WO2013016547A2 - Engineered comestible meat - Google Patents

Engineered comestible meat Download PDF

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WO2013016547A2
WO2013016547A2 PCT/US2012/048357 US2012048357W WO2013016547A2 WO 2013016547 A2 WO2013016547 A2 WO 2013016547A2 US 2012048357 W US2012048357 W US 2012048357W WO 2013016547 A2 WO2013016547 A2 WO 2013016547A2
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Prior art keywords
multicellular bodies
plurality
embodiments
layers
meat
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PCT/US2012/048357
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French (fr)
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WO2013016547A3 (en
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Gabor Forgacs
Francoise Marga
Karoly Robert JAKAB
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The Curators Of The University Of Missouri
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Priority to US61/511,948 priority
Application filed by The Curators Of The University Of Missouri filed Critical The Curators Of The University Of Missouri
Publication of WO2013016547A2 publication Critical patent/WO2013016547A2/en
Publication of WO2013016547A3 publication Critical patent/WO2013016547A3/en

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    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; THEIR TREATMENT, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A23B - A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L13/00Meat products; Meat meal; Preparation or treatment thereof
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; THEIR TREATMENT, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A23B - A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L13/00Meat products; Meat meal; Preparation or treatment thereof
    • A23L13/03Coating with a layer; Stuffing, laminating, binding, or compressing of original meat pieces
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062General methods for three-dimensional culture
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues
    • C12N5/0697Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00Coculture with; Conditioned medium produced by
    • C12N2502/13Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1347Smooth muscle cells
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00Coculture with; Conditioned medium produced by
    • C12N2502/28Vascular endothelial cells
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1307Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00Supports or coatings for cell culture, characterised by material
    • C12N2533/70Polysaccharides
    • C12N2533/76Agarose, agar-agar

Abstract

Provided are engineered meat products formed as a plurality of at least partially fused layers, wherein each layer comprises at least partially fused multicellular bodies comprising non-human myocytes and wherein the engineered meat is comestible. Also provided are multicellular bodies comprising a plurality of non-human myocytes that are adhered and/or cohered to one another; wherein the multicellular bodies are arranged adjacently on a nutrient- permeable support substrate and maintained in culture to allow the multicellular bodies to at least partially fuse to form a substantially planar layer for use in formation of engineered meat. Further described herein are methods of forming engineered meat utilizing said layers.

Description

ENGINEERED COMESTIBLE MEAT

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of U.S. Provisional Application No. 61/511,948, filed July 26, 2011, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[002] Protein is a nutrient needed by the human body for growth and maintenance. Aside from water, protein is the most abundant molecule in the body. According to U.S. and Canadian Dietary Reference Intake guidelines, women aged 19-70 need to consume 46 grams of protein per day, while men aged 19-70 need to consume 56 grams of protein per day to avoid deficiency. This recommendation, however, is for a sedentary person free of disease. Protein deficiency can lead to reduced intelligence or mental retardation as well as contribute to the prevalence of diseases such as kwashiorkor. Protein deficiency is a serious problem in developing countries, particularly, in countries affected by war, famine, and overpopulation. Animal sources of protein, such as meat, are often a source of the complete complement of all the essential amino acids in adequate proportions.

[003] The nutritional benefits of meat are tempered by potential associated environmental degradation. According to a 2006 report by the Livestock, Environment And Development Initiative, entitled Livestock's Long Shadow— Environmental Issues and Options, the livestock industry is one of the largest contributors to environmental degradation worldwide, and modern practices of raising animals for food contributes widely to air and water pollution, land degradation, climate change, and loss of biodiversity. The production and consumption of meat and other animal sources of protein is also associated with the clearing of rainforests and species extinction. Accordingly, there is a need for a solution to demands for alternative to meat produced from live animals.

SUMMARY OF THE INVENTION

[004] Tissue engineering technology offers new opportunities to produce edible sources of animal protein that are not associated with the environmental degradation of raising livestock. Tissue engineering has been defined as an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ. Langer R, Vacanti JP, Tissue Engineering, Science 260(5110):920-926 (May 1993). Despite the potential to apply tissue engineering technology to meet the nutritional needs of living beings, scientifically sound and industrially feasible processes have not been developed to produce comestible meat and engineered comestible meat products are not available.

[005] Disclosed herein are engineered meat products, layers comprising a plurality of multicellular bodies for use in production of said meat, and methods of producing the engineered meat products. In a first aspect, disclosed herein is engineered meat comprising a plurality of layers, wherein each layer comprises non-human myocytes and wherein the engineered meat is comestible and for ingestion.

[006] Also described herein are engineered meat products, the meat product comprising: a body having a volume, wherein the body comprises a plurality of stacked planar layers, wherein the layers are at least partially fused and each planar layer comprises a plurality of at least partially fused non-human multicellular bodies comprising myocytes; further wherein the body does not include any blood vessels, and wherein the engineered meat product is comestible and for ingestion.

[007] The body volume may be greater than some minimum volume (e.g., greater than 0.1 cm3, greater than 1 cm3, greater than 10 cm3, greater than 50 cm3, greater than 100 cm3, greater than 500 cm3, greater than 1000 cm3, etc., including any intermediate volume).

[008] The layered nature of the engineered meat may be visible upon examination of the volume or meat. For example, the meat may have planar layers in which the myocytes are differently oriented between layers. In some variations, the layered organization of the engineered meat may be apparent by looking at regions of cell death. For example, the planar layers within a middle region of the meat product may have experienced cell death before the outer layers of the engineered meat. As described in greater detail below, formation of the engineered meat by successively stacking fused (or partially fused) planar layer atop one another and allowing the stacked layers to fuse may result in regions of cell death as successively "deeper" layers are separated further from fresh nutrients in the culture media. This may result in a pattern of cell death in which deeper regions (which may be stratified into planar layers) further from the edges of the volume experience cell death before more newly applied layers. The stratified pattern of timing of cell death may be visualized by examining markers for cell death progression, including nuclear fragmentation, and other metabolic markers, known in the art. For example, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) may be used. The planar layers may be formed of multicellular bodies having additional non-human cell types. For example, each planar layer of the engineered meat volume may comprise a plurality of at least partially fused multicellular bodies comprising non-human myocytes and one or more of: non-human endothelial cells, non-human fibroblasts, and/or non-human adipose cellsor the like.

[009] In general, the engineered meat does not include any filler bodies, such as may be found in other forms of engineered tissues. For example, filler bodies that act as scaffolding (e.g., support structures) are not necessary, and may be detrimental to the final comestible product. Filler bodies may include biocompatible material that resists migration and ingrowth of cells from the multicellular bodies and that is resistant to adherence of cells to it. See, e.g., U.S. 8, 143.055, herein incorporated by reference in its entirety.

[010] The term "comestible," as used herein, means edible or suitable to be eaten by a human being or a non-human animal. The phrase "for ingestion," as used herein, means suitable and adapted to be consumed orally by a human being or a non-human animal. In some embodiments, each layer of the engineered meat further comprises non-human endothelial cells. In some embodiments, each layer of the engineered meat further comprises non-human adipose cells. In some embodiments, each layer further comprises non-human fibroblast cells. In some embodiments, the engineered meat disclosed herein comprises a plurality of layers, wherein each layer comprises non-human myocytes and wherein each layer is characterized by a thickness adapted to allow diffusion to sufficiently support the maintenance and growth of the non-human myocytes in culture. In various embodiments, the engineered meat comprises a plurality of layers, wherein each layer is about 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μιη thick. The term "about," as used herein when referring to a measurable value, and may mean within +/- 2%, +1-5%, or +/-10% of a given value or range. In some embodiments, the thickness of each layer is about 100 μι ίο about 1000 μιη. In further embodiments, the thickness of each layer is about 150 μιη to about 900 μιη. In still further embodiments, the thickness of each layer is about 200 μιη to about 800 μιη. In still further embodiments, the thickness of each layer is about 250 μιη to about 700 μιη. In still further embodiments, the thickness of each layer is about 300 μιη to about 600 μιη. In various embodiments, the engineered meat comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 layers. In other various embodiments, the engineered meat comprises 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 layers. In some

embodiments, the engineered meat disclosed herein comprises about 2 to about 100 layers. In further embodiments, the engineered meat comprises about 20 to about 80 layers. In still further embodiments, the engineered meat comprises about 40 to about 60 layers. In some embodiments, each layer comprises multicellular bodies of about 100, 200, 300, 400, 500 μιη in diameter. In some embodiments, the diameter of said multicellular bodies is about 100 μιη to about 500 μιη. In further embodiments, the diameter of said multicellular bodies is about 200 μιη to about 400 μιη. In further embodiments, the diameter of said multicellular bodies is about 200 μιη to about 300 μιη. In still further embodiments, the diameter of said multicellular bodies is about 250 μιη to about 400 μιη. In still further embodiments, the diameter of said multicellular bodies is about 300 μιη to about 400 μιη. In general, the diameter of a multicellular body may refers to the length of the longest line extending through the midpoint a cross-section through the elongate multicellular body from one side of the sectioned multicellular body to the opposite side.

[011] In some embodiments, the engineered meat disclosed herein is characterized by a composition that is substantially 60-80 percent aqueous fluid, 14-35 percent protein, 1-25 percent fat, 1-5 percent carbohydrates and 1-5 percent other substances. In further embodiments, the engineered meat has substantially the same composition with respect to percent proteins, fat, carobhydrates and the like as beef, veal, pork, chicken, or fish. In some embodiments, the engineered meat comprises a plurality of layers, wherein each layer comprises non-human myocytes and non-human endothelial cells and wherein each layer is bioprinted. In various embodiments, the engineered meat disclosed herein 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, mo Husks, and cephalopods, or combinations thereof. In some embodiments, the engineered meat comprises non-human myocytes, which are skeletal myocytes. In some embodiments, the myocytes are cardiac myocytes. In some embodiments the myocytes are smooth myocytes. In other embodiments, the myocytes are combinations of skeletal, cardiac, and smooth myocytes. In some embodiments, the engineered meat comprises endothelial cells, which are microvascular endothelial cells. In some embodiments, the engineered meat disclosed herein is characterized by a ratio of non-human myocytes to non- human endothelial cells of about 19: 1 to about 3: 1. In some embodiments, non-human endothelial cells comprise about 5% to about 15% of the total cell population of the engineered meat. In some embodiments, the engineered meat is substantially free of non-differentiated myocytes and/or non-differentiated endothelial cells. In some embodiments, the myocytes are aligned relative to each other. In further embodiments, the myocytes are aligned relative to a layer of the meat. In various embodiments, the engineered meat disclosed herein further comprises one or more substances that enhance the nutritional value of the meat, the culinary appeal of the meat, or the growth characteristics of the non-human cells. In some embodiments, the engineered meat further comprises one or more nutritional supplements. In further embodiments, the nutritional supplements are selected from: vitamins, minerals, fiber, fatty acids, and amino acids. In some embodiments, the engineered meat further comprises one or more flavorants and/ or colorants. In some embodiments, the engineered meat further comprises one or more of: matrix proteins, proteoglycans, antioxidants, perfluorocarbons, and growth factors. In some embodiments, the engineered meat is suitable for human consumption. In other embodiments, the engineered meat is suitable for non-human animal consumption. In still other embodiments, the engineered meat is suitable for both human and non-human animal consumption.

[012] In a another aspect, disclosed herein is a plurality of multicellular bodies comprising a plurality of living non-human myocytes wherein the cells are adhered and/or cohered to one another; wherein the multicellular bodies are arranged adjacently on a support substrate to form a substantially planar layer for use in formation of engineered comestible meat. In certain embodiments, a multicellular body is substantially spherical in shape. In certain embodiments, a multicellular body is substantially cylindrical. In some embodiments, a multicellular body has a substantially circular cross section. In some embodiments, a multicellular body has an elongate shape with a square, rectangular, triangular, or other non-circular cross-sectional shape. In some embodiments, a multicellular body has a non-elongate cylindrical shape or a cuboidal shape. The term "adjacent," as used herein when referring to arrangement of multicellular bodies, means in contact and on top of, under, or next to, either horizontally or vertically relative to the support substrate.

[013] In a another aspect, disclosed herein is a plurality of multicellular bodies comprising a plurality of living non-human myocytes wherein the cells are adhered and/or cohered to one another; wherein the multicellular bodies are arranged adjacently on a support substrate and maintained in culture to allow the multicellular bodies to fuse to form a substantially planar layer for use in formation of engineered comestible meat.

[014] In a another aspect, disclosed herein is a plurality of multicellular bodies comprising a plurality of living non-human myocytes wherein the cells are adhered and/or cohered to one another; wherein the multicellular bodies are arranged adjacently on a support substrate and maintained in culture to allow the multicellular bodies to fuse to form a substantially planar layer for use in formation of engineered comestible meat; wherein the support substrate is permeable to fluids and nutrients and allows cell culture media to contact all surfaces of said layer. In some embodiments, the engineered layers disclosed herein further comprise non-human endothelial cells and/or non-human adipose cells. In some embodiments, the layers further comprise non-human fibroblast cells. In some embodiments, the engineered layers are characterized by a ratio of non-human myocytes to non-human endothelial cells of about 19: 1 to about 3: 1. In some embodiments, the non-human endothelial cells comprise about 5% to about 15% of the total cell population. In some embodiments, the engineered layers disclosed herein are characterized by a thickness adapted to allow diffusion to sufficiently support the maintenance and growth of said non-human myocytes and non-human endothelial cells in culture. In various embodiments, the engineered layers are about 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μιη thick. In some embodiments, the thickness of the layers is about 100 μιη to about 1000 μιη. In further embodiments, the thickness of the layers is about 150 μιη to about 900 μιη. In still further embodiments, the thickness of the layers is about 200 μιη to about 800 μιη. In still further embodiments, the thickness of the layers is about 250 μιη to about 700 μιη. In still further embodiments, the thickness of the layers is about 300 μιη to about 600 μιη. In various embodiments, the engineered layers disclosed herein further comprise one or more substances that enhance nutritional value, culinary appeal, or growth characteristics. In some embodiments, the engineered layers further comprise one or more of: matrix proteins, proteoglycans, antioxidants, perfluorocarbons, and growth factors. In some embodiments, the plurality of multicellular bodies comprising a plurality of living non-human myocytes and non- human endothelial cells, wherein the cells are adhered and/or cohered to one another, are arranged adjacently on a support substrate to form a substantially planar layer for use in formation of engineered comestible meat and the layer is bioprinted. In some embodiments, each layer comprises multicellular bodies of about 100, 200, 300, 400, 500 μιη in diameter. In some embodiments, the diameter of said multicellular bodies is about 100 μιη to about 500 μιη. In further embodiments, the diameter of said multicellular bodies is about 200 μιη to about 400 μιη. In further embodiments, the diameter of said multicellular bodies is about 200 μιη to about 300 μιη. In still further embodiments, the diameter of said multicellular bodies is about 250 μιη to about 400 μιη. In still further embodiments, the diameter of said multicellular bodies is about 300 μι ίο about 400 μιη.

[015] Also described herein are methods of forming a comestible engineered meat product, the method comprising: forming a plurality of planar layers, wherein each layer is formed by adjacently positioning a plurality of multicellular bodies in a plane, further wherein each multicellular body comprises a plurality of cohered non-human myocytes; culturing each of the planar layers at least until the plurality of multicellular bodies within each layer begin to fuse; stacking the plurality of layers to form a layered volume of engineered meat; and culturing the volume of meat at least until the stacks begin to fuse.

[016] In some variations, the method may also include a step of preparing the plurality of multicellular bodies by culturing a plurality of non-human myocyte cells and non-human endothelial cells at least until the cells are cohered to one another. As mentioned above, any other appropriate non-human cell type may be included as part of some or all of the multicellular bodies forming the layers, including endothelial cells and/or adipose cells, and/or fibroblast cells.

[017] During the formation of the engineered meat product, the layers maybe individually or collectively stacked atop other layer to create the volume of engineered meat. In some variations each successive layer is differently oriented with respect to the adjacent layer(s). For example, as they are stacked, the new layers may be rotated relative to the other layers in the volume. In some variations, each layer is rotated approximately 90° relative to the other layers as it is stacked.

[018] In any of the engineered meat described herein the layers may be exercised as they are formed. As described in greater detail below, exercising the layers may enhance the formation of extracellular matrix (ECM). This may also orient the cells (e.g., myocytes) within a layer as it is formed. Thus, in some variations of the method of forming the engineered meat may include a step of applying mechanical, electrical or electromechanical force to exercise the myocytes in each layer.

[019] As mentioned, any appropriate number of layers may be included. For example, the step of stacking the layers may include stacking more than about 10 layers, more than about 50 layers, more than about 100 layers, or the like.

[020] In a another aspect, disclosed herein are methods of forming engineered meat , comprising: preparing a plurality of multicellular bodies comprising a plurality of living non- human myocytes wherein the cells are adhered and/or cohered to one another; laying more than one multicellular body adjacently onto a support substrate; allowing said multicellular bodies to fuse to form a layer; laying more than one layer adjacently onto that layer; allowing said layers to fuse to form engineered meat; and optionally, freezing said meat; provided that the engineered meat is comestible and for ingestion. In some embodiments, disclosed herein are methods of forming engineered meat , comprising: preparing a plurality of multicellular bodies comprising a plurality of non-human myocytes wherein the cells are adhered and/or cohered to one another; laying more than one multicellular body adjacently onto a support substrate; fusing said multicellular bodies to form a layer; laying more than one layer adjacently onto the first layer; and fusing said layers to form engineered meat; provided that the engineered meat is comestible. In some embodiments, the methods provided herein further comprise freezing said meat.

[021] In a another aspect, disclosed herein are methods of forming engineered meat , comprising: preparing a plurality of elongate multicellular bodies comprising a plurality of living non-human myocytes wherein the cells are adhered and/or cohered to one another;

preparing a plurality of substantially spherical multicellular bodies comprising a plurality of living non-human myocytes wherein the cells are adhered and/or cohered to one another; laying more than one elongate multicellular body and more than one substantially spherical multicellular body adjacently onto a support substrate; allowing said multicellular bodies to fuse to form a layer; laying (e.g., stacking) more than one layer adjacently onto the first layer;

allowing said layers to fuse to form engineered meat; and optionally, freezing said meat;

provided that the engineered meat is comestible and for ingestion. In some embodiments, disclosed herein are methods of forming engineered meat, comprising: preparing a plurality of elongate multicellular bodies comprising a plurality of non-human myocytes wherein the cells are adhered and/or cohered to one another; preparing a plurality of substantially spherical multicellular bodies comprising a plurality of non-human myocytes wherein the cells are adhered and/or cohered to one another; laying more than one elongate multicellular body and more than one substantially spherical multicellular body adjacently onto a support substrate; fusing said multicellular bodies to form a layer; laying more than one layer adjacently onto the first layer; and fusing said layers to form a volume of engineered meat; provided that the engineered meat is comestible and for ingestion. In some embodiments, the methods provided herein further comprise freezing said meat.

[022] In some embodiments, the ratio of the elongate multicellular bodies and the substantially spherical multicellular bodies is about 0: 100, 1 : 100, 2: 100, 3: 100, 4: 100, 5: 100, 6: 100, 7: 100, 8: 100, 9: 100, 1 : 10, 11 : 100, 12: 100, 13: 100, 14: 100, 15: 100, 16: 100, 17: 100, 18: 100, 19: 100, 1 :5, 21 : 100, 22: 100, 23: 100, 24: 100, 25: 100, 26: 100, 27: 100, 28: 100, 29: 100, 3: 10, 31 : 100, 32: 100, 33: 100, 34: 100, 35: 100, 36: 100, 37: 100, 38: 100, 39: 100, 2:5, 41 : 100, 42: 100, 43: 100, 44:100, 45:100, 46:100, 47:100, 48:100, 49:100, 1:2, 51:100, 52:100, 53:100, 54:100, 55:100, 56:100, 57:100, 58:100, 59:100, 3:5, 61:100, 62:100, 63:100, 64:100, 65:100, 66:100, 67:100, 68:100, 69:100, 7:10, 71:100, 72:100, 73:100, 74:100, 75:100, 76:100, 77:100, 78:100, 79:100, 4:5, 81:100, 82:100, 83:100, 84:100, 85:100, 86:100, 87:100, 88:100, 89:100, 9:10, 91:100, 92:100, 93:100, 94:100, 95:100, 96:100, 97:100, 98:100, 99:100, or 1:1. In some embodiments, the ratio of the substantially spherical multicellular bodies and the elongate multicellular bodies is about 0:100, 1:100, 2:100, 3:100, 4:100, 5:100, 6:100, 7:100, 8:100, 9:100, 1:10, 11:100,

12:100, 13 100, 14:100, 15:100, 16:100, 17:100, 18:100, 19:100, 1:5,21:100, 22:100, 23:100, 24:100, 25100, 26:100, 27:100, 28:100, 29:100, 3:10, 31:100, 32:100, 33:100, 34:100, 35:100, 36:100, 37 100, 38:100, 39:100, 2:5, 41:100, 42:100, 43:100, 44:100, 45 100, 46:100, 47:100, 48:100, 49100, 1:2, 51:100, 52:100, 53:100, 54:100, 55:100, 56:100, 57100,58:100,59:100,

3:5, 61:100, 62:100, 63:100, 64:100, 65:100, 66:100, 67:100, 68:100, 69100, 7:10,71:100,

72:100, 73 100, 74:100, 75:100, 76:100, 77:100, 78:100, 79:100, 4:5, 81: 100, 82:100, 83:100, 84:100, 85100, 86:100, 87:100, 88:100, 89:100, 9:10, 91:100, 92:100, 93:100, 94:100, 95:100, 96:100, 97100, 98:100, 99:100, or 1:1.

[023] In a another aspect, disclosed herein are methods of forming engineered meat, comprising: preparing a plurality of substantially spherical multicellular bodies comprising a plurality of living non-human myocytes wherein the cells are adhered and/or cohered to one another; laying more than one substantially spherical multicellular body adjacently onto a support substrate; allowing said substantially spherical multicellular bodies to fuse to form a layer; laying more than one layer adjacently onto the first layer; allowing the layers to fuse to form a volume of engineered meat; and optionally, freezing said meat; provided that the engineered meat is comestible and for ingestion. In some embodiments, disclosed herein are methods of forming engineered meat, comprising: preparing a plurality of substantially spherical multicellular bodies comprising a plurality of non-human myocytes wherein the cells are adhered and/or cohered to one another; laying more than one substantially spherical

multicellular body adjacently onto a support substrate; fusing said substantially spherical multicellular bodies to form a layer; laying more than one layer adjacently onto the first layer; and fusing said layers to form a volume of engineered meat; provided that the engineered meat is comestible. In some embodiments, the methods provided herein further comprise freezing said meat.

[024] In some embodiments, the methods of forming engineered meat disclosed herein comprise preparing a plurality of multicellular bodies comprising a plurality of living non- human myocytes wherein the cells are adhered and/or cohered to one another, wherein the multicellular bodies further comprise living, non-human adipose cells, and/or endothelial cells. In some embodiments, the multicellular bodies further comprise living, non-human fibroblast cells. In some embodiments, the methods of forming engineered meat disclosed herein comprise laying more than one multicellular body adjacently onto a support substrate, wherein the multicellular bodies are laid horizontally adjacent and/or vertically adjacent. In some

embodiments, the methods of forming engineered meat disclosed herein comprise laying more than one layer adjacently onto a support substrate, wherein the layers are laid horizontally adjacent and/or vertically adjacent. In some embodiments, the support substrate is permeable to fluids and nutrients and allows cell culture media to contact all surfaces of said multicellular bodies and/or layers. In some embodiments, the methods of forming engineered meat disclosed herein comprise allowing multicellular bodies to fuse to form a layer, wherein the multicellular bodies fuse to form a layer in a cell culture environment. In some embodiments, fusing of multicellular bodies takes place over about 2 hours to about 36 hours. In some embodiments, the methods comprise allowing layers to fuse to form engineered meat, wherein the layers fuse to form engineered meat in a cell culture environment. In some embodiments, fusing of layers takes place over about 2 hours to about 36 hours. In some embodiments, the elongate

multicellular bodies of non-human myocytes and non-human endothelial cells are of differing lengths. In various embodiments, the elongate multicellular bodies have a length of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mm. In various embodiments, the elongate multicellular bodies have a length of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cm. In some embodiments, the elongate

multicellular bodies have a length ranging from about 1 mm to about 10 cm. In further embodiments, the elongate multicellular bodies have a length ranging from about 1 cm to about 8 cm. In still further embodiments, the elongate multicellular bodies have a length ranging from about 2 cm to about 6 cm. In some embodiments, the methods of forming engineered meat disclosed herein comprise laying more than one layer adjacently onto a support substrate and allowing the layers to fuse to form engineered meat. In various embodiments, the meat comprises about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 layers. In some embodiments, the meat comprises about 10 to about 100 layers. In further embodiments, the meat comprises about 20 to about 80 layers. In still further embodiments, the meat comprises about 40 to about 60 layers. In some embodiments, the methods of forming engineered meat disclosed herein comprise preparing a plurality of multicellular bodies comprising a plurality of living non-human myocytes wherein the cells are adhered and/or cohered to one another, wherein the multicellular bodies have a diameter adapted to allow diffusion to sufficiently support the maintenance and growth of the non-human myocytes and non-human endothelial cells in culture. In various embodiments, the multicellular bodies have a diameter of about 100, 200, 300, 400, or 500 μιη. In some embodiments, the multicellular bodies have a diameter of about 100 μιη to about 500 μιη. In further embodiments, the multicellular bodies have a diameter of about 200 μιη to about 400 μιη. In some embodiments, the diameter applies to multicellular bodies with substantially rod or sphere shape. In some embodiments, the methods of forming engineered meat disclosed herein comprise preparing a plurality of multicellular bodies comprising a plurality of living non-human myocytes wherein the cells are adhered and/or cohered to one another, wherein the multicellular bodies are bioprinted.

BRIEF DESCRIPTION OF THE DRAWINGS

[025] Fig. 1 depicts a non-limiting example of a multicellular body; in this case, a multicellular body 1 with width Wl that is approximately equal to height HI and length LI that is

substantially greater than width Wl or height HI.

[026] Fig. 2 depicts a non-limiting example of a substantially spherical multicellular body; in this case, a substantially spherical multicellular body 2 with width Wl that is approximately equal to height HI.

[027] Fig. 3 depicts a non-limiting example of a multicellular body; in this case, a multicellular body 1 on a support substrate 3.

[028] Fig. 4 depicts a non-limiting example of a substantially spherical multicellular body; in this case, a substantially spherical multicellular body 2 on a support substrate 3.

[029] Fig. 5 depicts a non-limiting example of one method of making the multicellular bodies illustrated in Figs. 1-4; in this case, a method involving transferring a mixed cell pellet 4 into a capillary tube 5.

[030] Fig. 6 depicts a non-limiting example of a plurality of multicellular bodies; in this case, a plurality of multicellular bodies 1 laid adjacently onto a support substrate 3 such that they are allowed to fuse.

[031] Fig. 7 depicts a non-limiting example of a plurality of substantially spherical

multicellular bodies; in this case, a plurality of substantially spherical multicellular bodies 2 laid adjacently onto a support substrate 3 such that they are allowed to fuse.

[032] Fig. 8 depicts a non-limiting example of one method of making a layer comprising a plurality of multicellular bodies; in this case, a method involving extruding multicellular bodies 6 from a pressure-operated mechanical extruder comprising a capillary tube 5 onto a support substrate 3.

[033] Fig. 9 depicts a non-limiting example of one method of making engineered meat; in this case, a method involving laying more than one layer, comprising a plurality of multicellular bodies 7, 8, adjacently onto a support substrate 3.

[034] Fig. 10 depicts a non-limiting example of one method of making engineered meat; in this case, a method involving laying more than one layer, comprising a plurality of multicellular bodies 9 and a plurality of substantially spherical multicellular bodies 10, adjacently onto a support substrate 3.

[035] Fig. 11 depicts a non- limiting example of one method of making engineered meat; in this case, a method involving stacking more than one layer, wherein layers subsequent to the first are rotated 90 degrees with respect to the layer below.

DETAILED DESCRIPTION OF THE INVENTION

[036] Tissue engineered products made using traditional materials and methods are limited in size due to the short distances gases and nutrients can diffuse to nourish interior cells. Also, existing techniques fail to provide adequate speed and throughput for mass production of engineered products. As a result, existing tissue engineering methods, used to produce meat products, result in unappealing thin sheets and pastes on a commercially infeasible scale.

[037] Thus, an objective of the comestible meat products, layers, multicellular bodies, and methods of making the same disclosed herein is to provide commercially viable and appealing meat products. Another objective is to provide high-throughput methods that reliably, accurately, and reproducibly scale up to commercial levels. Advantages of the comestible meat products, layers, multicellular bodies, and methods of making the same disclosed herein include, but are not limited to, production of customized tissues in a reproducible, high throughput and easily scalable fashion while keeping precise control of pattern formation, particularly in cases of multiple cell types, which may result in engineered meat products with appealing flavor, texture, thickness, and appearance.

[038] Disclosed herein, in various embodiments, is engineered meat comprising a plurality of layers, wherein each layer comprises non-human myocytes, wherein the engineered meat is comestible and for ingestion. Also disclosed herein, in various embodiments, is a plurality of multicellular bodies comprising a plurality of living non-human myocytes wherein the cells are adhered and/or cohered to one another; wherein the multicellular bodies are arranged adjacently on a support substrate to form a substantially planar layer for use in formation of engineered, comestible meat.

[039] Also disclosed herein, in various embodiments, is a plurality of multicellular bodies comprising a plurality of living non-human myocytes wherein the cells are adhered and/or cohered to one another; wherein the multicellular bodies are arranged adjacently on a support substrate and maintained in culture to allow the multicellular bodies to fuse to form a

substantially planar layer for use in formation of engineered, comestible meat.

[040] Also disclosed herein, in various embodiments, is a plurality of multicellular bodies comprising a plurality of living non-human myocytes wherein the cells are adhered and/or cohered to one another; wherein the multicellular bodies are arranged adjacently on a support substrate and maintained in culture to allow the multicellular bodies to fuse to form a

substantially planar layer for use in formation of engineered, comestible meat; wherein the support substrate is permeable to fluids and nutrients and allows cell culture media to contact all surfaces of said layer.

[041] Also disclosed herein, in various embodiments, are methods of forming engineered meat, comprising: a) preparing a plurality of multicellular bodies comprising a plurality of living non- human myocytes wherein the cells are adhered and/or cohered to one another; b) laying more than one multicellular body adjacently onto a support substrate; c) allowing said multicellular bodies to fuse to form a layer; d) stacking more than one layer adjacently onto each other; e) allowing said layers to fuse to form engineered meat; and f) optionally, freezing said meat;

provided that the engineered meat is comestible and for ingestion. Also disclosed herein, in various embodiments, are methods of forming engineered meat , comprising: a) preparing a plurality of multicellular bodies comprising a plurality of non-human myocytes wherein the cells are adhered and/or cohered to one another; b) laying more than one multicellular body adjacently onto a support substrate; c) fusing said multicellular bodies to form a layer; d) laying more than one layer adjacently onto the first layer; and e) fusing said layers to form engineered meat; wherein the engineered meat is comestible. In some embodiments, the methods further comprise freezing said meat. [042] Also disclosed herein, in various embodiments, are methods of forming engineered meat, comprising: a) preparing a plurality of elongate multicellular bodies and/or a plurality of substantially spherical multicellular bodies comprising a plurality of living non-human myocytes wherein the cells are adhered and/or cohered to one another; b) laying more than one elongate multicellular body and more than one substantially spherical multicellular body adjacently onto a support substrate; c) allowing said multicellular bodies to fuse to form a layer; d) stacking more than one layer adjacently onto each other on a support substrate; e) allowing said layers to fuse to form engineered meat; and f) optionally, freezing said meat; provided that the engineered meat is comestible and for ingestion. Also disclosed herein, in various embodiments, are methods of forming engineered meat , comprising: a) preparing a plurality of elongate multicellular bodies and/or a plurality of substantially spherical multicellular bodies comprising a plurality of non-human myocytes wherein the cells are adhered and/or cohered to one another; b) laying more than one elongate multicellular body and more than one substantially spherical multicellular body adjacently onto a support substrate; c) fusing said multicellular bodies to form a layer; d) stacking more than one layer adjacently onto each other on a support substrate; and e) fusing said layers to form a volume of engineered meat; provided that the engineered meat is comestible. In some embodiments, the methods comprise laying more than one elongate multicellular body and more than one substantially spherical multicellular body in different ratios adjacently onto a support substrate. In some embodiments, the methods further comprise freezing said meat.

[043] Also disclosed herein, in various embodiments, are methods of forming engineered meat , comprising: a) preparing a plurality of substantially spherical multicellular bodies comprising a plurality of living non-human myocytes wherein the cells are adhered and/or cohered to one another; b) laying more than one substantially spherical multicellular body adjacently onto a support substrate; c) allowing said substantially spherical multicellular bodies to fuse to form a layer; d) laying more than one layer adjacently onto each other on a support substrate; e) allowing said layers to fuse to form engineered meat; and f) optionally, freezing said meat;

provided that the engineered meat is comestible and for ingestion. Also disclosed herein, in various embodiments, are methods of forming engineered meat , comprising: a) preparing a plurality of substantially spherical multicellular bodies comprising a plurality of non-human myocytes wherein the cells are adhered and/or cohered to one another; b) laying more than one substantially spherical multicellular body adjacently onto a support substrate; c) fusing said substantially spherical multicellular bodies to form a layer; d) stacking more than one layer adjacently onto each other on a support substrate; and e) fusing said layers to form engineered meat; provided that the engineered meat is comestible and for ingestion. In some embodiments, the methods further comprise freezing said meat.

[044] A basic idea underlying classical tissue engineering is to seed living cells into biocompatible and eventually biodegradable scaffold, and then culture the system in a bioreactor so that the initial cell population can expand into a tissue. Classical tissue engineering harbors several shortcomings, especially when applied to the production of meat products. First, the process of seeding cells generally involves contacting a solution of cells with a scaffold such that the cells are trapped within pores, fibers, or other micro structure of the scaffold. This process is substantially random with regard to the placement of cells within the scaffold and the placement of cells relative to each other. Therefore, seeded scaffolds are not immediately useful for production of three-dimensional constructs that exhibit planned or pre-determined placement or patterns of cells or cell aggregates. Second, selection of the ideal biomaterial scaffold for a given cell type is problematic and often accomplished by trial and error. Even if the right biomaterial is available, a scaffold can interfere with achieving high cell density. Moreover, scaffold-based tissue engineering does not easily or reliably scale up to industrial levels.

[045] In some embodiments, the engineered meat products, layers, and multicellular bodies, are made with a method that utilizes a rapid prototyping technology based on three-dimensional, automated, computer-aided deposition of multicellular bodies (e.g., cylinders and spheroids) and a biocompatible support structure (e.g., composed of agarose) by a three-dimensional delivery device (e.g., a bioprinter). The term "engineered," typically means man-made or arranged when used to refer to the layers and the meat products described herein. One example of an engineered meat may include arranging or placing multicellular bodies and/or layers to form engineered meat products by a computer-aided device (e.g., a bioprinter) according to a computer script. In further embodiments, the computer script is, for example, one or more computer programs, computer applications, or computer modules. In still further embodiments, three-dimensional tissue structures form through the post-printing fusion of the multicellular bodies similar to self-assembly phenomena in early morphogenesis.

[046] Unlike other engineered tissues, the engineered meat described herein if formed by stacking layers of two-dimensional planar sheets of at least partially fused multicellular bodies. Thus, methods for forming even large volumes of engineered meat described herein may not require simultaneous three dimensional patterning, but may be performed by culturing (in parallel) multiple two-dimensional layers that may be later assembled into a three-dimensional assembly, or sub-assemblies that can then be stacked together. This advantageous method of forming the engineered meats described herein may permit the volume of engineered meat to be formed without requiring the need for scaffolding or three-dimensional support structures, such as filler bodies. Further, the two-dimensional layers may be formed in parallel at a relatively thin thickness that allows for diffusion of nutrients from a culture medium into the planar layer during culture (e.g., while fusing the component multicellular bodies into the layer). It is only after the component layers are stacked to form the volume that diffusion of nutrients may be limiting, resulting in cell death.

[047] Thus, while a number of methods are available to arrange the multicellular bodies on a support substrate to produce a three-dimensional structure including manual placement, including positioning by an automated, computer-aided machine such as a bioprinter, such methods may be useful but are not required. Advantages of delivery of multicellular bodies with bioprinter technology include rapid, accurate, and reproducible placement of multicellular bodies to produce constructs exhibiting planned or pre-determined orientations or patterns of multicellular bodies and/or layers of various compositions. Advantages also include assured high cell density, while minimizing cell damage often associated with other solid freeform

fabrication-based deposition methods focused on printing cells in combination with hydrogels.

[048] The embodiments disclosed herein include methods of manufacture or making of engineered meats, and may also include business methods. In some embodiments, the speed and scalability of the techniques and methods disclosed herein are utilized to design, build, and operate industrial and/or commercial facilities for the production of comestible, engineered meat products. In further embodiments, the engineered meat products are produced, packaged, frozen, stored, distributed, marketed, advertised, and sold as, for example, food products for human beings, components or ingredients of food products for human beings, food products for non-human animals, or components or ingredients of food products for non-human animals.

Cells

[049] Many self-adhering cell types may be used to form the multicellular bodies, layers, and engineered meat products described herein. In some embodiments, the engineered meat products are designed to resemble traditional meat products and the cell types are chosen to approximate those found in traditional meat products. In further embodiments, the engineered meat products, layers, and multicellular bodies include non-human myocytes. In still further embodiments, the engineered meat products, layers, and multicellular bodies include non-human myocytes, and/or endothelial cells, and/or adipose cells, and/or fibroblasts.

[050] In general, the engineered meats described herein may differ from natural meats and other engineered meats by lacking blood vessels, and also lacking in nerve enervation. Even in variations in which endothelial cells are included as a component of one or more multicellular body, the engineered meat will not include blood vessels competent to transmit blood. Thus, even the large volumes of engineered meat formed by the methods described herein may not have any blood vessels. Further, the engineered meats described herein may lack any nerve compnents (e.g., axons, dendrites, nerve cell bodies), as they may be gown without such componenets.

[051] Human beings traditionally eat several types of animal muscle tissue. Therefore, in some embodiments, the myocytes are skeletal myocytes. In some embodiments, the myocytes are cardiac myocytes. In some embodiments, the myocytes are smooth myocytes. In some embodiments, the endothelial cells are microvascular endothelial cells.

[052] In other embodiments, the engineered meat products include neural cells, connective tissue (including bone, cartilage, cells differentiating into bone forming cells and chondrocytes, and lymph tissues), epithelial cells (including endothelial cells that form linings in cavities and vessels or channels, exocrine secretory epithelial cells, epithelial absorptive cells, keratinizing epithelial cells, and extracellular matrix secretion cells), and undifferentiated cells (such as embryonic cells, stem cells, and other precursor cells), among others.

[053] In some embodiments, the cells used to form a multicellular body are obtained from a live animal and cultured as a primary cell line. For example, in further embodiments, the cells are obtained by biopsy and cultured ex vivo. In other embodiments, the cells are obtained from commercial sources.

[054] The engineered meat products and the layers comprising a plurality of multicellular bodies for use in production of said meat disclosed herein are comestible and intended for consumption by human beings, non-human animals, or both. In some embodiments, the engineered meat products are human food products. In other embodiments, the engineered meat products are animal feed such as feed for livestock, feed for aquaculture, or feed for domestic pets. Therefore, in light of the disclosure provided herein, those of skill in the art will recognize that non-human cells from a plethora of sources are suitable for use in production of such products and with the methods disclosed herein. In various embodiments, the multicellular bodies, layers comprising multicellular bodies, and engineered meat products comprise non- human cells derived from, by way of non-limiting examples, mammals, birds, reptiles, fish, crustaceans, mo Husks, cephalopods, insects, non-arthropod invertebrates, and combinations thereof.

[055] In some embodiments, suitable cells are derived from mammals such as antelope, bear, beaver, bison, boar, camel, caribou, cattle, deer, elephant, elk, fox, giraffe, goat, hare, horse, ibex, kangaroo, lion, llama, moose, peccary, pig, rabbit, seal, sheep, squirrel, tiger, whale, yak, and zebra, or combinations thereof. In some embodiments, suitable cells are derived from birds such as chicken, duck, emu, goose, grouse, ostrich, pheasant, pigeon, quail, and turkey, or combinations thereof. In some embodiments, suitable cells are derived from reptiles such as turtle, snake, crocodile, and alligator, or combinations thereof. In some embodiments, suitable cells are derived from fish such as anchovy, bass, catfish, carp, cod, eel, flounder, fugu, grouper, haddock, halibut, herring, mackerel, mahi mahi, marlin, orange roughy, perch, pike, pollock, salmon, sardine, shark, snapper, sole, swordfish, tilapia, trout, tuna, and walleye, or

combinations thereof. In some embodiments, suitable cells are derived from crustaceans such as crab, crayfish, lobster, prawn, and shrimp, or combinations thereof. In some embodiments, suitable cells are derived from mo Husks such as abalone, clam, conch, mussel, oyster, scallop, and snail, or combinations thereof. In some embodiments, suitable cells are derived from cephalopods such as cuttlefish, octopus, and squid, or combinations thereof. In some

embodiments, suitable cells are derived from insects such as ants, bees, beetles, butterflies, cockroaches, crickets, damselflies, dragonflies, earwigs, fleas, flies, grasshoppers, mantids, mayflies, moths, silverfish, termites, wasps, or combinations thereof. In some embodiments, suitable cells are derived from non-arthropod invertebrates (e.g., worms) such as flatworms, tapeworms, flukes, threadworms, roundworms, hookworms, segmented worms (e.g., earthworms, bristle worms, etc.), or combinations thereof.

Multicellular bodies

[056] Disclosed herein are multicellular bodies including a plurality of living non-human cells wherein the cells are adhered and/or cohered to one another. Also disclosed herein are methods comprising: preparing a plurality of multicellular bodies comprising a plurality of living non- human myocytes wherein the cells are adhered and/or cohered to one another; laying more than one multicellular body adjacently onto a support substrate; and allowing the multicellular bodies to fuse to form a substantially planar layer for used in forming engineered meat. In some embodiments, a multicellular body comprises a plurality of cells adhered and/or cohered together in a desired three-dimensional shape with viscoelastic consistency and sufficient integrity for easy manipulation and handling during a bioengineering process, such as tissue engineering. In some embodiments, sufficient integrity means that the multicellular body, during the subsequent handling, is capable of retaining its physical shape, which is not rigid, but has a viscoelastic consistency, and maintaining the vitality of the cells.

[057] In some embodiments, a multicellular body is homocellular. In other embodiments, a multicellular body is hetero cellular. In homocellular multicellular bodies, the plurality of living cells includes a plurality of living cells of a single cell type. Substantially all of the living cells in a homocellular multicellular body are substantially cells of the single cell type. In contrast, a hetero cellular multicellular body includes significant numbers of cells of more than one cell type. The living cells in a hetero cellular body may remain unsorted or can "sort out" (e.g., self- assemble) during the fusion process to form a particular internal structure for the engineered tissue. The sorting of cells is consistent with the predictions of the Differential Adhesion Hypothesis (DAH). The DAH explains the liquid-like behavior of cell populations in terms of tissue surface and interfacial tensions generated by adhesive and cohesive interactions between the component cells. In general, cells can sort based on differences in the adhesive strengths of the cells. For example, cell types that sort to the interior of a heterocellular multicellular body generally have a stronger adhesion strength (and thus higher surface tension) than cells that sort to the outside of the multicellular body.

[058] In some embodiments, the multicellular bodies disclosed herein also include one or more extracellular matrix (ECM) components or one or more derivatives of one or more ECM components in addition to the plurality of cells. For example, the multicellular bodies may contain various ECM proteins including, by way of non-limiting examples, gelatin, fibrinogen, fibrin, collagen, fibronectin, laminin, elastin, and proteoglycans. The ECM components or derivatives of ECM components can be added to a cell paste used to form a multicellular body. The ECM components or derivatives of ECM components added to a cell paste can be purified from an animal source, or produced by recombinant methods known in the art. Alternatively, the ECM components or derivatives of ECM components can be naturally secreted by the cells in the multicellular body.

[059] In some embodiments, a multicellular body includes tissue culture medium. In further embodiments, the tissue culture medium can be any physiologically compatible medium and will typically be chosen according to the cell type(s) involved as is known in the art. In some cases, suitable tissue culture medium comprises, for example, basic nutrients such as sugars and amino acids, growth factors, antibiotics (to minimize contamination), etc.

[060] The adhesion and/or cohesion of the cells in a multicellular body is suitably sufficiently strong to allow the multicellular body to retain a three-dimensional shape while supporting itself on a flat surface. For instance, in some cases, a multicellular body supporting itself on a flat substrate may exhibit some minor deformation (e.g., where the multicellular body contacts the surface), however, the multicellular body is sufficiently cohesive to retain a height that is at least one half its width, and in some cases, about equal to the width. In some embodiments, two or more multicellular bodies placed in side-by-side adjoining relation to one another on a flat substrate form a void space under their sides and above the work surface. See, e.g., Figs. 3 and 4. In further embodiments, the cohesion of the cells in a multicellular body is sufficiently strong to allow the multicellular body to support the weight of at least one similarly sized and shaped multicellular body when the multicellular body is assembled in a construct in which the multicellular bodies are stacked on top of one another. See, e.g., Figs. 9 and 10. In still further embodiments, the adhesion and/or cohesion of the cells in a multicellular body is also suitably sufficiently strong to allow the multicellular body to be picked up by an implement (e.g., a capillary micropipette).

[061] In light of the disclosure provided herein, those of skill in the art will recognize that multicellular bodies having different sizes and shapes are within the scope of the embodiments provided herein. In some embodiments, a multicellular body is substantially cylindrical and has a substantially circular cross section. For example, a multicellular body, in various

embodiments, has an elongate shape (e.g., a cylindrical shape) with a square, rectangular, triangular, or other non-circular cross-sectional shape. Likewise, in various embodiments, a multicellular body has a generally spherical shape, a non-elongate cylindrical shape, or a cuboidal shape.

[062] In various embodiments, the diameter of a multicellular body is about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 μιη, or quantifiable increments therein. In some embodiments, a multicellular body is configured to limit cell necrosis caused by inability of oxygen and/or nutrients to diffuse into central portions of the multicellular body. For example, a multicellular body is suitably configured such that none of the living cells therein is more than about 250 μιη from an exterior surface of the multicellular body, and more suitably so none of the living cells therein is more than about 200 μιη from an exterior of the multicellular body.

[063] In some embodiments, the multicellular bodies have differing lengths. In other embodiments, multicellular bodies are of substantially similar lengths. In various embodiments, the length of a multicellular body is about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10 mm, or quantifiable increments therein. In other various embodiments, the length of a multicellular body is about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10 cm, or quantifiable increments therein. In some embodiments, the length of multicellular bodies is chosen to result in a shape and/or size of engineered meat product that approximates that of a traditional meat product, for example, a strip of bacon, a hamburger patty, a fish fillet, a chicken breast, or a steak.

[064] Referring to Fig. 1, in some embodiments, a multicellular body 1 is substantially cylindrical with a width Wl roughly equal to a height HI and has a substantially circular cross section. In further embodiments, a multicellular body 1 is elongate with a length of LI. In still further embodiments, Wl and HI are suitably about 300 to about 600 μιη and LI is suitably about 2 cm to about 6 cm.

[065] Referring to Fig. 2, in some embodiments, a multicellular body 2 is substantially spherical with a width Wl roughly equal to a height HI. In further embodiments, Wl and HI are suitably about 300 to about 600 μιη.

Layers

[066] The engineered meat disclosed herein, includes a plurality of layers. In some

embodiments, a layer includes a plurality of multicellular bodies comprising a plurality of living non-human cells wherein the cells are adhered and/or cohered to one another. Also disclosed herein are methods comprising the steps of laying multicellular bodies adjacently onto a support substrate and allowing the multicellular bodies to fuse to form a substantially planar layer for use in formation of engineered comestible meat products. In some embodiments, each layer is bioprinted, using techniques described herein.

[067] In some embodiments, a layer includes homocellular multicellular bodies. In other embodiments, a layer includes heterocellular multicellular bodies. In yet other embodiments, a layer includes both homocellular and heterocellular multicellular bodies. In further

embodiments, a layer includes non-human myocytes. In still further embodiments, a layer includes non-human myocytes, non-human endothelial cells, and adipose cells and/or fibroblast cells. In still further embodiments, a layer includes non-human myocytes, non-human

endothelial cells, and other cell types disclosed herein.

[068] In embodiments including both non-human myocytes and non-human endothelial cells, a layer may include non-human myocytes and non-human endothelial cells in a ratio of about 30: 1, 29: 1, 28: 1, 27:1, 26: 1, 25: 1, 24: 1, 23: 1, 22: 1, 21 : 1, 20: 1, 19: 1, 18: 1, 17: 1, 16: 1, 15: 1, 14: 1, 13: 1, 12: 1, 11 : 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, and 1 : 1, or increments therein. In some embodiments, a layer contains non-human myocytes and non-human endothelial cells in a ratio of about 19: 1 to about 3: 1. In various embodiments, a layer includes non-human

endothelial cells that comprise about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, and 25%, or increments therein, of the total cell population. In some embodiments, a layer includes non-human endothelial cells that comprise about 5% to about 15% of the total cell population. In further embodiments, the presence of endothelial cells contributes to endothelialization, described further herein.

[069] In various embodiments, the thickness of each layer is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, or 5000 μιη, or quantifiable increments therein. In some embodiments, the thickness of each layer is chosen to allow diffusion to sufficiently support the maintenance and growth of substantially all the cells in the layer in culture.

[070] In various embodiments, the plurality of layers includes about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 layers, or increments therein. In some embodiments, the number of layers is chosen to result in an engineered meat product with thickness that approximates that of a traditional meat product, for example, a strip of bacon, a hamburger patty, a fish fillet, a chicken breast, or a steak.

[071] In some embodiments, the engineered layers are designed to resemble traditional meat products and design parameters (e.g., cell types, additives, size, shape, etc.) are chosen to approximate those found in traditional meat products. In further embodiments, a layer is characterized by a nutritional composition that is substantially similar to traditional meat products. In still further embodiments, a layer is characterized by a nutritional composition that is substantially 60-80 percent aqueous fluid, 14-35 percent protein, 1-25 percent fat, 1-5 percent carbohydrates and 1-5 percent other substances. In some embodiments, myocytes of the engineered layers or endothelialized meat are aligned. In some embodiments, myocytes are aligned by application of an electrical field as is known in the art. In some embodiments, myocytes are aligned by application of a mechanical stimulus, such as cyclical stretching and relaxing the substratum, as is known in the art. In further embodiments, aligned (e.g., electro- oriented and mechano -oriented) myocytes have substantially the same orientation with regard to each other as is found in many animal muscle tissues. In some embodiments, layers of multicellular bodies provided herein are exposed to electrical and/or mechanical stimulation to facilitate the formation of physiological arrangement of muscle cells.

Additives

[072] In some embodiments, the engineered meat products, engineered layers, and/or multicellular bodies include one or more nutritional supplements. In further embodiments, one or more nutritional supplements are selected from: vitamins, minerals, fiber, fatty acids, and amino acids. In some embodiments, the engineered meat products, layers, and/or multicellular bodies include one or more additives to enhance the commercial appeal (e.g., appearance, taste, color, odor, etc.). In further embodiments, the engineered meat products, layers, and/or multicellular bodies include one or more flavorants, one or more colorants, and/or one or more odorants.

[073] In some embodiments, the engineered meat products, engineered layers, and/or multicellular bodies include one or more of: matrix proteins, proteoglycans, antioxidants, perfluorocarbons, and growth factors. The term "growth factor," as used herein, refers to a protein, a polypeptide, or a complex of polypeptides, including cytokines, that are produced by a cell and which can affect itself and/or a variety of other neighboring or distant cells. Typically growth factors affect the growth and/or differentiation of specific types of cells, either developmentally or in response to a multitude of physiological or environmental stimuli. Some, but not all, growth factors are hormones. Exemplary growth factors are insulin, insulin- like growth factor (IGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), fibroblast growth factors (FGFs), including basic FGF (bFGF), platelet-derived growth factors (PDGFs), including PDGF-AA and PDGF-AB, hepatocyte growth factor (HGF), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-β), including TGFpi and TGFP3, epidermal growth factor (EGF), granulocyte-macrophage colony- stimulating factor (GM-CSF), granulocyte colony- stimulating factor (G-CSF), interleukin-6 (IL-6), IL-8, and the like.

[074] In some embodiments, the engineered meat products, engineered layers, and/or multicellular bodies include one or more food preservatives known to the art. In some embodiments, the preservatives are antimicrobial preservatives including, by way of non- limiting examples, calcium propionate, sodium nitrate, sodium nitrite, sulfites (e.g., sulfur dioxide, sodium bisulfite, potassium hydrogen sulfite, etc.) and disodium

ethylenediaminetetraacetic acid (EDTA). In some embodiments, the preservatives are antioxidant preservatives including, by way of non-limiting examples, butylated hydro xyanisole (BHA) and butylated hydroxytoluene (BHT).

Support substrate

[075] Disclosed herein, in some embodiments, is a plurality of multicellular bodies arranged adjacently on a support substrate to form a substantially planar layer for use in formation of engineered comestible meat. Also disclosed herein, in some embodiments, are methods comprising arranging multicellular bodies adjacently on a support substrate to form substantially planar layers, laying more than one layer adjacently onto a single support substrate, and allowing the layers to fuse to form engineered meat. For example, a plurality of layers may be formed as described above at the same time on different substrates then removed from their substrate when the multicellular bodies have fused sufficiently to allow them to be removed and stacked atop one another or atop a single substrate.

[076] In general, each layer includes non-human myocytes . The cells in the central portions of such constructs are typically supplied with oxygen and nutrients by diffusion; however, gasses and nutrients typically diffuse approximately 200-300 microns into three-dimensional cellular constructs.

[077] In some embodiments, the multicellular bodies disclosed herein have a diameter adapted to allow diffusion to sufficiently support the maintenance and growth of said non-human myocytes in culture. As a result, in further embodiments, the layers disclosed herein have a thickness adapted to allow diffusion to sufficiently support the maintenance and growth of said non-human myocytes in culture.

[078] To facilitate and enhance diffusion, in some embodiments, a support substrate is permeable to fluids, gasses, and nutrients and allows cell culture media to contact all surfaces of multicellular bodies and/or layers during, for example, growth, maturation, and fusion. In various embodiments, a support substrate is made from natural biomaterials, synthetic biomaterials, and combinations thereof. In some embodiments, natural biomaterials include, by way of non-limiting examples, collagen, fibronectin, laminin, and other extracellular matrices. In some embodiments, synthetic biomaterials may include, by way of non-limiting examples, hydroxyapatite, alginate, agarose, polyglycolic acid, polylactic acid, and their copolymers. In some embodiments, a support substrate is solid. In some embodiments, a support substrate is semisolid. In further embodiments, a support substrate is a combination of solid and semisolid support elements.

[079] In some embodiments, the support substrate is raised or elevated above a non-permeable surface, such as a portion of a cell culture environment (e.g., a Petri dish, a cell culture flask, etc.) or a bioreactor. In still further embodiments, an elevated support substrate further facilitates circulation of cell culture media and enhances contact with all surfaces of the multicellular bodies and/or layers.

Methods of forming multicellular bodies

[080] There are various ways to make multicellular bodies having the characteristics described herein. In some embodiments, a multicellular body can be fabricated from a cell paste containing a plurality of living cells or with a desired cell density and viscosity. In further embodiments, the cell paste can be shaped into a desired shape and a multicellular body formed through maturation (e.g., incubation). In a particular embodiment, a multicellular body is produced by shaping a cell paste including a plurality of living cells into a desired shape (e.g., a cylinder, a sphere). In further embodiments, the cell paste is incubated in a controlled environment to allow the cells to adhere and/or cohere to one another to form the multicellular body. In another particular embodiment, a multicellular body is produced by shaping a cell paste including a plurality of living cells in a device that holds the cell paste in a three-dimensional shape. In further embodiments, the cell paste is incubated in a controlled environment while it is held in the three dimensional shape for a sufficient time to produce a body that has sufficient cohesion to support itself on a flat surface, as described herein.

[081] In various embodiments, a cell paste is provided by: (A) mixing cells or cell aggregates (of one or more cell types) and a cell culture medium (e.g., in a pre-determined ratio) to result in a cell suspension, and (B) compacting the cellular suspension to produce a cell paste with a desired cell density and viscosity. In various embodiments, compacting is achieved by a number of methods, such as by concentrating a particular cell suspension that resulted from cell culture to achieve the desired cell concentration (density), viscosity, and consistency required for the cell paste. In a particular embodiment, a relatively dilute cell suspension from cell culture is centrifuged for a determined time to achieve a cell concentration in the pellet that allows shaping in a mold. Tangential flow filtration ("TFF") is another suitable method of concentrating or compacting the cells. In some embodiments, compounds are combined with the cell suspension to lend the extrusion properties required. Suitable compounds include, by way of non-limiting examples, collagen, hydrogels, Matrigel, nanofibers, self-assembling nano fibers, gelatin, fibrinogen, etc.

[082] In some embodiments, the cell paste is produced by mixing a plurality of living cells with a tissue culture medium, and compacting the living cells (e.g., by centrifugation). One or more ECM component (or derivative of an ECM component) is optionally included by, resuspending the cell pellet in one or more physiologically acceptable buffers containing the ECM component(s) (or derivative(s) of ECM component(s)) and the resulting cell suspension centrifuged again to form the cell paste.

[083] In some embodiments, the cell density of the cell paste desired for further processing may vary with cell types. In further embodiments, interactions between cells determine the properties of the cell paste, and different cell types will have a different relationship between cell density and cell-cell interaction. In still further embodiments, the cells may be pre-treated to increase cellular interactions before shaping the cell paste. For example, cells may be incubated inside a centrifuge tube after centrifugation in order to enhance cell-cell interactions prior to shaping the cell paste.

[084] In various embodiments, many methods are used to shape the cell paste. For example, in a particular embodiment, the cell paste is manually molded or pressed (e.g., after

concentration/compaction) to achieve a desired shape. By way of a further example, the cell paste may be taken up (e.g., aspirated) into a preformed instrument, such as a micropipette (e.g., a capillary pipette), that shapes the cell paste to conform to an interior surface of the instrument. The cross sectional shape of the micropipette (e.g., capillary pipette) is alternatively circular, square, rectangular, triangular, or other non-circular cross-sectional shape. In some

embodiments, the cell paste is shaped by depositing it into a preformed mold, such as a plastic mold, metal mold, or a gel mold. In some embodiments, centrifugal casting or continuous casting is used to shape the cell paste. [085] Referring to Fig. 5, in a particular example, the shaping includes retaining the cell paste 4 in a shaping device 5 (e.g., a capillary pipette) to allow the cells to partially adhere and/or cohere to one another in the shaping device. By way of further example, cell paste can be aspirated into a shaping device and held in the shaping device for a maturation period (also referred to herein as an incubation period) to allow the cells to at least partially adhere and/or cohere to one another. In some embodiments, the shaping device (e.g., capillary pipette) is part of a printing head of a bioprinter or similar apparatus operable to automatically place the multicellular body in a three-dimensional construct. However, there is a limit to the amount of time cells can remain in a shaping device such as a capillary pipette, which provides the cells only limited access at best to oxygen and/or nutrients, before viability of the cells is impacted.

[086] In some embodiments, a partially adhered and/or cohered cell paste is transferred from the shaping device (e.g., capillary pipette) to a second shaping device (e.g., a mold) that allows nutrients and/or oxygen to be supplied to the cells while they are retained in the second shaping device for an additional maturation period. One example of a suitable shaping device that allows the cells to be supplied with nutrients and oxygen is a mold for producing a plurality of multicellular bodies (e.g., substantially identical multicellular bodies). By way of further example, such a mold includes a biocompatible substrate made of a material that is resistant to migration and ingrowth of cells into the substrate and resistant to adherence of cells to the substrate. In various embodiments, the substrate can suitably be made of Teflon®, (PTFE), stainless steel, agarose, polyethylene glycol, glass, metal, plastic, or gel materials (e.g., agarose gel or other hydrogel), and similar materials. In some embodiments, the mold is also suitably configured to allow supplying tissue culture media to the cell paste (e.g., by dispensing tissue culture media onto the top of the mold).

[087] In a particular embodiment, a plurality of elongate grooves is formed in the substrate. In a further particular embodiment, the depth of each groove is in the range of about 500 microns to about 1000 microns and the bottom of each groove has a semicircular cross-sectional shape for forming elongate cylindrical multicellular bodies that have a substantially circular cross- sectional shape. In a further particular embodiment, the width of the grooves is suitably slightly larger than the width of the multicellular body to be produced in the mold. For example, the width of the grooves is suitably in the range of about 300 microns to about 1000 microns.

[088] Thus, in embodiments where a second shaping device is used, the partially adhered and/or cohered cell paste is transferred from the first shaping device (e.g., a capillary pipette) to the second shaping device (e.g., a mold). In further embodiments, the partially adhered and/or cohered cell paste can be transferred by the first shaping device (e.g., the capillary pipette) into the grooves of a mold. In still further embodiments, following a maturation period in which the mold is incubated along with the cell paste retained therein in a controlled environment to allow the cells in the cell paste to further adhere and/or cohere to one another to form the multicellular body, the cohesion of the cells will be sufficiently strong to allow the resulting multicellular body to be picked up with an implement (e.g., a capillary pipette). In still further embodiments, the capillary pipette is suitably be part of a printing head of a bioprinter or similar apparatus operable to automatically place the multicellular body into a three-dimensional construct.

[089] In some embodiments, the cross-sectional shape and size of the multicellular bodies will substantially correspond to the cross-sectional shapes and sizes of the first shaping device and optionally the second shaping device used to make the multicellular bodies, and the skilled artisan will be able to select suitable shaping devices having suitable cross-sectional shapes, cross-sectional areas, diameters, and lengths suitable for creating multicellular bodies having the cross-sectional shapes, cross-sectional areas, diameters, and lengths discussed above.

[090] As discussed herein, a large variety of cell types may be used to create the multicellular bodies of the present embodiments. Thus, one or more types of cells or cell aggregates including, for example, all of the cell types listed herein, may be employed as the starting materials to create the cell paste. For instance, cells such as non-human myocytes, endothelial cells, adipose cells, and fibroblasts are optionally employed. As described herein, a multicellular body is homocellular or heterocellular. For making homocellular multicellular bodies, the cell paste suitably is homocellular, i.e., it includes a plurality of living cells of a single cell type. For making heterocellular multicellular bodies, on the other hand, the cell paste will suitably include significant numbers of cells of more than one cell type (i.e., the cell paste will be heterocellular). As described herein, when heterocellular cell paste is used to create the multicellular bodies, the living cells may "sort out" during the maturation and cohesion process based on differences in the adhesive strengths of the cells, and may recover their physiological conformation.

[091] In some embodiments, in addition to the plurality of living cells, one or more ECM components or one or more derivatives of one or more ECM components (e.g., gelatin, fibrinogen, collagen, fibronectin, laminin, elastin, and/or proteoglycans) can suitably be included in the cell paste to incorporate these substances into the multicellular bodies, as noted herein. In further embodiments, adding ECM components or derivatives of ECM components to the cell paste may promote cohesion of the cells in the multicellular body. For example, gelatin and/or fibrinogen are optionally added to the cell paste. More particularly, a solution of 10-30% gelatin and a solution of 10-80 mg/ml fibrinogen are optionally mixed with a plurality of living cells to form a cell suspension containing gelatin and fibrinogen.

[092] Various methods are suitable to facilitate the further maturation process. In one embodiment, the cell paste may be incubated at about 37°C for a time period (which may be cell-type dependent) to foster adherence and/or coherence. Alternatively or in addition, the cell paste may be held in the presence of cell culture medium containing factors and/or ions to foster adherence and/or coherence.

Arranging multicellular bodies on a support substrate to form layers

[093] A number of methods are suitable to arrange multicellular bodies on a support substrate to produce a desired three-dimensional structure (e.g., a substantially planar layer). For example, in some embodiments, the multicellular bodies are manually placed in contact with one another, deposited in place by extrusion from a pipette, nozzle, or needle, or positioned in contact by an automated machine such as a bioprinter.

[094] As described herein, in some embodiments, the support substrate is permeable to fluids, gasses, and nutrients and allows cell culture media to contact all surfaces of the multicellular bodies and/or layers during arrangement and subsequent fusion. As further described herein, in some embodiments, a support substrate is made from natural biomaterials such as collagen, fibronectin, laminin, and other extracellular matrices. In some embodiments, a support substrate is made from synthetic biomaterials such as hydroxyapatite, alginate, agarose, polyglycohc acid, polylactic acid, and their copolymers. In some embodiments, a support substrate is solid. In some embodiments, a support substrate is semisolid. In further embodiments, a support substrate is a combination of solid and semisolid support elements. In further embodiments, a support substrate is planar to facilitate production of planar layers. In some embodiments, the support substrate is raised or elevated above a non-permeable surface, such as a portion of a cell culture environment (e.g., a Petri dish, a cell culture flask, etc.) or a bioreactor. Therefore, in some embodiments, a permeable, elevated support substrate contributes to prevention of premature cell death, contributes to enhancement of cell growth, and facilitates fusion of multicellular bodies to form layers.

[095] As described herein, in various embodiments, multicellular bodies have many shapes and sizes. In some embodiments, multicellular bodies are elongate and in the shape of a cylinder. See e.g., Figs. 1 and 3. In some embodiments, multicellular bodies provided herein are of similar lengths and/or diameters. In other embodiments, multicellular bodies provided herein are of differing lengths and/or diameters. In some embodiments, multicellular bodies are substantially spherical. See e.g., Figs. 2 and 4. In some embodiments, layers include substantially spherical multicellular bodies that are substantially similar in size. In other embodiments, layers include substantially spherical multicellular bodies that are of differing sizes.

[096] Referring to Fig. 6, in some embodiments, multicellular bodies 1 are arranged on a support substrate 3 horizontally adjacent to, and in contact with, one or more other multicellular bodies to form a substantially planar layer.

[097] Referring to Fig. 7, in some embodiments, substantially spherical multicellular bodies 2 are arranged on a support substrate 3 horizontally adjacent to, and in contact with, one or more other substantially spherical multicellular bodies. In further embodiments, this process is repeated to build up a pattern of substantially spherical multicellular bodies, such as a grid, to form a substantially planar layer.

[098] Referring to