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WO2024243271A1 - Systèmes de cryoconservation comestibles et de qualité alimentaire - Google Patents

Systèmes de cryoconservation comestibles et de qualité alimentaire Download PDF

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Publication number
WO2024243271A1
WO2024243271A1 PCT/US2024/030498 US2024030498W WO2024243271A1 WO 2024243271 A1 WO2024243271 A1 WO 2024243271A1 US 2024030498 W US2024030498 W US 2024030498W WO 2024243271 A1 WO2024243271 A1 WO 2024243271A1
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human
cryopreservation
medium
cell
aspects
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PCT/US2024/030498
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English (en)
Inventor
Seyedvahid HOSSEINI
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Hatchless Inc
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Hatchless Inc
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Publication of WO2024243271A1 publication Critical patent/WO2024243271A1/fr
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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/122—Preservation or perfusion media
    • A01N1/125—Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators

Definitions

  • cultured meat has the capacity to significantly reduce land and water use, emit fewer greenhouse gases, and reduce agriculture-related pollution and eutrophication. It also provides a more ethical way of producing food, avoiding animal confinement, mistreatment of animals and animal slaughter.
  • cultured meat can be obtained from animal-based components, but with the advantage of sustainably increased yields, similarity of underlying structure and nutrition to factory farmed animals.
  • cultured animal cells can also be used for production of other animal products like leather, horns, or feathers.
  • Industrialized animal cell culture has also proven to be useful for medical purposes, for example for production of recombinant proteins, tissues, organs and organoids.
  • Cryopreservation methods are key to any successful cellular agriculture enterprise. Cryopreservation can be used at several key operational points in the system, for example for storage of cells and cell lines for consistent product production. Cryopreservation is also useful during bioreactor operations and cell culture stage because systems can be unreliable, and batch freezing may be required at any step during the process to prevent wastage. Cryopreservation is also necessary to ensure proper storage and transport of the final product. However, currently available cryopreservation methods use harmful chemicals or have high costs that make them unsuitable for use in cellular agriculture.
  • the current disclosure encompasses a cryopreservation medium for use in cellular agriculture applications, the medium comprising egg yolk and at least one additional edible cryoprotectant.
  • edible cryoprotectants include glycerin, sorbitol, trehalose, polyvinylpyrrolidone(PVP), betaine, carboxylated poly-l-lysine, glucose, dextran, proline, peptides, sucrose or any combination thereof.
  • the cryoprotection medium does not contain dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • the cryoprotection medium comprises about 5% to about 30% of egg yolk. In some aspects, the cryoprotection medium comprises about 20% of egg yolk. In some aspects, the cryoprotection medium comprises about 5% to about 25% glycerin. In some aspects, the cryoprotection medium comprises about 10% glycerin.
  • the cryoprotection medium comprises egg yolk obtained from an oviparous animal, non-limiting examples of which include fish, amphibian, reptile, or any combination thereof. In some aspects, the egg yolk is from an avian animal, for example a chicken, ostrich, quail, partridge, turkey etc.
  • the medium is for cryopreservation of a population of non-human animal cells, non-human cell lines, non-human cell population, non-human cell cultures, non-human tissue, non-human organ or non-human cellular biomass.
  • 2 95004041.1 Attorney Docket No.121430-791898
  • the non-human animal cell is a fibroblast, a myoblast, an adipocyte, a stem cell, an endothelial cell, or a mesodermal cell or any combination thereof.
  • the non-human cell cultures, or non-human cellular biomass is preserved in a bioreactor container.
  • the temperature range of the cryopreservation is ⁇ 20° C. to ⁇ 196° C.
  • the medium is a ready-to-use medium. In some aspects, the medium is a concentrate. In some aspects, the current disclosure also encompasses a method of cryopreservation for cellular agriculture applications, the method comprising contacting at least one non-human animal cell with a medium comprising egg yolk and at least one additional edible cryoprotectant. [0014] In some aspects, the current disclosure also encompasses a method of cryopreservation for cellular agriculture applications, the method comprising contacting at least one non-human animal cell with the cryopreservation medium as disclosed herein.
  • the non- human animal cell is a fibroblast, a myoblast, an adipocyte, a stem cell, an endothelial cell, or a mesodermal cell or any combination thereof.
  • the at least one non-human cell is part of a non-human cell population, non-human cell cultures, non-human tissue, non-human organ, non-human cellular biomass, or non-human cell grown on microcarrier and/or scaffolds.
  • the non-human cell may be in any vessel, for example a dish, tube, plate, jar or a bioreactor.
  • the recovery survival rate of the non-human cell after cryopreservation is at least 50% and preferably 70% or more.
  • the non-human cells are suspended in the cryopreservation medium at a concentration that 1 ⁇ 10 2 to 1 ⁇ 10 9 non-human cells/mL of said cell cryopreservation protective solution.
  • the temperature range of the cryopreservation is ⁇ 20° C. to ⁇ 196° C.
  • the cryopreservation is ultrarapid cryopreservation.
  • the cryopreservation is gradual cryopreservation.
  • the current disclosure also encompasses a method of cellular agriculture, the method comprising; a) growing non-human animal cells in a bioreactor container; b) adding a cryopreservation medium as disclosed herein to the bioreactor container; c) freezing the non-human animal cells in the bioreactor container.
  • step (c) uses ultrarapid freezing.
  • the non-human animal cells are grown to a density of about 1 ⁇ 10 2 to 1 ⁇ 10 9 non-human cells/mL.
  • the recovery survival rate of the non-human animal cells is at 50% and preferably 70% or more.
  • the method further comprises 3 95004041.1 Attorney Docket No.121430-791898 thawing the non-human animal cells in the bioreactor container. In some aspects, the method further comprises resuming the growth of the non-human cells. In some aspects, prior to freezing, the cells are concentrated using a filtration or centrifugation method. In some aspects, the cells are for production of a cultured meat product. In some aspects, the method is used for production of cultured meat products, recombinant proteins, or structured animal products. DETAILED DESCRIPTION [0018] The following detailed description illustrates various aspects of the present disclosure. The description is intended to describe aspects of the present disclosure in sufficient detail to enable those skilled in the art to practice the present inventive concept.
  • the present disclosure is based, in part, on the discovery that media comprising egg- yolk is an efficient cryopreservative. Traditionally, cell preservation is done using media that contain dimethyl sulfoxide or similar chemicals that can be harmful for human and animal consumption. These chemicals need to be removed before the products can be consumed. This makes the process of cryopreservation unnecessarily cumbersome and costly and may pose unknown risks to humans if consumed over long periods.
  • Edible preservatives like salts and sugars add unwanted flavor and change food texture, thus making them unsuitable for cellular agriculture type applications.
  • the current disclosure provides cryopreservatives compositions comprising egg yolk and at least one other edible cryoprotectant, the combination of which makes large scale cryopreservation feasible.
  • Egg yolks are naturally resistant to low temperature fluctuations as would be experienced by an embryo in the wild and function as membrane protectors. Additionally, eggs are inexpensive, easily available, and easy to manipulate thus making them desirable additives to food formulations.
  • egg-based cryopreservatives have not been used in cellular agriculture application.
  • Other applications that use egg yolks as cryopreservatives usually depend on eggs to provide membrane protection, while other components like DMSO are used to maintain internal structure and viability after freezing.
  • compositions and methods disclosed herein do not use DMSO.
  • the compositions and methods disclosed herein use egg yolk as the primary cryopreservative. 4 95004041.1 Attorney Docket No.121430-791898 I.
  • the current disclosure encompasses compositions comprising egg yolks and at least one additional edible cryoprotectant, for cryopreservation in applications related to cellular agriculture.
  • cellular agriculture refers to the field of growing animal agricultural products directly from cell cultures instead of using poultry and livestock farming.
  • egg yolk or “deutoplasm” refers to the nutrient-bearing portion of the egg whose primary function is to supply food for the development of the embryo.
  • Egg yolks can be uniformly distributed in an egg, as in isolecithal or homolecithal eggs of invertebrates, or concentrated in one hemisphere of the egg, as in telolecithal eggs or may be centrally placed as in centrolecithal eggs.
  • the egg yolk is rich in fatty acids, vitamins, proteins and minerals.
  • Egg yolks are usually separated from the surrounding portion of an egg by a vitelline membrane.
  • compositions of the current disclosure may comprise egg yolks from any oviparous animal including but not limited to fish, amphibians, reptiles or birds.
  • the egg is a bird egg.
  • Non-limiting examples of birds from which the egg protein may be derived include chicken, duck, emu, goose, guinea fowl, gull, ostrich, pheasant, pigeon, quail, and turkey.
  • the egg is a fish egg.
  • Non-limiting examples include eggs from fishes that are used as common sources of tobiko, masago, ikura, or caviar for example salmon, paddlefish, bowfin, whitefish, trout, capelin or flying fish, bottarga, lumpfish etc.
  • the egg maybe a reptile egg for example a crocodile, alligator, snake, or lizard egg.
  • the egg yolk as disclosed herein may be isolated from a fertilized or an unfertilized egg. In some aspects, the egg yolk may be isolated from a fertilized egg. In some aspects, the egg yolk may be isolated from a fertilized egg in the first trimester. In some aspects, the egg yolk may be from a naturally occurring or recombinant animal. In some aspects, the egg yolk may be produced in vivo or in vitro. [0022] In some aspects, the compositions of the current disclosure comprise at least one additional edible and food safe cryoprotectant.
  • the additional cryoprotectant for use in compositions disclosed herein is not dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • the compositions as disclosed herein may comprise about 5% to about 30% egg yolk by weight of the composition.
  • the composition comprises about 5 95004041.1 Attorney Docket No.121430-791898 1% to about 5% or about 5% to about 10%, or about 10% to about 15%, or about 15% to about 20%, or about 20% to about 25%, or about 25% to about 30% egg yolk by weight of the composition.
  • the composition comprises about 20% of egg yolk.
  • the compositions disclosed herein may comprise at least one additional cryoprotectant as disclosed above.
  • the compositions comprise about 1% to about 5%, or about 5% to about 10%, or about 10% to about 15%, or about 15% to about 20%, or about 20% to about 25% by weight of the edible cryoprotectant disclosed herein.
  • the edible cryopreservative is glycerin.
  • the composition comprises about 1% to about 5%, or about 5% to about 10%, or about 10% to about 15%, or about 15% to about 20%, or about 20% to about 25% of glycerin by weight. In some aspects, the composition comprises about 10%, about 11%, about 12%, about 13%, about 14% or about 15% of glycerin. In some aspects, the composition comprises 12% glycerin.
  • the cryopreservation medium as disclosed herein may further comprise one or more additional ingredients including but not limited to a salt, a sugar, a fat, a basal medium, a growth factors, amino acids, vitamins, minerals, edible surfactants, preservatives, antibiotics, pectin, alginate, agarose, elastin, chitin, chitosan, fibrin, fibrinogen, polysaccharides, alginates, collagen, gelatin, poly(amino acids), peptides, polypeptides, fibers, buffering agents or any combinations thereof.
  • each of the one or more additives may be included at about 0.001% to about 10% by weight of the composition.
  • each of the one or more additives may be included at about 0.001% to about 0.01%, 0.01% to about 0.05%, 0.05% to about 0.1%, 0.1% to about 0.5%, 0.5% to about 1%, 1% to about 5%, or 5% to about 10% by weight of the composition.
  • the cryopreservation media composition as disclosed herein may be formulated as a ready-to-use formulation such that it can be added directly to the sample to be cryopreserved.
  • the cryopreservation medium may be formulated as a concentrate that is diluted with the culture medium, cell culture or water, depending on application and the exact composition of the cryopreservation media.
  • the concentrate may be anywhere between a 2 X to a 100 X concentrate.
  • Methods for concentrating media components are well known in the art and may comprise for example, lyophilization, freeze drying, spray drying micro or nanofiltration steps.
  • the compositions disclosed herein may also be formulated as powder or pastes to be added to the culture medium as desired.
  • the compositions of the current disclosure may be formulated to work at different temperature ranges and for different applications. For example, in some aspects, the 6 95004041.1 Attorney Docket No.121430-791898 relative ratios of egg yolk to the edible cryoprotectants may be varied depending on the desired temperature for freezing.
  • the composition is formulated to be protective when a sample is frozen and stored at a temperature range of about ⁇ 20° C to ⁇ 196° C. In some aspects, the composition is protective when a sample is frozen and stored at a temperature range of about ⁇ 20° C to about ⁇ 40° C, or about ⁇ 40° C to about ⁇ 60° C, or about ⁇ 60° C to about ⁇ 80° C, or about ⁇ 80° C to about ⁇ 100° C, or about ⁇ 100° C to about ⁇ 120° C, or about ⁇ 120° C to about ⁇ 140° C.
  • the disclosed compositions may be formulated for use at any stage, and any system of cellular agriculture.
  • the compositions, as disclosed herein may be formulated for preservation of non-human cells, cell lines, cell populations, tissues, organs, organoids, or cellular biomass.
  • any type of cell may be preserved using the formulations disclosed herein, including fibroblast, a myoblast, an adipocyte, a stem cell, an endothelial cell, or a mesodermal cell or any combination thereof.
  • the disclosed compositions may be added directly to a bioreactor for preserving biomass.
  • the ratio of the egg yolk to the edible cryoprotectant can be varied depending on the sample type, for example cells, tissues, biomass, organs, to maximize cryopreservation. As such the ratio of the egg yolk to the edible cryoprotectant may vary from about 0.01:1 to about 5:1, for example 0.01:1, or 0.5:1, or 1:1, or 2:1, or 3:1, or 4:1, or 5:1.
  • the current disclosure encompasses a method of cryopreservation for cellular agriculture applications, the method comprising contacting at least one non-human animal cell with a medium comprising egg yolk and at least one additional edible cryoprotectant.
  • the current disclosure encompasses a method of cryopreservation for cellular agriculture applications, the method comprising contacting at least one non-human animal cell with a composition has provided herein.
  • the current disclosure encompasses the use of the compositions as disclosed herein for cryopreservation of non-human animal cells.
  • non- human cell encompasses any cell from a non-human source or a part thereof (tissue, organ, 7 95004041.1 Attorney Docket No.121430-791898 system or embryo).
  • derivative of a non-human cell encompasses isolated cells, cell lines, or recombinant cell lines.
  • the cell is an embryonic cell.
  • the cell is a somatic cell.
  • the cell is a differentiated cell.
  • the cell is an adherent cell, for example a fibroblast, a myoblast, an adipocyte, an endothelial cell, or a mesodermal cell.
  • the cell is a stem cell for example an embryonic stem cell, bone marrow derived stem cell, adipose derived stem cell, or induced pluripotent stem cell.
  • the non-human cell is a vertebrate cell.
  • vertebrate cell suitable for use in the current disclosure include cells derived from fish, bird, amphibian, reptile, or mammals or recombinant cell lines thereof.
  • the non-human cell is an invertebrate cell.
  • Non-limiting examples of invertebrate cells suitable for the current disclosure include cells derived from oysters, mussels, clams, scallop, jelly fishes, squids, prawns, octopus, sea cucumbers, sea squirts or recombinant cell lines thereof.
  • the current disclosure encompasses, obtaining a non-human animal cell, and cryopreserving it using the compositions disclosed herein, for future use in a cellular agriculture related application.
  • the process requires addition for the cryopreservation media as disclosed herein to an adherent cell culture.
  • the process requires mixing of the cryopreservation medium with a cell suspension at a desired concentration.
  • the concentration of the cryopreservation medium can vary based on the various factors, like the composition of the suspension medium in which the cells are present. In some aspects, the exact formulation and concentration of the medium to be used for cryopreservation can be easily determined by a person of ordinary skill in the art, either empirically or based on manufacturer’s instructions. [0031] In some aspects, the current disclosure encompasses cryopreservation of cells, wherein the non-human animal cells are suspended in the cryopreservation medium at a concentration that 1 ⁇ 10 2 to 1 ⁇ 10 9 non-human cells/mL.
  • the concentration of cells in the cryopreservation compositions disclosed herein may vary from about 1 ⁇ 10 2 to about 1 ⁇ 10 3 , about 1 ⁇ 10 3 to about 1 ⁇ 10 4 , about 1 ⁇ 10 4 to about 1 ⁇ 10 5 , about 1 ⁇ 10 5 to about 1 ⁇ 10 6 , about 1 ⁇ 10 6 to about 1 ⁇ 10 7 , about 1 ⁇ 10 7 to about 1 ⁇ 10 8 , or about 1 ⁇ 10 8 to about 1 ⁇ 10 9 non-human cells/mL.
  • the compositions of the current disclosure can also be used cryopreserve the middle or end-product of a cellular agriculture process.
  • the compositions as disclosed herein may be used to cryopreserve a population of non- human animal cells, non-human tissue, non-human organ, non-human organoids, non-human cellular biomass, or non-human cell grown on microcarrier or scaffold.
  • the composition can be used to preserve a population of non-human animal cells.
  • 8 95004041.1 Attorney Docket No.121430-791898 such a population of cells may be present in an adherent or a suspension culture or a combination thereof.
  • the population of cells is a homogenous population.
  • the population of cells is a heterogenous population.
  • the population of cells is derived directly from an animal.
  • the population of cells is a result of a cell culture process.
  • the cell population is frozen in the culture medium supplemented with a composition as disclosed herein, for the purpose of cryopreservation.
  • the culture medium comprise any medium known in the art.
  • Non-limiting exemplary cell culture media that may be purchased from commercial vendors (e.g., Gibco, Sartorius, etc.), or synthesized, include SAFC Excell media, BME (basal Eagle Medium), MEM (minimum Eagle Medium), medium 199, DMEM (Dulbecco's modified Eagle Medium), GMEM (Glasgow modified Eagle medium), DMEM-HamF12, Ham-F12 (Gibco) and Ham-F10 (Gibco), IMDM (Iscove's Modified Dulbecco's medium), MacCoy's 5A medium, RPMI 1640, and GTM3.
  • SAFC Excell media BME (basal Eagle Medium), MEM (minimum Eagle Medium), medium 199, DMEM (Dulbecco's modified Eagle Medium), GMEM (Glasgow modified Eagle medium), DMEM-HamF12, Ham-F12 (Gibco) and Ham-F10 (Gibco), IMDM (Iscove
  • the cell population may be cryopreserved and stored in a dish, tube, plate, jar or a bioreactor.
  • the cell population is separated from the cell culture medium, for example using a technique like filtration, centrifugation, and then resuspended in the cryopreservation medium.
  • the population of cells are cultured in a bioreactor and frozen within the bioreactor without concentration or removal of media.
  • the cryopreservation medium as disclosed herein may be added directly to the bioreactor comprising the cell culture. In some aspects, these measures make it possible to halt cell culture operations at any stage in a cellular agriculture system.
  • the current disclosure also encompasses a method of cellular agriculture, the method comprising; a) growing non-human animal cells in a bioreactor container; b) adding a cryopreservation medium as disclosed herein to the bioreactor container; c) freezing the non- human animal cells in the bioreactor container.
  • step (c) is an ultrarapid freezing.
  • the non-human animal cells are grown to a density of about 1 ⁇ 10 2 to 1 ⁇ 10 9 non- human cells/mL.
  • the recovery survival rate of the non-human animal cells is at least 50% and preferably 70% or more.
  • the method further comprises thawing the non-human animal cells in the bioreactor container.
  • the method further comprises resuming the growth of the non-human cells.
  • the cryopreservation can be used for shipping large amounts of cellular biomass from one facility to another facility for further cultivation. 9 95004041.1 Attorney Docket No.121430-791898 [0034]
  • the current compositions can also be used to cryopreserve adherent cell populations, on plates, scaffolds or microcarriers.
  • the term “microcarrier” refers to a support matrix allowing for adherence/attachment, maturation, differentiation, and proliferation of cells in a culture, with at least one of it dimensions being in the micrometer range.
  • the microcarrier is for growth of adherent cells in a non-adherent container.
  • the microcarrier is for growth in a bioreactor.
  • the non-adherent container is a bioreactor.
  • growth while adhered to a microcarrier is not anchorage- independent growth, as the cell is anchored to the microcarrier.
  • the microcarriers as disclosed herein are edible microcarriers.
  • the term “scaffold” refers to a structure comprising a material that provides a surface suitable for adherence/attachment, maturation, differentiation, and proliferation of cells.
  • a scaffold may further provide mechanical stability and support.
  • a scaffold may be in a particular shape or form to influence or delimit a three-dimensional shape or form assumed by a population of proliferating cells.
  • a scaffold of the invention is three-dimensional.
  • the scaffold may comprise microcarriers as disclosed herein.
  • the microcarriers, scaffolds or a combination thereof can be used for production of cell culture based cultured meat products.
  • the microcarriers or scaffolds can be suspended in the cryopreservation media as disclosed herein and frozen.
  • the current disclosure also encompasses the use of the disclosed compositions for cryopreserving products manufactured using cellular agriculture techniques.
  • these products may be in the form of tissue mass, organs, organoids, cellular biomass, cultured meat, and processed cultured meat products.
  • the cultured meat for cryopreservation has substantially the same composition with respect to percent proteins, fat, carbohydrates and the like, such as beef, veal, pork, chicken, or fish.
  • the cultured meat comprises a plurality of layers, wherein each layer comprises non- human myocytes and non-human endothelial cells.
  • the cultured meat comprises a plurality of layers, wherein each layer comprises myocytes, and may include one or more of endothelial cells, adipose cells, and/or fibroblasts, wherein the cells are derived from sources including, but not limited to, mammals, birds, reptiles, fish, crustaceans, mollusks, and cephalopods, or combinations thereof.
  • the myocytes are aligned relative to each other.
  • the myocytes are aligned relative to a layer of the meat.
  • the cultured meat is suitable for human consumption.
  • the cultured meat is suitable for non-human animal consumption.
  • the cultured meat is suitable for both human and non-human animal consumption.
  • the cultured meat is further 10 95004041.1 Attorney Docket No.121430-791898 processed to make food products for example, cuts, fillets, mince, meat balls, sausages and comprise the compositions as disclosed herein for cryopreservation.
  • the compositions do not need to be removed prior to consumption. In some aspects, these compositions may be removed prior to consumption.
  • any freezing method or technology can be used for freezing samples in the disclosed cryopreservation media. Methods of freezing and thawing cultures are well known in the art.
  • samples can be frozen using liquid nitrogen, dry ice, ethanol dry ice mix or any other suitable method.
  • the temperature of the culture may be brought down gradually.
  • the culture may be frozen using ultrarapid freezing technologies.
  • the sample can be frozen and stored at a temperature range of about ⁇ 20° C to ⁇ 196° C.
  • the sample is frozen and stored at a temperature range of about ⁇ 20° C to about ⁇ 40° C, or about ⁇ 40° C to about ⁇ 60° C, or about ⁇ 60° C to about ⁇ 80° C, or about ⁇ 80° C to about ⁇ 100° C, or about ⁇ 100° C to about ⁇ 120° C, or about ⁇ 120° C to about ⁇ 140° C.
  • cryopreservation methods as disclosed herein enables the revival of at least 50% and preferably 70% or more of live cells in the sample, wherein the sample may be cells, cell lines, tissue mass, organs, organoids, cellular biomass, cultured meat, and processed cultured meat products.
  • the phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. For example, the use of a singular term, such as, “a” is not intended as limiting of the number of items.
  • references to the terms “aspect,” “aspects,” and/or the like in the description mean that the feature and/or features being referred 11 95004041.1 Attorney Docket No.121430-791898 to are included in, at least, one aspect of the description.
  • Separate references to the terms “aspect,” “aspects,” and/or the like in the description do not necessarily refer to the same aspect and are also not mutually exclusive unless so stated and/or except as will be readily apparent to those skilled in the art from the description.
  • a feature, structure, process, step, action, or the like described in one aspect may also be included in other aspects but is not necessarily included.
  • the present inventive concept may include a variety of combinations and/or integrations of the aspects described herein.
  • the terms “about” or “approximately,” as used in the description and the appended claims, should be understood to include the recited values or a value that is three times greater or one third of the recited values.
  • about 3 mm includes all values from 1 mm to 9 mm
  • approximately 50 degrees includes all values from 16.6 degrees to 150 degrees.
  • they can refer to less than or equal to ⁇ 5%, such as less than or equal to ⁇ 2%, such as less than or equal to ⁇ 1%, such as less than or equal to ⁇ 0.5%, such as less than or equal to ⁇ 0.2%, such as less than or equal to ⁇ 0.1%, such as less than or equal to ⁇ 0.05%.
  • EXAMPLE 1 Provided are some exemplary formulations: Formulations 1: ⁇ 20% Chicken egg yolk ⁇ 10% glycerin ⁇ DMEM/F12 culture media up to 100% Formulations 2: ⁇ 15% Chicken egg yolk ⁇ 10% glycerin ⁇ 1% dextrose ⁇ 1% sodium chloride ⁇ Water up 100% Formulation 3: ⁇ 20% Chicken egg yolk ⁇ 5% PVP ⁇ DMEM culture media up to 100% [0045] These exemplary formulations were tested against three commercially available formulations, provided here as comparative formulations 4-6.
  • the samples were then frozen in a deep freezer (-80 oC) using freezing container for gradual cool down at the rate of 1 oC per minute.
  • the samples were stored in -80 oC for 1 day.
  • the samples were thawed and cell viability was studied using live dead immunostaining assay. The results with 50% and preferably 70% or more viable cells were considered successful.

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Abstract

L'invention concerne des compositions avec du jaune d'oeufs et des cryoconservateurs comestibles destinés à être utilisés dans l'agriculture cellulaire et des applications associées. L'invention concerne diverses formulations destinées à être utilisées dans ces applications. L'invention concerne également des procédés de cryoconservation utilisant les compositions.
PCT/US2024/030498 2023-05-23 2024-05-22 Systèmes de cryoconservation comestibles et de qualité alimentaire Pending WO2024243271A1 (fr)

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WO2014089924A1 (fr) * 2012-12-10 2014-06-19 山东天龙牧业科技有限公司 Agent antigel à base de jaune d'œuf de canard pour la cryoconservation de sperme d'âne et son procédé de préparation
US20150037783A1 (en) * 2012-03-14 2015-02-05 Membrane Protective Technologies, Inc. System and Substances for Cryopreservation of Viable Cells

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