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WO2021007359A1 - Fabrication de viande comestible cultivée à l'aide d'une bio-impression - Google Patents

Fabrication de viande comestible cultivée à l'aide d'une bio-impression Download PDF

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Publication number
WO2021007359A1
WO2021007359A1 PCT/US2020/041265 US2020041265W WO2021007359A1 WO 2021007359 A1 WO2021007359 A1 WO 2021007359A1 US 2020041265 W US2020041265 W US 2020041265W WO 2021007359 A1 WO2021007359 A1 WO 2021007359A1
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WO
WIPO (PCT)
Prior art keywords
bio
ink
cells
layer
pattern
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2020/041265
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English (en)
Inventor
Sharon Fima
Idan GAL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Meatech 3d Ltd
IP Law Offices of Guy Levi LLC
Original Assignee
Meatech 3d Ltd
IP Law Offices of Guy Levi LLC
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Publication of WO2021007359A1 publication Critical patent/WO2021007359A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
    • A23P20/00—Coating of foodstuffs; Coatings therefor; Making laminated, multi-layered, stuffed or hollow foodstuffs
    • A23P20/20—Making of laminated, multi-layered, stuffed or hollow foodstuffs, e.g. by wrapping in preformed edible dough sheets or in edible food containers
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
    • A23P30/00—Shaping or working of foodstuffs characterised by the process or apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y70/00—Materials specially adapted for additive manufacturing
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
    • A23P20/00—Coating of foodstuffs; Coatings therefor; Making laminated, multi-layered, stuffed or hollow foodstuffs
    • A23P20/20—Making of laminated, multi-layered, stuffed or hollow foodstuffs, e.g. by wrapping in preformed edible dough sheets or in edible food containers
    • A23P20/25—Filling or stuffing cored food pieces, e.g. combined with coring or making cavities
    • A23P2020/253—Coating food items by printing onto them; Printing layers of food products
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29L—INDEXING SCHEME ASSOCIATED WITH SUBCLASS B29C, RELATING TO PARTICULAR ARTICLES
    • B29L2031/00—Other particular articles
    • B29L2031/753—Medical equipment; Accessories therefor
    • B29L2031/7532—Artificial members, protheses
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y10/00—Processes of additive manufacturing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y80/00—Products made by additive manufacturing

Definitions

  • bio-printing of biostructures having predetermined two- and three dimensional pattern of viable cells that is cells which can then be cultured to form edible protein such as meat.
  • methods for bio-printing of biostructures having a predetermined three dimensional structure with cells incorporated therein in a non-random two- and three dimensional pattern are provided herein.
  • a three dimensional (3D) computer aided design (CAD) model of the biostmcture to be made can be assembled, from which a suitable (in other words, a file format that can be read and executed by the processor to achieve the desired results) format file is generated within a CAD package.
  • the file can then be processed and in effect sliced along the Z-axis at a thickness matching the thickness of the systems’ capabilities or other requirements. This creates a series of plan cross sections layer of the part and at any particular height, each having a simple two dimensional (2D) profile.
  • the various scaffolding, cell types and ECM can be designated and the corresponding pattern dispensed.
  • bio-ink comprising a certain type, and or several types of viable cells or other materials, each which can be manipulated to multiply and/or change properties using a trigger.
  • a computer software, hardware and firmware controlling the functionality of the printer, directing the fabrication of the 3D biostmcture by a drop on demand digital printing
  • An bio-ink printer equipped with a designated software and bio-inks, which deposits the bio-ink according to the directing of the software, to provide with the desired bio-structure.
  • a method of bio-printing using a 3D printer comprising: providing a 3D bio-ink printer, the printer having: a library to store printer operation parameters; a processor in communication with the library; a memory storage device storing a set of operational instmctions for execution by the processor; a micromechanical bio-ink dispenser(s) in communication with the processor and with the library; and a bio-ink dispenser’ s(‘) interface circuit in communication with the library, the memory and the micromechanical bio-ink dispenser(s), the library configured to provide printer operation parameters specific for a substantially 2D layer or portion of a specific layer of the bioprinted biostmcture to be fabricated, thereby obtaining a plurality of vector data models and/or bitmaps, each vector data model and/or bitmap specific for a predetermined layer or their interface and/or cross section and/or a portion thereof; loading the plurality of bitmaps and/or vector data models processed in the step of pre-processing onto the library
  • files used to recreate the Computer-Aided Design/Computer-Aided Manufacturing (CAD/CAM) generated information compositions of the substantially 2D layer(s) or portions thereof associated with the bioprinted structure to be fabricated for use in the printer can be converted from MRI’s files (e.g., *.dcm, *.ima), as well as computer tomography (CT) DICOM files e.g., *.SEQ. It is further noted that other sources that can be converted in any way to a raster representation.
  • a DICOM file as used in the methods and systems described herein, means a high-resolution digital pathology image.
  • the 2D images uploaded from the compositions stored are used to form a portion of the first layer and can be used as well to define the printing sequence and order of the second bio-ink on a portion of the first layer, such that the first bio-ink and the second bio ink are alternated or sequenced according to the predetermined 3D structure, followed in certain implementation, by the third bio-ink.
  • the methods described further comprising, subsequent to the step of functionalizing the first layer of predetermined pattern of the second bio-ink: following uploading the substantially 2D image representing a subsequent layer or portion thereof, using the first bio-ink dispenser, forming a second layer of cells’ pattern (or portion thereof) on, and/or adjacent to the first layer of cells’ pattern, and/or the first layer of predetermined pattern of the second bio-ink composition; and functionalizing the second layer of cells pattern, wherein the cells in the second layer are different than the cells in the first layer.
  • Portions of the second layer can be formed similar to the layers in the first layer.
  • the methods, systems and compositions for use in the bio-printing of non-random biostmctures described herein further comprise, using the second bio-ink dispenser, forming a second layer of predetermined pattern of the second bio-ink on and/or adjacent to the first layer of cells’ pattern and/or the first layer of predetermined pattern of the second bio-ink composition; and functionalizing the second layer of predetermined pattern of the second bio-ink, wherein a triggering compounds in the second layer of predetermined pattern of the second bio-ink, is different than the triggering compounds in the first layer of predetermined pattern of the second bio-ink.
  • biostmcture is to be understood as any structure/tissue/organ, which is composed of more than one cell, having some kind of cellular organization and/or complexity and/or functionality. A mere random cluster of cells therefore would not fall under this definition.
  • Biostmcture may also comprise a scaffolding structure providing mechanical support and/or conduits for the flow of nutrients and/or other fluids (e.g., functionalizing fluids) to the biostmcture. It is noted, that the whole biostmcture is edible.
  • a first bio-ink can comprise a dispersing medium (e.g., Eagle Medium) with a first polymer, monomer or oligomer without any cells suspended in it, which can be used to form the scaffolding for the biostmcture.
  • the first bio-ink dispenser may be associated with a dedicated functionalizing print head which can be used to stiffen the polymer to a desired degree.
  • the scaffolding can be printed and configured to form lumens with spaced openings.
  • a second bio-ink dispenser with a second bio ink can be used, wherein the second bio-ink can comprise dispersing medium with cells which has been triggered, or otherwise manipulated to undergo a specific differentiation and/or alternatively, cells and either the same or different biocompatible polymer, monomer or oligomer, associated with another functionalizing print head.
  • a third bio-ink dispenser can be used with a third bio-ink comprising a dispersing medium with triggering compounds therein and either the same or different biocompatible polymer, monomer or oligomer.
  • Another (fourth) print head can be used with a fourth bio-ink, comprising dispersing medium with viable cells, associated with a dedicated functionalizing print head. Adding and/or removing of the various print head can be done based on the printed biostructure.
  • a first scaffold layer or portion thereof, as defined in the vector data model, or raster images provided can be functionalized using a first method (e.g., photopolymerization), followed alternatively by printing of a pattern of other tissue or organ cells using another print-head and then followed by printing of a pattern of extracellular matrix compositions; for example, manipulation triggering compounds, growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), or epidermal growth factor (EGF) , allowing the cells to multiply or undergo a specific manipulation or additionally or alternatively, retaining native growth of the tissue or organ; and then, using a dedicated print head, functionalizing the patterns defined by the 2D images (e.g., using spray nozzles, of KC1, and/or CaCh).
  • VEGF vascular endothelial growth factor
  • FGF fibroblast growth factor
  • PDGF platelet-derived growth factor
  • IGF insulin
  • bio-ink dispensers can then be used with additional bio-inks, each which can provide different cells and based on the 2D images stored in the libraries, and be used to create a biostructure that emulates a single organ (e.g., liver), a tissue (e.g., cartilage, bone), or an interface between tissues and organs (e.g., connective muscle tissue).
  • a single organ e.g., liver
  • a tissue e.g., cartilage, bone
  • an interface between tissues and organs e.g., connective muscle tissue
  • FIG. 2A depicting the growing cell colonies at 18 days in brightfield, with the same stained to determine the live cells portions in FIF. 2B;
  • FIG. 5 is a graph showing the increase in thickness of the tissue as a function of the number of layers
  • bio-printing of biostructures having predetermined, non-random two- and three dimensional pattern of cells.
  • the exemplary implementations of bio-printing described provide edible biostmctures having a predetermined three dimensional structure that emulate tissue and/or organs for food consumption.
  • a computerized bio-printing method for forming a composite biostmcture (e.g., tissue, organ, tissue scaffold, connective tissue) having predetermined 2D or 3D pattern of cells (e.g., myocytes fibroblasts, chondrocytes, hepatocytes, osteocytes and the like) therein, the method comprising: providing a computerized bio-printing system in communication with a central processing module (CPM) having a non-volatile storage device with a library of images corresponding to layers of the biostructures or portions thereof sought to be printed, the system further comprising: a first bio-ink dispenser having: at least one aperture, a first bio-ink reservoir, and a first actuator operable to dispense the first bio-ink through the aperture, wherein the first bio-ink is a composition comprising cells (e.g., myocytes, hepatocytes, adipocytes and the like) suspended in
  • the biostructures described herein may be tissue substitute including but not limited to a bone cartilage, liver, epithelial, muscular, fatty tissue and the like, or other tissue substitute for either a portion of a tissue or an entire tissue and/or organ.
  • the biostructure, or its corresponding matrix may have dimensions which may be customized for a particular application (e.g., fish cross section, T-Bone steak and the like) and which may be based on suitable imaging data and may further include geometric features not present in the suitable imaging data.
  • the biostructure may be used in culturing cells in-vitro, in a separate bio reactor.
  • imaging data can be an imaging file of a tissue or organ sough to be bio-printed, which can be converted to a raster file or image vector file and used in the printing libraries by the CPM.
  • imaging files can be, for example MRI files, PET files CAT scan files, histology files and the like.
  • the term“scaffold”, or“scaffolding” refers in an exemplary implementation, to an engineered platform having a predetermined three dimensional structure, which mimic the 3D environment of the natural extracellular matrix (ECM), provide short term mechanical support of the biostructure, and provide an increased surface area for cells adhesion, proliferation, migration, and differentiation, eventually leading to accelerated tissue formation.
  • “scaffolding” refers to a fabricated systems of conduits, sized adapted configured, once printed, to maintain fluid communication within the growing biostructure to nutrients, buffer fluids, functionalizing fluids and other similar functional liquids.
  • the scaffold can also be a composite scaffold.
  • A“composite scaffold” refers to a scaffold platform which is engineered in order to support colonization and/or proliferation of two or more tissue types which together comprise a“heterogeneous tissue”.
  • the systems and methods described herein can be used to form a composite scaffold comprising a first 3D, chondrocytes-embedded biostructure (e.g., tissue, organ, tissue scaffold, connective tissue) for supporting formation of a first tissue type (e.g., cartilage) thereupon and a second 3D, myocytes-embedded biostructure (e.g., muscle tissue, meat replacement, tissue scaffold) for supporting formation of a second tissue type thereupon (e.g., muscle).
  • chondrocytes-embedded biostructure e.g., tissue, organ, tissue scaffold, connective tissue
  • myocytes-embedded biostructure e.g., muscle tissue, meat replacement, tissue scaffold
  • the methods, systems and compositions for use in the bio-printing or forming the 3D, cell-embedded biostructure can comprise a step of optionally providing a substrate (e.g., a film).
  • the bio-ink dispensing actuator (and derivatives thereof; are to be understood to refer to any device or technique that deposits, dispenses, transfers or creates material on a surface in a controlled accretive or additive manner) depositing the first, and/or second bio-ink, can be configured to provide the bio-ink droplet(s) upon demand, in other words, as a function of various process parameters such as conveyor speed, desired cells layer thickness, cell concentration, layer type (e.g., certain type of cell or several types of cells, either manipulated (differentiated) or not ) and the like.
  • process parameters such as conveyor speed, desired cells layer thickness, cell concentration, layer type (e.g., certain type of cell or several types of cells, either manipulated (differentiated) or not ) and the like.
  • the substrate can also be a relatively rigid material, for example, glass or crystal (e.g., sapphire), Alternatively, the substrate may be a flexible (e.g., Tollable) substrate (or film) and can be, for example, poly(ethylenenaphthalate) (PEN), polyimide (e.g. KAPTONE ® by DuPont), silicon films etc. Moreover, the substrate does not need necessarily to be solid and printing can take place into a vessel containing a liquid into which the biostmcture is printed.
  • PEN poly(ethylenenaphthalate)
  • KAPTONE ® by DuPont
  • other functional heads may be located before, between or after the first, second and/or third bio-ink dispenser.
  • These may include a source of electromagnetic radiation (such as UV lamps) used as means for functionalizing the biocompatible polymers, monomers and/or oligomers used in the bio-inks and be configured to emit electromagnetic radiation at a predetermined wavelength (l), for example, between 190 nm and about 450nm, e.g. 420 nm which in an exemplary implementation, can be used to accelerate and/or modulate and/or facilitate a photopolymerizable monomer/oligomer/polymer or otherwise, to sterilize surfaces before, during or after the layers are dispensed.
  • l predetermined wavelength
  • Other functional heads can be heating elements, additional printing heads with various inks (e.g., dispensing actuators for accurately dispensing different ionic solution for chemical functionalizing, for example by spraying, dispensing, nebulizing, aerosolizing, jetting, injecting and the like) and a combination of the foregoing.
  • dispensing actuators in the method of forming the 3D, Cell-embedded biostructure e.g., tissue, organ, tissue scaffold, connective tissue
  • the steps of using the first bio-ink dispenser and depositing the first bio-ink onto the substrate, thereby forming a first layer of cells and/or the step of using the second bio-ink dispenser - depositing the second bio-ink onto and/or adjacent to the first layer of cells is preceded, followed or takes place simultaneously with a step of heating, EMR exposure, drying, cross linking, annealing, depositing ionic solution or a combination of steps comprising one or more of the foregoing.
  • Formulating the first bio-ink composition may take into account the requirements, if any, imposed by the dispensing print head and the surface characteristics (e.g., hydrophilic or hydrophobic, and the surface energy of and optionally provided substrate).
  • the viscosity of either the first bio-ink and/or the second bio-ink (measured at the dispensing temperature and pressure) can be, for example, between about 1 cP and about 10,000 cP, or between about 500 cP and about 8,000 cP, for example, between about 1500 cP and about 5,000 cP or between about 1,600 cP and about 2,500 cP.
  • the first bio-ink composition used to form the layer of cells, and/or the second bio-ink comprising a composition comprising bio compatible and/or manipulation triggering compounds can comprise biocompatible polymers, for example; PEGilated- methacrylate, PEG-dimethacrylate (PEGDMA), carrageenan, poly(lactic) acid, poly (lactic -co- glycolic acid), (poly(lysine), their methacrylate conjugates, co-polymers, interpenetrating networks or a composition comprising one or more of the foregoing.
  • biocompatible polymers for example; PEGilated- methacrylate, PEG-dimethacrylate (PEGDMA), carrageenan, poly(lactic) acid, poly (lactic -co- glycolic acid), (poly(lysine), their methacrylate conjugates, co-polymers, interpenetrating networks or a composition comprising one or more of the foregoing.
  • biocompatible polymer refers to any polymer which when in contact with the cells, tissues or body fluid of an organism; does not induce adverse effects such as immunological reactions and/or rejections and the like.
  • first and/or second bio-ink used in the methods and systems described herein can be a biodegradable polymer, referring in an exemplary implementation, to any polymer which can be degraded in the physiological environment such as by proteases.
  • biodegradable polymers are; collagen, fibrin, hyaluronic acid, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polydioxanone (PDO), trimethylene carbonate (TMC), polyethyleneglycol (PEG), alginate, chitosan or mixtures thereof.
  • a carrageenan hydrocolloid can be used in the first and/or second bio ink, for example kappa carrageenan (k-CA) and functionalizing comprises heating and using the third bio-ink dispenser - depositing ionic solution comprising KC1, CaCh, and their combination.
  • the first bio-ink composition comprises poly(ethylenoxide)-methacrylate (PEODMA) and functionalizing comprises exposure to electromagnetic radiation (EMR).
  • the PEO can be, for example Poly(ethylenglycol).
  • the physico-chemical properties of the first and/or second bio-inks can be controlled by varying for example molecular weight, chemical composition, the amount and type of functionalizing agent used and the degree of functionalization, which modifies their mass transport properties, physico-chemical properties and biological properties.
  • poly(ethylene glycol)-diacrylate (PEG-DA) hydrogels have been shown to be compatible in vivo with porcine islet cells and poly(ethylene glycol) -dimethacrylate (PEG-DMA) hydrogels to be compatible with chondrocytes.
  • the first and/or second bio-inks can be used to form 3D cell-embedded biostructure (e.g., tissue, organ, tissue scaffold, connective tissue) with bio-compatible compositions from PEG-DMA monomers suspended in aqueous solution and be gelled by radical polymerization in the presence of a photoinitiator. The polymerization reaction starts when the solution is exposed to UV light.
  • PEG-DMA monomer has two methacrylate groups which can react with up to two other methacrylate groups to make covalent bonds in other words, cross linking forming a covalently crosslinked branched network.
  • the concentration of the photoinitiator e.g., Phenylglyoxylate, benzophenone
  • the concentration of the photoinitiator e.g., Phenylglyoxylate, benzophenone
  • the concentration of the photoinitiator e.g., Phenylglyoxylate, benzophenone
  • the concentration of the photoinitiator e.g., Phenylglyoxylate, benzophenone
  • the velocity of the substrate depends in certain implementations, for example, on the number of dispensing actuators used in the process, the desired 3D biostructure (e.g., tissue, organ, scaffold, connective tissue,) printed (interchangeable with dispensed), the functionalizing time of the bio-ink, the evaporation rate of any solvents present, the distance between the bio-ink dispensing actuator containing the first bio-ink and the second bio ink dispenser comprising the second bio-ink, the desired spatial distribution of the cells in the embedded portion (see e.g., FIG.s 3, 4), whether the biostructure is a composite scaffold, stand-alone tissue or organ and the like or a combination of factors comprising one or more of the foregoing.
  • the desired 3D biostructure e.g., tissue, organ, scaffold, connective tissue,
  • the apparent viscosity of the first, second, or third bio-ink composition(s), can each be (before functionalizing) for example, between about 1 cP and about 10,000 cP, or between about 500 cP and about 8,000 cP, for example, between about 1500 cP and about 5,000 cP or between about 1,600 cP and about 2,500 cP, at the working temperature and pressure, which can be controlled.
  • cells’ dispersion, solution, emulsion, suspension, hydrogel or liquid composition comprising the foregoing, or the second bio-ink comprising suspended cells can each be between about 1,000 and about 5,000 cP, for example between about 1,500 cP and about 2,500 cP.
  • the volume of each droplet of the first and/or second and/or third bio-ink(s) can range of about 5 nL to about 250 microLiter (pL), for example between about 50 pL and about 200 pL, depended on the dispensing actuators’ parameters and the properties of the bio-ink.
  • the waveform to expel a single droplet can be a 10V to about 170 V pulse, or about 16V to about 90V, and can be expelled at frequencies between about 1 kHz and about 25 kHz.
  • polymer concentration e.g., PEGDMA, Chitosan
  • the suspending polymer can be different
  • the scaffold support pattern formed can exhibit compressive modulus (in other words, the ratio between the load and strain needed to achieve irreversible deformation of the gel), of no less than 0.5 MPa, for example, between 0.5 and 1.5 MPa or between about 0.6 MPa and 1.0 MPa.
  • Other compressive moduli can be designed for the biostructure itself, which can be between about 0.08 MPa and about 1.0 MPa.
  • a computerized bio-printing method for forming a composite biostructure (e.g., tissue, organ, tissue scaffold, connective tissue, and their combination) having predetermined 2d and/or 3D pattern of cells (e.g., one or more of endothelial cells, muscle cells, fibroblast cells, mesothelial cells, pericyte cells, monocyte cells, plasma cells, mast cells, adipocyte cells, chondrocyte cells, bone cells, or a cell population cultured from a specific cell type) therein, the method comprising: providing a bio-printing system in communication with a microprocessor coupled to a non-volatile storage device storing thereon a processor readable media with an executable set of instructions configured, when executed, to cause the (at least one, but potentially more) processor(s) to perform the method, as well being in communication with a database having a library of 2D images of the various layers to be printed, the system comprising: a first bio ink
  • the predetermined 3D pattern of the second bio-ink, embedded in the layer of cells and biostmcture described herein, can be non-random.
  • the cell-laden biostmcture has a substantial variation in the spatial distribution and/or density of the cells, forming a predetermined 2D (gleaned for example, from MRI and/or CT images converted to raster and/or vector data models and converted to bio-printing instructions) and/or 3D pattern of cells.
  • the predetermined three dimensional pattern of the second bio-ink can be configured to; accelerate cell adhesion, retain native growth of the cells, and/or organ and/or tissue and other similar functions.
  • the 3D scaffolding support and the cell-laden scaffold can be configured to emulate an edible animal tissue, for example, a muscle tissue and can comprise, for example, myocytes, satellite cells and other myogenic cells dispensed in the first bio-ink, while nanofibers of at least one of, PCL and collagen are dispensed in the second bio-ink, with the third bio-ink comprising RGD peptide.
  • an edible animal tissue for example, a muscle tissue
  • the 3D scaffolding support and the cell-laden scaffold can be configured to emulate an edible animal tissue, for example, a muscle tissue and can comprise, for example, myocytes, satellite cells and other myogenic cells dispensed in the first bio-ink, while nanofibers of at least one of, PCL and collagen are dispensed in the second bio-ink, with the third bio-ink comprising RGD peptide.
  • the second bio-ink can further comprise other additives that affect colonization, proliferation, adherence, inhibit apoptosis or other manipulation of the cells, retain native growth of the cells, and/or organ and/or tissue and other similar functions.
  • the second bio-ink used in the methods, systems and compositions for use in the direct bio-printing of a composite biostmcture can further comprise: cells manipulation triggering compounds, (for example, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), bone morphogenic protein (BMP), insulin-like growth factor (IGF), glucoseaminoglycan (GAG), Transforming growth factor (TGF) or signaling compound composition comprising the foregoing), as well as therapeutically effective compounds, antimicrobial compounds, immunosuppressing compounds and the like.
  • EGF epidermal growth factor
  • bFGF basic fibroblast growth factor
  • BMP bone morphogenic protein
  • IGF insulin-like growth factor
  • GAG glucoseaminoglycan
  • TGF Transforming growth factor
  • Bovine mesenchymal stem cell (MSCs) Isolation MSCs were isolated from intact umbilical cord samples of a newborn calf. Cells were isolated using one cycle (37 °C, 60 min, with agitation every 10 min) of 4 enzymes digestion (e.g., Collagenase type I (FS004196), Collagenase type II (FS004176), Collagenase type IV, and hyaluronidase (FS002592)), Worthington biochemical corporation, NJ, USA) in Dulbecco's Phosphate Buffered Saline (DPBS) Without Calcium and Magnesium (02-023-1 A, Biological Industries, Beit-Haemek, Israel) as well as penicillin- streptomycin solution (03-031- IB , Biological Industries, Beit-Haemek, Israel) and amphotericin B (BP928-250, Fisher Bioreagents, USA).
  • DPBS Dulbecco's Phosphat
  • the digested tissue was then transferred through an 8- folded (PBS wet-) gauze paper over a 70-100 um strainer into a 50 ml tube and centrifuged at 400g for 10 min. The supernatant was discarded and the cells were resuspend in 5 ml Dulbecco's
  • DMEM Modified Eagle Medium
  • Bovine MSCs/mL within 0.5% alginate (Ag) + 0.25% collagen were dispensed as described above. Growth media was dispensed on the printed layer, comprising:
  • FIG. 1 depicts the cell proliferation and growth over a 18-day period. As illustrated, colonies of the printed cells printed increase in size and continue to spread within the hydrogel media. As further illustrated in FIG.2A and 2B, at 18 days 10 xlO 6 Bovine MSCs/mL show high viability both in brightfield microscopy (FIG. 2A) as well as staining as described in the viability assay (FIG. 2B).
  • the term "operable" means the system and/or the device (e.g., the dispensing actuator) and/or the program, or a certain element, component or step is/are fully functional sized, adapted and calibrated, comprising elements for, having the proper internal dimension to accommodate, and meets applicable operability requirements to perform a recited function when activated, coupled or implemented, regardless of being powered or not, coupled, implemented, effected, actuated, realized or when an executable program is executed by at least one processor associated with the system, method, and/or the device.
  • operable also means the system and/or the circuit is fully functional and calibrated, comprises logic for, and meets applicable operability requirements to perform a recited function when executed by at least one processor
  • a computerized bio printing method for forming an edible biostructure having predetermined 3D pattern of cells therein, the method comprising: providing a bio-printing system comprising: a microprocessor in communication with: a non-volatile storage device having thereon a microprocessor-readable medium with set of executable instructions configured to, when executed, to cause the microprocessor to execute the method of bio-printing bio-printing; and an image library corresponding to a two dimensional (2D) layer of at least one of: cells, scaffolding, and connective tissue, within the biostructure having predetermined 3D pattern of cells therein; a first bio-ink dispenser in communication with the microprocessor having: at least one aperture, a first bio-ink reservoir, and a first actuator operable to dispense the first bio-ink through the aperture, wherein the first bio-ink is a composition comprising viable cells suspended in at least one of: a bio-compatible dispersing medium and bio-compatible polymer, monomer
  • an edible food product fabricated using the computerized bio-printing method for forming an edible biostmcture having predetermined 3D pattern of cells therein, the method comprising: providing a bio-printing system comprising: a microprocessor in communication with: a non-volatile storage device having thereon a microprocessor-readable medium with set of executable instmctions configured to, when executed, to cause the microprocessor to execute the method of bio-printing bio-printing; and an image library corresponding to a two dimensional (2D) layer of at least one of: cells, scaffolding, and connective tissue, within the biostmcture having predetermined 3D pattern of cells therein; a first bio-ink dispenser in communication with the microprocessor having: at least one aperture, a first bio-ink reservoir, and a first actuator operable to dispense the first bio-ink through the aperture, wherein the first bio-ink is a composition comprising viable cells suspended in at least one of: a

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  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Manufacturing & Machinery (AREA)
  • Materials Engineering (AREA)
  • Health & Medical Sciences (AREA)
  • Nutrition Science (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Materials For Medical Uses (AREA)

Abstract

L'invention concerne des procédés, des systèmes et des compositions destinés à être utilisés dans la bio-impression de biostructures ayant un motif bidimensionnel (2D) et/ou tridimensionnel (3D) de cellules. Plus particulièrement, l'invention concerne des procédés et des compositions pouvant être mis en œuvre dans des systèmes de bio-impression informatisés pour la fabrication de biostructures comestibles à l'aide de gouttes à la demande, présentant une structure tridimensionnelle prédéterminée qui peut être assemblée à partir de motifs en 2D avec des cellules et un matériau extracellulaire (ECM) incorporés dans celle-ci, en présence d'un support biocompatible ou non, dans un motif bidimensionnel ou tridimensionnel non aléatoire.
PCT/US2020/041265 2019-07-08 2020-07-08 Fabrication de viande comestible cultivée à l'aide d'une bio-impression Ceased WO2021007359A1 (fr)

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US201962871438P 2019-07-08 2019-07-08
US62/871,438 2019-07-08

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CN108795867A (zh) * 2018-06-05 2018-11-13 华东理工大学 用于构建结肠癌细胞腹膜转移体外三维模型的方法
US12471608B2 (en) 2023-07-13 2025-11-18 Nstx Industries Inc. Process of producing a food analogue precursor comprising of a plurality of phases

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US20140093618A1 (en) * 2011-07-26 2014-04-03 The Curators Of The University Of Missouri Engineered comestible meat
US20160009029A1 (en) * 2014-07-11 2016-01-14 Southern Methodist University Methods and apparatus for multiple material spatially modulated extrusion-based additive manufacturing
WO2018035282A1 (fr) * 2016-08-17 2018-02-22 Nano-Dimension Technologies, Ltd. Procédés et bibliothèques destinés à être utilisés dans la bioimpression par jet d'encre de biostructures
WO2018165613A1 (fr) * 2017-03-10 2018-09-13 Prellis Biologics, Inc. Procédés et systèmes d'impression d'un matériau biologique
WO2018164672A1 (fr) * 2017-03-07 2018-09-13 Nano-Dimension Technologies, Ltd. Fabrication de composant composite utilisant une impression à jet d'encre
WO2019079292A1 (fr) * 2017-10-16 2019-04-25 President And Fellows Of Harvard College Procédés de formation d'échafaudages tissulaires tridimensionnels à l'aide d'encres biologiques à base de fibres et procédés d'utilisation associés

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Publication number Priority date Publication date Assignee Title
US20140093618A1 (en) * 2011-07-26 2014-04-03 The Curators Of The University Of Missouri Engineered comestible meat
US20160009029A1 (en) * 2014-07-11 2016-01-14 Southern Methodist University Methods and apparatus for multiple material spatially modulated extrusion-based additive manufacturing
WO2018035282A1 (fr) * 2016-08-17 2018-02-22 Nano-Dimension Technologies, Ltd. Procédés et bibliothèques destinés à être utilisés dans la bioimpression par jet d'encre de biostructures
WO2018164672A1 (fr) * 2017-03-07 2018-09-13 Nano-Dimension Technologies, Ltd. Fabrication de composant composite utilisant une impression à jet d'encre
WO2018165613A1 (fr) * 2017-03-10 2018-09-13 Prellis Biologics, Inc. Procédés et systèmes d'impression d'un matériau biologique
WO2019079292A1 (fr) * 2017-10-16 2019-04-25 President And Fellows Of Harvard College Procédés de formation d'échafaudages tissulaires tridimensionnels à l'aide d'encres biologiques à base de fibres et procédés d'utilisation associés

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108795867A (zh) * 2018-06-05 2018-11-13 华东理工大学 用于构建结肠癌细胞腹膜转移体外三维模型的方法
US12471608B2 (en) 2023-07-13 2025-11-18 Nstx Industries Inc. Process of producing a food analogue precursor comprising of a plurality of phases

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