tissue can be seared to form a seared fatty tissue morsel.
  • the cultured adipose tissue is cooked or fried.
  • the cultured adipose tissue is shaped to a particular three- dimensional configuration as desired.
  • the food products described herein both those that integrate the culture adipose tissue and those that are composed entirely of cultured adipose tissue, can be adapted in the ways described above with respect to altering the nutritional or flavor contents, either by way of genetic manipulation or process manipulation. In this fashion, a previously unhealthy product can be adapted to provide a healthier alternative.
  • a cultured adipose tissue chip that is flavored to taste like pork rinds could be adapted to have a different fatty acid blend, thereby producing fewer negative health consequences to a consumer than a comparison conventional pork rind.
  • comparisons regarding the health consequences of food may require larger studies and statistical averages rather than direct measurements of individual cases.
  • Example 1 Timeline of 3T3-L1 adipogenic differentiation
  • FIG. 5 shows a timeline for differentiation of 3T3-L1 adipogenic cells. Days 0 (dO), 2 (t/2), 15 and 30 (d3O) are indicated on the timeline. Confluent pre-adipocytes were grown in adipogenic induction media for the first two days and then switched to lipid accumulation media until harvest for cultured fat tissue formation on day 15 (see Example 2). Additional samples were grown in lipid accumulation media for 30 days to analyze lipid accumulation over longer-term culture.
  • Lipidomics and immunostaining were carried out on day 13. Additional samples were grown in lipid accumulation media for 1 month to analyze lipid accumulation over longer-term culture.
  • Example 2 Harvest of lipid-laden adipocytes and formation of 3D cultured fat constructs
  • lipid-filled adipocytes were detached using a cell scraper. The adipocytes were then drained of non-cell liquid using a 0.22 micrometer vacuum filter. After detaching and draining, the in vitro adipocytes were then combined with transglutaminase or alginate and formed into discrete macroscale tissues in a 3D printed mold. Finally, 3D cultured fat constructs were mechanically tested for compressive strength, fluorescently stained for lipid and analyzed for volatile compounds.
  • Example 3 Methods for generating 3D cultured fat using alginate or transglutaminase
  • Transglutaminase aggregation Cultured fat was produced by mixing a 15% solution of transglutaminase with drained adipose tissue at a 2:8 volumetric ratio in a 3D printed mold.
  • Example 4 Generating Macroscale Cultured Fat via the Aggregation of In Vitro Grown Adipocytes
  • Native (in vivo) adipose tissue is largely a dense packing (aggregation) of lipid-filled adipocytes, held together by a sparse extracellular matrix (ECM) network. This is opposed to muscle tissue, which is comprised of aligned fibers in a multi -hi erar chi cal structure. Since native adipose is an aggregation of adipocyte globules with less structural features than in muscle it is possible to recapitulate it on an organoleptic basis by aggregating separately grown adipocytes or adipocyte clusters.
  • ECM extracellular matrix
  • adipocytes separately, or in small clusters, is desirable because it is currently infeasible to directly grow large tissues on the macroscale (millimeter scale and up) using contemporary tissue engineering techniques.
  • By growing individual or small clusters of cells we are able to produce-at-scale a general mass of adipose cells, followed by the formation of actual adipose tissue through various methods of binding and aggregating the cells into a solid 3D construct.
  • Methods of binding and aggregating the fat cells include enzymatic cross-linking with transglutaminase, as well as embedding in hydrogels such as alginate (a material which is already used as a fat replacer in the food industry).
  • An adipose aggregation approach circumvents the mass transport issue of macroscale 3D culture/tissue engineering by deliberately culturing individual adipocytes or small adipocyte clusters via 2D culture or various scalable bioreactors. Only after the separated adipocytes have grown and accumulated sufficient lipid does cell culture end, followed by the manual aggregation of the cultured cells into denser 3D tissues without the need for vascularization and perfusion. This is uniquely possible with cultured fat because for food purposes, for example, once the tissue is formed and harvested the cultured cells do not need to stay alive. This process is analogous to meat production in conventional animal agriculture where muscle and fat cells gradually cease to be viable after slaughter. For conventional tissue engineering (e.g., medical applications etc.) cells in 3D tissues are expected to remain viable to be used for implantation into the body, or for use and testing in an in vitro tissue model.
  • tissue engineering e.g., medical applications etc.
  • Example 5 Avoiding lift-off issues faced with adherent adipocytes
  • Applicant has also observed that a large subpopulation of the cultured adipocytes adhere strongly to tissue culture plates and do not float away, avoiding issues of lift-off of adherent adipocytes in vitro due to increasing buoyancy as the adipocytes become fatty.
  • the 2D culture systems self-sort for adherent cell populations.
  • techniques have been developed for dealing with the non-adherent cell populations.
  • An assessment of such benchmark threshold or property/properties allows for the processing/harvesting decision to select an appropriate time for processing/harvesting cells (or cell). This decision can be made based on cell growth ((i.e., cell size and/or mass and/or density, number of cells or cell count).
  • lipid droplet size is an important factor in determining when an adipose cell is ready for harvesting. This lipid droplet size is optically visible and can be interrogated by known optical methods. As such, monitoring of lipid droplet size within a cell or within a population of cells could be used for harvesting decisions.
  • the cell secretome can also be analyzed. Specifically, the level of adipokine or adiponectin surrounding the cells can be used as a measure of readiness for harvest.
  • monitoring can be performed on non-adhered (or “floating”) cells.
  • floating cells In the case where multiple different density gradients are deployed and/or where multiple different populations of floating cells are collected, these cells can be divided into quality grades based on one or more properties as discussed above.
  • Floating cells can be delivered to a number of useful devices including chromatographic systems, microfluidics systems, cell sorting systems, cell pickers, and cell skimmers, to name a few examples.
  • the microfluidics system can be tailored specifically for handling floating cells described herein.
  • One example of tailoring the microfluidics system for floating cells is to adjust the density of the carrier fluid prior to entry into the microfluidics system, such that the cells are neutral buoyance and are no longer "floating" in the medium.
  • the harvested cells might be rinsed to remove/minimize/dilute undesired culture media components. Rinsing can be done by way of counter-flow centrifugation or conventional filtration. As another example, cells can be clarified by removing undesirable components, which are not removed by rinsing. This is a process that is regularly used in the pharmaceutical arts, but it is not conventionally used in cellular agriculture. In one specific example, clarification of cells to remove cell debris or excess nucleic acids is expressly contemplated. Other methods such as flocculation via pH or the use of polymers such as chitosan which can induce flocculation are contemplated. Other methods of collecting non-adhered cells may comprise collecting non-clustered floating cells, collecting only non-adhered cells, and/or not collecting adhered cells.
  • a specific stationary phase and mobile phase may be selected for a desired result. For instance, if size selection is an important criterion, then a stationary phase that has porosity that provides differentiated retention properties based on size could be useful. As another example, if surface chemistry of the adipose cells is an important criterion, then a stationary and/or mobile phase could be selected based on those desirable surface chemistries. In one specific example, certain cells may be more amenable to embedding into a hydrogel if they have a specific surface chemistry, so a chromatography system could be used to isolate cells that are more or less amenable to embedding into the hydrogel.
  • certain cells may be more amenable to direct cross-linking to other cells if they have a specific surface chemistry, so a chromatography system could be used to isolate cells that are more or less amenable to direct cross-linking with one another.
  • the cell sorting system can be used to differentiate and categorize the cells based on measurable properties, as would be appreciated by a skilled artisan. Applicant does not intend to provide specific inventive contribution with respect to the cell sorting itself, but rather integrates and utilizes the understood concept of cell sorting into inventive concepts that form a part of the broader disclosure described herein.
  • the cells are sorted through multiple different centrifuges.
  • the cells that make it through multiple centrifuges will have different properties from those that do not and those different categories of cells can be divided into grades, in a similar fashion as USDA grading.
  • the cells could be graded as superior or inferior relative to one another.
  • the systems and methods of this disclosure provide approaches to collecting and utilizing these floating cells, which have traditionally been discarded as waste. By making productive use of these previously wasted products, much higher efficiency is achieved by virtue of not wasting this large population of cells.
  • sedimented cells may be inversely applicable to sedimented cells.
  • the bottom of a reaction tank could be monitored for sedimented cells.
  • Sedimented cells may be preferred for certain applications and those sedimented cells can be collected and further processed for those applications.
  • adipose tissue which is generally buoyant in aqueous environments such as growth cultures, the sedimented cells are typically waste products and can be processed as such.
  • Sedimented cells can be isolated and further processed as biomass or recycled for raw materials and reintroduced into the system and/or method at various points in the system/process. For example, sedimented cells can be harvested and have components for culture media extracted from them. In certain cases sedimented cells are harvested separately from non-adhered cells. Other uses may include filler material in animal feedstock, starting material for a further culture, and/or a single cell protein for growth of bacteria or fungus.
  • sedimented cells are typically very high in nucleic acid content.
  • the sedimented cells can have nucleic acid content reduced before further use.
  • the cells can still be useful without reducing the nucleic acid content, for example, as livestock feed.
  • the sedimented cells, with or without reducing the nucleic acid content can be mixed into one or more of the food products described herein for the purpose of adding more of a "meat" flavor.
  • Example 7 Advantages in culturing adipocytes
  • the techniques described herein can provide advantageous ability to administer growth factors to the cells, in the interest of providing them with more optimal receipt and uptake of the growth factors.
  • Growth factors and other additives can be incorporated into the hydrogel or binder in which harvested adipose cells 12 are aggregated. These additives can include flavorants, colorants, texturizers, vitamins, minerals, amino acids, proteins/peptides, and fatty acids and can be added to the hydrogel or binder in amounts that are tuned to the desired outcome.
  • Growth factors that are suitable for use as additives can include fibroblast growth factor 2, transforming growth factor beta 3, and insulin-like growth factor. It is believed that cultured cells are generally deficient in oleic acid, linoleic acid, and/or arachidonic acid. Thus, these can all be additives, thereby obviating the deficiency.
  • One particularly useful additive for the present disclosure is a fatty acid. It is believed that cell lines that are generally lacking in the production of a desirable fatty acid can be supplemented in the development process to include more of the desirable fatty acid and that in vitro fat cells have historically been lacking in certain fatty acids.
  • the most abundantly missing fatty acid is linoleic acid (18:2), as mammals take this from the diet rather than synthesizing it themselves.
  • the lack of 20:4 can be both good and bad, as it's been reported to be important for flavor but also a precursor molecule to pro-inflammatory compounds (eicosanoids).
  • Jn vitro mammalian cells are also typically lacking in omega 3 fatty acids as these are also dietary. These would be alphalinolenic acid (18:3), eicosapentaenoic acid (EP A, 20:5), and docosahexaenoic acid (DHA, 22:6). Omega 3s are generally less abundant in terrestrial (land) animals so a lack of them is not that detrimental. However, they can make up a significant proportion of fish lipids, so it may be important to have for fish adipocytes. In a general sense, when multiple different fatty acids are supplemented, the supplementing concentrations can be chosen to provide desirable end concentrations.
  • soybean oil can be added. Soybean oil has been shown to increase lipid drop size in in vitro adipose tissue. By adding soybean oil, a desired droplet size can be achieved more quickly, thereby enhancing efficiency of the overall process.
  • Certain additives may be present in the form of an uptake-enhancing additive for the explicit purpose of enhancing uptake of other additives.
  • the uptake of triglycerides was enhanced by administering an agent that induced the release of a relevant lipase, which allowed the breakdown of the triglycerides for entry into the cells.
  • Co-administered agents may be required to be administered with a given additive in the interest of improving uptake of the additive.
  • the uptake-enhancing additive can be an enzyme that modifies a different additive to make that different additive more suitable for uptake (e.g., a lipase with large lipids).
  • the uptakeenhancing additive can be a permeability-enhancing agent, which enhances the permeability of the cell membrane of the adipose cells.
  • One specific avenue for introducing growth factors and/or agents and/or additive into the inventive compositions is by way of adding the growth factors and/or agents and/or additives into the first culture media or the second culture media at the time that those culture media are made and/or when those culture media are introduced into their respective tanks.
  • the culture media can be tailored for proliferation and differentiation, as well for other steps of the process.
  • the various steps can use more than one culture media.
  • one culture media could be used for a first part of a proliferation process and the cells can be transferred to a different culture media for a later part of the proliferation process.
  • the culture media is typically comprised of a basal medium containing basic nutrients (sugars/carbohydrates, lipids/fatty acids, amino acids, vitamins, minerals, salts, water) combined with a growth factor or complex component containing proteins, peptides, hormones, or other bioactive compounds responsible for directing cell behavior, (e.g., to promote proliferation/cell survival, or to promote adipogenesis, or to suppress other cell pathways such as osteogenesis).
  • basic nutrients sucgars/carbohydrates, lipids/fatty acids, amino acids, vitamins, minerals, salts, water
  • a growth factor or complex component containing proteins, peptides, hormones, or other bioactive compounds responsible for directing cell behavior, (e.g., to promote proliferation/cell survival, or to promote adipogenesis, or to suppress other cell pathways such as osteogenesis).
  • Basal media will likely be best optimized for the specific adipogenic progenitor cell that is used but may be similar to or based off of existing formulations such as Dulbecco’s modified eagle medium (DMEM), Ham’s F12, Ham’s MCDB 131, Iscove’s modified Dulbecco’s medium (IMDM).
  • DMEM Dulbecco modified eagle medium
  • IMDM Iscove’s modified Dulbecco’s medium
  • Growth factors for adipogenic progenitor proliferation media formulations may include non-exhaustively: serum albumins, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), fetuin, bone morphogenic proteins (BMPs, e.g., BMP4), Wnt proteins, leukemia inhibitor factor (LIF), hydrocortisone, testosterone, progesterone, estrogen, hypoxia inducible factors (HIFs), stem cell growth factor-P (SCGF-P), tumor necrosis factor alpha (TNFa), interleukin- 1 beta (IL- 1 ) and other interleukins (ILs), transforming growth factor beta (TGFP), insulin-like growth factor 1 (IGF-1), insulin, transferrin, myoglobin, vitronectin, truncated vitronectin peptides, laminin, laminin peptides (e.g., 511-E8), fibronectin,
  • Growth factors specifically for adipogenic progenitor cell differentiation include non- exhaustively: Phospholipids, cholesterols (e.g., very low density lipoprotein, VLDL), fatty acids (e.g., oleic acid, linoleic acid), triacylglycerides (e.g., soybean oil, olive oil, rapeseed oil, fish oil), cyclodextrins (to solubilize non-polar ingredients), PPARy agonists (e.g., rosiglitazone, pioglitazone, pristanic acid, phytanic acid, lutein, beta-carotene, all-trans retinoic acid, 9-cis retinoic acid), ascorbic acid, isobutylmethylxanthine (IBMX), dexamethasone, hydrocortisone, insulin, insulin-like growth factor 1 (IGF-1). In some cases, components that promote differentiation may overlap with ingredients in the basal medium formulation
  • Example 8 A system for culturing adipocytes
  • a system for culturing adipocytes includes one or more raw material sources feeding into a first bioreactor.
  • the first bioreactor can provide a first output to optionally proceed to a second bioreactor and can provide waste via a waste output to be collected for reuse and recycling.
  • a second bioreactor may not be present and the first output is moved on to other portions of the system for further processing.
  • the system can include any number of bioreactors. Those bioreactors can be arranged to operate in series, in parallel, or in any combination of the two.
  • the system includes various extraction, filtration, and monitoring systems.
  • the raw material sources can be generally conventional material storage and supply technologies as would be appropriate for cell agriculture applications.
  • the raw material sources need to be sterile when they are eventually introduced into the system for executing the methods described above.
  • Another optional feature of the raw material sources is temperature control. Particularly for the cellular material, the temperature control is important.
  • the conduits and other means of transport for the various material flows also require sterility and optionally include temperature controls.
  • the raw material sources can have quality control facilities, cellular testing facilities (e.g., PCR, morphology assessment, genetic testing, etc.), and/or appropriate facilities to confirm sterility, such as sampling and testing facilities.
  • the raw material sources can be adapted to sterilize the raw materials. At least one portion of the raw material sources is adapted to provide a supply of cells for use in the methods described herein.
  • the raw material sources can take any physical form that meets the requirements of the applications described, including but not limited to, storage tanks, hoppers, reservoirs, silos, and other means of storing raw materials onsite for use in the methods described herein. Receiving raw materials into the raw materials sources can be handled in generally conventional ways, with the understanding that the ordering and delivery of cells is an emerging field.
  • the system is adapted to receive commercial-scale delivery of cells as raw material for the methods described herein.
  • sterility is of paramount importance.
  • the raw material sources may be fed by an on-site supply of raw materials and/or with locally sourced cells.
  • the system can include the necessary processing facilities to isolate cells of interest from livestock or other local sources of cells.
  • the raw material sources for cells may include cells that are embedded in a matrix, such as the natural tissue matrix from which they originated and may be provided to the system in the form of agricultural silage mixtures.
  • the cells themselves may be extracted from agricultural waste products.
  • the system may include an entire sub-facility dedicated to producing cells for use in the methods described herein.
  • This sub-facility can include all of the various processing systems that are required for processing agricultural and livestock products to isolate cells
  • the systems may be paired with conventional farming and food distributions systems, such as butcheries or meat packing facilities. It should be appreciated that the initial stages of the growth of cellular agriculture are unlikely to supplant traditional meat sources, so the systems described herein may benefit from being integrated into a conventional food supply chain. In these cases, products that are typically viewed as waste could provide useful cells for use in the methods described herein.
  • the system can be divided into raw material production facilities, where cells and other reagents are produced, and end product production facilities, where the desired adipose tissue is produced. At least one portion of the raw material sources is adapted to provide culture media for use in the methods described herein. These raw material sources can include powdered storage media that is combined with purified water to make culture media or they can include the completed culture media itself.
  • the system is adapted to receive commercial-scale quantities of material, so the system includes receiving docks and pipe infrastructure to receive trailer trucks and tanker trucks of raw materials. Similarly, rail, air, and sea transport features are contemplated. As outlined above, pipelines are also a viable option for receiving raw material.
  • the raw materials can be recycled materials, including recycled agricultural materials and other recycled materials that can be useful in the processes described herein. In some cases, the raw materials are recycled from the system or method described herein.
  • the raw materials are delivered from the raw material sources into the first bioreactor.
  • the means for delivery into the first bioreactor and for the transfer between bioreactors are conventional, with sterility maintained.
  • Waste streams can be moved in conventional ways. However, the systems and methods described herein make improved use of waste. For example, most of the waste streams in this system and method can be recycled within the system.
  • Cellular material can be isolated from the culture media in ways understood to those having ordinary skill in the art, including centrifugation and filtration.
  • a device for separating cells from culture media is a counter flow centrifuge.
  • the cells can be isolated from culture media by using micro-carrier, such as those made out of pectin.
  • the cells can be harvested using techniques made popular in different industries, such as the pharmaceutical industry and brewing industry, where cellular processes are used widely. As discussed in Example 7, the cells can be harvested as floating cells. In these cases, certain specific harvesting devices can be used, which may not be applicable in other environments. For example, a skimmer could be used to skim the surface of a bioreactor to remove surface cells.
  • a screen or mesh could be located beneath the surface of the culture media and then raised out of the culture media to harvest the floating cells while the culture media is drained and left behind.
  • the cells can be harvested as sedimented cells.
  • certain specific harvesting devices can be used, which may not be applicable in other environments. For example, suction filtration of a bottom portion of the bioreactor may be one suitable way to harvest sedimented cells.
  • the culture media within a given bioreactor is replaced with every usage. In some cases, the culture media is retained for some length of time before being replaced. In some cases, the culture media is slowly regenerated over time by supplementing the portions of the media that are removed in the process.
  • the system can include culture media filters, which filter culture media for reintroduction into the bioreactors.
  • the system can include an analytical device, such as optical spectrophotometer, a gas chromatography system, a mass spectrometry system, other analytical devices known in the art to useful for assessing quality control of liquid compositions such as culture media, or a combination thereof. This analytical device can be used to assess new or used culture media to determine if it is appropriate for further use.
  • the analytical device may be used to search for contaminants, such as undesirable bacterial growth or harsh chemical solvents. If contamination is identified, then the culture media can be routed to waste or to a refining facility for removal of the contaminants.
  • the analytical device may be used to confirm that certain desirable components are present. For example, the analytical device can confirm the presence or amount of one of the growth factors discussed above. If the analytical device identifies one or more desirable components are missing from a new or used culture media, then the culture media can be supplemented with the missing desirable components. In some cases, the analytical device inspects for both contaminants and desirable components and directs the system to make the necessary remedial corrections, if needed.
  • the facility may be equipped with the necessary scanning and tracking devices that would be required to keep track of the sourcing of the various components that go into the methods described herein.
  • one of the powerful advantages that may be provided with cellular agriculture is the reduction in transportation costs that are required to transport meat from places where the animals are harvests to the consumer.
  • the system can be located nearer to the consumer, so the bulk of the shipping cost and environmental impact is related to raw material shipping.
  • the system includes the necessary computing facilities to generate and/or modify blockchains in ways that are understood by those in the art to be useful for tracking the authenticity or sourcing of food products. Again, without wishing to be bound by any particular theory, it is believed that there may be advantage to providing consumers with evidence of how little shipping was required to produce a given food product.
  • blockchain sourcing authentication techniques or other methods known in the art to provide similar capabilities
  • the culture adipose tissue produced by the methods described herein can have corresponding entries into a blockchain regarding the cultured adipose tissue's provenance.
  • the system and method described herein has the full capability to acquire and record any data that is necessary for complying with food regulatory authorities.
  • Logs of processing parameters, test results, and other information that is relevant to regulators can be created and saved.
  • the logs can be made manually or automatically.
  • the digital records can be utilized to improve the system and method. In some cases, the improvements can be based on measured properties of the products, but in other cases, the improvements can be based on user feedback.
  • the data can include both information about the cell source material and the other formulation information from the process. If a certain cell line, process, and set of formulations produces a superior product, then the formula can be retrieved for reproduction. Data can be collected from multiple facilities, thereby providing a global data set, from which broader conclusions can be drawn. For example, if all plants around the world show an inefficiency in the use of a given reagent, then this flags the problem as likely being related to the portions of the system or method that are deployed in all facilities.
  • Example 9 A technique for harvesting cells from the surface
  • Applicant has developed a technique for harvesting cells from the surface, which is different from existing techniques.
  • a layer of fat cells or otherwise
  • the layer is pipetted off the top by placing the tip of the pipette into the fat layer and introducing suction.
  • this conventional process causes problems with certain samples and the fat layer cannot be isolated.
  • the inventors surprisingly discovered that removing fat cells by maintaining suction on a pipette as the tip is drawn close to the fat layer, contacting the fat layer with the tip of the pipette, then lifting the pipette away from the surface, thereby removing a small section of the layer.
  • This process is repeated for different locations on the layer, until most or all of the layer has been removed.
  • This process can be manual or automated. If automated, a robotic arm with sensors can bring the pipette tip to the surface with adequate control to selectively remove the fat layer.
  • Example 10 Aggregation of adipose cells by flocculation
  • Applicant proposes the aggregation of non-adhered adipose cells by flocculation.
  • Flocculation often refers to the aggregation of unstable and small particles through surface charge neutralization, electrostatic patching and/or bridging after addition of flocculants (or agents that make fine and sub-fine solids or colloids suspended in the solution form large loose flocs through bridging thus achieving solid-liquid separation).
  • Flocculation may be performed by one or more of several techniques, including auto-flocculation, bio-flocculation, chemical flocculation, particle-based flocculation, and electrochemical flocculation.
  • Various flocculation techniques have been reviewed by Matter, etal (2019, Applied Sciences).
  • Auto-flocculation may be performed at acidic pH (for example, a pH of 4.0). Autoflocculation may be performed at alkaline pH (for example, a pH of 10.4, 11.0, 1.5, 11.6, 12.0, or 12.5). Auto-flocculation may be performed over several days (for example, 16 days).
  • Chemical flocculation may be performed with inorganic flocculants such as aluminum sulfate, calcium oxide, iron(III) chloride, and magnesium hydroxide. Chemical flocculation may be performed with organic flocculants such as inulin, starches, chitosan, tannins, alginates, and lysines. Food-safe flocculants (such as chitosan and other long polymers) may be preferred.
  • inorganic flocculants such as aluminum sulfate, calcium oxide, iron(III) chloride, and magnesium hydroxide.
  • Chemical flocculation may be performed with organic flocculants such as inulin, starches, chitosan, tannins, alginates, and lysines. Food-safe flocculants (such as chitosan and other long polymers) may be preferred.
  • Particle-based flocculation may be performed using aminoclay-based nanoparticles such as 3-aminopropyltriethoxysilane (APTES) conjugated to magnesium and aminoclays conjugated to aluminum, titanium oxide, and humic acid.
  • Particle-based flocculation may be performed using magnetic particles including ferrous oxide nanoparticles and composites.
  • Adipose cells aggregated through means discussed in the present example may be further processed or used in the various methods, food products, systems (etc.) described in the present application.
  • Example 11 Food product prepared by adipose tissue made by the methods herein
  • a food product may be prepared directly from the culture adipose tissue that is made by the method described herein. This product stands in contrast to the other food products described herein, where the cultured adipose tissue is integrated with other components to make a food product that includes the cultured adipose tissue.
  • the disclosed food product is a shaped and cooked piece of the cultured adipose tissue described herein. It is believed that the cultured adipose tissue disclosed herein can be treated as a food product with similar properties to certain animal products.
  • slices of the cultured adipose tissue can be fried to form cultured adipose chips, which may resemble pork rinds in certain embodiments.
  • slices of the culture adipose tissue can be seared to form a seared fatty tissue morsel.
  • a method for producing cultured adipose tissue comprising: growing adipogenic precursor cells in a first culture media; differentiating the adipogenic precursor cells to adipose cells in a second culture media; harvesting non-adhered adipose cells; and aggregating the harvested non-adhered adipose cells to provide the cultured adipose tissue.
  • a method for producing cultured adipose tissue comprising: growing adipogenic precursor cells in a culture media; differentiating the adipogenic precursor cells to adipose cells in the culture media; harvesting non-adhered adipose cells; and aggregating the harvested non-adhered adipose cells to provide the cultured adipose tissue.
  • bioreactor is selected from the group consisting of a stirred suspension tank bioreactor, airlift bioreactor, bubble column bioreactor, fluidized bed bioreactor, packed bed bioreactor, vertical wheel bioreactor, and wave bag bioreactor .
  • a method for producing cultured adipose tissue comprising: growing adipogenic precursor cells on a two-dimensional (2D) substrate; differentiating the adipogenic precursor cells to adipose cells on the 2D substrate; harvesting non-adhered adipose cells; and aggregating the harvested non-adhered adipose cells to provide the cultured adipose tissue.
  • 2D two-dimensional
  • a method for producing cultured adipose tissue comprising: culturing adipose cells from adipogenic precursor cells in culture media; harvesting non-adhered adipose cells after a predetermined or desired amount of adipose cells are produced; and aggregating the harvested non-adhered adipose cells to provide the cultured adipose tissue.
  • adipogenic precursor cells are selected from the group consisting of mesenchymal stem cells, dedifferentiated fat cells, fibroblasts, and fibroadipogenic progenitor cells.
  • hydrogel or binder is selected from the group consisting of alginate, cellulose, gelatin, starch, tara gum, agar, agarose, carrageenan, cornstarch, gellan gum, lecithin, maltodextrin, methylcellulose, carboxymethylcellulose, cellulose gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose tragacanth, karaya, acacia, ghatti, beta-glucan, psyllium husk, inulin, chitosan, curdlan, dextran, potato starch, tapioca/arrowroot starch, hyaluronic acid, fibrin, carrageenan, guar gum, inulin, konjac, oat bran, pectin, locust bean gum, xanthan gum, soy protein, wheat protein, pea protein, chickpea protein, almond protein, rice protein, hemp protein, quinoa protein, sunflower
  • cross-linking the harvested non-adhered adipose cells comprises cross-linking the harvested non-adhered adipose cells using an enzyme selected from the group consisting of a transglutaminase, a tyrosinase, a peroxidase, a laccase, a sortase, a subtilisin, and lysyl oxidase.
  • cross-linking the harvested non-adhered adipose cells comprises enzymatically cross-linking the harvested non-adhered adipose cells using transglutaminase.
  • cross-linking the harvested non-adhered adipose cells with transglutaminase comprises mixing a solution of transglutaminase with the harvested non-adhered adipose cells at a predetermined volumetric ratio.
  • cross-linking the harvested non-adhered adipose cells comprises cross-linking the harvested non-adhered adipose cells using a cross-linker selected from the group consisting of polymers functionalized with aldehyde groups, genipin, and phenolic compounds.
  • flocculation comprises a flocculation technique selected from the group comprising auto-flocculation, bio-flocculation, chemical flocculation, particle-based flocculation, and electrochemical flocculation
  • monitoring comprises optical or electrical monitoring.
  • monitoring comprises measuring lipid droplet size within a cell or within a population of cells.
  • the at least one additive is a fatty acid selected from the group comprising linoleic acid, arachidonic acid, alpha-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid.
  • a cultured adipose tissue comprising adipose cells embedded in a hydrogel or binder, wherein the cultured adipose tissue has a three-dimensional (3D) shape and a size on the macroscale.
  • a cultured adipose tissue comprising adipose cells cross-linked together, wherein the cultured adipose tissue has a three-dimensional (3D) shape and a size on the macroscale.
  • centrifugation comprises counter flow centrifugation.

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Abstract

La présente divulgation concerne un tissu adipeux de culture. Dans un mode de réalisation, le tissu adipeux de culture est produit en cultivant des cellules adipeuses dans un milieu de culture in vitro, en récoltant les cellules adipeuses en suspension après la production d'une quantité souhaitée de cellules adipeuses en suspension, et en agrégeant les cellules adipeuses en suspension récoltées pour obtenir le tissu adipeux de culture. Dans certains modes de réalisation, l'agrégation des cellules adipeuses en suspension récoltées consiste à mélanger les cellules adipeuses en suspension récoltées avec un hydrogel ou un liant dans un moule en trois dimensions (3D). Dans d'autres modes de réalisation, l'agrégation des cellules adipeuses en suspension récoltées consiste à réticuler les cellules adipeuses en suspension récoltées dans un moule en 3D. Dans certains modes de réalisation, l'agrégation des cellules adipeuses en suspension récoltées consiste à précipiter par floculation ou faire coaguler les cellules adipeuses en suspension récoltées. Le tissu adipeux de culture a une forme en 3D définie et une taille macroscopique. Dans certains modes de réalisation, le tissu adipeux de culture peut être un produit alimentaire.
PCT/US2023/071704 2022-08-05 2023-08-04 Systèmes et procédés de production de tissu adipeux de culture à l'échelle commerciale Ceased WO2024151321A2 (fr)

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