RU2778255C2 - Compositions out of cultured meat - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: present invention relates to the field of cell biology and the food industry, in particular to a method for obtaining a usable composition and a composition obtained using this method. To implement the method, many cell types of a non-human animal and a three-dimensional porous matrix suitable for human consumption are first incubated. After that, differentiation of myoblasts or their progenitor cells is induced into muscle tubules. Moreover, the specified set of cell types contains myoblasts or their progenitor cells, including satellite cells, and secreting extracellular matrix (SEM) cells, including fibroblasts, as well as, optionally, a small number of endothelial cells or their progenitor cells, no more than 15% of the specified set.

EFFECT: present invention makes it possible to improve the survival, proliferation and formation of muscle tubules when obtaining a composition suitable for consumption, a product out of cultured meat.

24 cl, 167 dwg, 3 tbl, 15 ex

Description

CROSS REFERENCE

[001] This application claims priority under U.S. Provisional Application No. 62/532,998, filed July 15, 2017, the contents of which are incorporated herein by reference in their entirety.

FIELD OF TECHNOLOGY

[002] The present invention relates, inter alia, to cultured meat compositions and methods for their preparation.

BACKGROUND OF THE INVENTION

негативное воздействие на окружающую среду по сравнению с продуктами растительного происхождения с точки зрения потребности в почве и воде, а также выбросов парниковых газов (ПГ). Согласно докладу Продовольственной и сельскохозяйственной организации Объединенных Наций, сектор животноводства отвечает за 18% выбросов ПГ, использует 30% территории земли или 70% пахотных земель, и 8% мировой пресной воды. В дополнение ожидается, что к 2050 году мировой спрос на мясо удвоится, что означает, что традиционные системы получения мяса небезопасны экологически и не рассчитаны на долгую перспективу. По сравнению с несколькими источниками мяса, культивируемое мясо, по оценкам, уменьшает потребление энергии на 7-45%, выбросы ПГ на 78-96%, использование земли на 99% и использование воды на 82-96%.[003] Cultured meat, also known as artificial (synthetic) or pure meat, is obtained from cell culture using tissue engineering methods, it is a significant alternative to traditional meat production using live animals. In the past decade, this discovery has received increased attention in public opinion, popular media, investors and the scientific community, especially after the first laboratory-cultivated beef burger. Animal-derived food is considered unsustainable because animals consume large amounts of food throughout their lives, with 80-90% of the calories expended on the animal's metabolism and tissue production. When comparing different industries, beef has the highest environmental impact, but in general, all animal products have

negative environmental impact compared to plant-based products in terms of soil and water requirements, as well as greenhouse gas (GHG) emissions. According to a report by the Food and Agriculture Organization of the United Nations, the livestock sector is responsible for 18% of GHG emissions, uses 30% of the land or 70% of arable land, and 8% of the world's fresh water. In addition, global demand for meat is expected to double by 2050, which means traditional meat production systems are not sustainable and sustainable. Compared to multiple sources of meat, cultured meat is estimated to reduce energy consumption by 7-45%, GHG emissions by 78-96%, land use by 99% and water use by 82-96%.

[004] Intensive industrial farming and poor animal welfare are the cause of foodborne diseases such as swine and bird flu, as well as the spread of E. coli, Salmonella and Campylobacter, which can be found in meat. Obtaining meat in a sterile controlled environment can improve food safety. In addition, 70% of all antibiotics used in the United States are given to farm animals as a food additive that promotes the selection of antimicrobial-resistant strains and increases the likelihood of multidrug-resistant bacteria. Antibiotic abuse is a key cause of antibiotic-resistant bacteria, which in the US alone is blamed for $55 billion in economic losses, 2 million infections, 250,000 hospitalizations, and at least 23,000 deaths a year. Bacteria resistant to colistin, the latest antibiotic drug, have recently emerged on Chinese pig farms.

[005] Current meat culture technology focuses on satellite cell culture. Cells are grown in two-dimensional (2D) balloons or on microcarriers in suspension, isolated, differentiated and harvested. However, tissues are not exclusively composed of cells; most of the tissue is the extracellular matrix (ECM), which consists of macromolecules such as glycoproteins and oligosaccharides and gives the tissue its biochemical and biomechanical properties. ECM regulates the behavior of cells and influences their composition. Thus, ECM-producing cells are essential for cultured meat and processes such as cell harvesting should be avoided. In addition, three-dimensional (3D) cell culture mimics the natural cellular environment and is critical for proper cell behavior, which affects the biochemical content of cells.

BRIEF DESCRIPTION OF THE INVENTION

[006] In some embodiments, the present invention relates to edible compositions containing myotubes and to a method for preparing edible compositions.

[007] The invention is based in part on the findings that a cell culture containing multiple cell types (e.g., satellite cells, ECM-secreting cells, and endothelial cells) showed better survival, proliferation, and myotube formation compared to a control group, e.g. , when grown on three-dimensional porous matrices.

[008] The invention is further based in part on the unexpected findings that satellite cells, such as non-human satellite cells, had better differential activity when co-cultured with low concentration of endothelial cells (EC) instead of high concentration of EC.

[009] In accordance with one aspect, a method for preparing an edible composition is provided, comprising the steps of: (a) incubating a three-dimensional porous matrix and a plurality of cell types containing: (i) myoblasts or progenitor cells thereof; and at least one of: (ii) at least one cell type that secretes extracellular matrix (ECM); or (iii) endothelial cells or their progenitors, wherein the myoblasts or their progenitors and their endothelial cells or their progenitors are incubated at a ratio between 10:1 and 1:10; and (b) inducing the differentiation of myoblasts or their progenitor cells into myotubes, resulting in an edible composition suitable for consumption.

[010] In some embodiments, the plurality of cell types comprises myoblasts or progenitor cells thereof, at least one extracellular matrix (ECM) secreting cell type, and endothelial cells or progenitor cells thereof.

[011] In some embodiments, the ECM-secreting cell is selected from the group consisting of: stromal cells, fibroblasts, pericytes, smooth muscle cells, and their precursor cells.

[012] In some embodiments, the plurality of cell types comprise myoblasts, ECM-secreting cells, and endothelial cells.

[013] In some embodiments, the myoblast progenitor cell is a satellite cell.

[014] In some embodiments, the endothelial cells are selected from skeletal microvascular endothelial cells, aortic smooth muscle cells, or combinations thereof.

[015] In some embodiments, the plurality of cell types comprises satellite cells, ECM-secreting cells, and endothelial cells.

[016] In some embodiments, myoblasts or progenitor cells and ECM-secreting cells are incubated at a ratio between 10:1 and 1:1.

[017] In some embodiments, ECM-secreting cells and endothelial cells are incubated at a ratio between 1:10 and 1:1.

[018] In some embodiments, satellite cells, ECM-secreting cells, and endothelial cells are incubated at a ratio ranging between 10:1:1 and 2:1:10.

[019] In some embodiments, the ECM-secreting cell is a fibroblast, its progenitor cell, or a combination thereof.

[020] In some embodiments, the three-dimensional porous matrix is selected from the group consisting of a structured protein, an unstructured protein, and a polysaccharide. In some embodiments, the structured protein is a structured soy protein. In some embodiments, the three-dimensional porous matrix contains pores with an average diameter ranging from 20 to 1000 micrometers.

[021] In some embodiments, the plurality of cell types are non-human cells. In some embodiments, the plurality of cells are from mammalian livestock.

[022] In some embodiments, the myoblasts or progenitor cells thereof and the 3D porous matrix are incubated at a ratio in the range of 10 ³ to 10 ⁷ myoblasts or progenitor cells thereof to 10 mg of the 3D porous matrix. In some embodiments, the three-dimensional porous matrix further comprises an extracellular matrix.

[023] In accordance with another aspect, a composition is provided comprising: (a) a three-dimensional porous matrix; (b) muscle tubules containing 100,000-250,000 muscle tubule nuclei per mm ³ three-dimensional porous matrix; and (c) a plurality of cell types selected from the group consisting of: (i) myoblasts or their precursor cells; and at least one of: (ii) at least one type of ECM-secreting cells; or (iii) endothelial cells or progenitor cells thereof, wherein endothelial cells or progenitor cells thereof comprise less than 15% of the plurality of cells.

[024] In some embodiments, the composition is edible.

[025] Unless otherwise indicated, all technical and/or scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the invention pertains. While methods and materials similar or equivalent to those described herein may be used in the practice or testing of embodiments of the invention, examples of methods and/or materials are described below. In the event of a conflict, the patent description, including definitions, will be controlled. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[026] Additional implementation options and the full scope of applicability of the present invention will become apparent from the detailed description below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, as various changes and modifications will be apparent to those skilled in the art within the spirit and scope of the invention from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[027] In FIG. 1A-1C are photographs showing commercial TBP (structured soy protein). ( A ) shows large, medium and small pieces of TSB products. ( B ) and ( C ) show 2 other commercial TSB flake products labeled TVP (left) and Arcon (right).

[028] In FIG. 2A-2B are photographs showing the preparation of the TSB matrix ( A ) and the cage containing the TSB matrices ( B ).

[029] In FIG. 3A-3B are 100X magnification SEM images of large TSB ( A ) and medium TSB ( B ).

[030] In FIG. 4A-4B are 2×10 ⁵ magnification SEM images of a large TSB ( A ) and a medium TSB ( B ).

[031] In FIG. 5A-5C are confocal microscopy images of TSB arrays populated with fibroblasts (red) and endothelial (green) cells at days 8 ( A ), 18 ( B ) and 21 ( C ) after fibroblast seeding.

[032] In FIG. Figure 6 shows confocal microscopy images taken 14 days after seeding 200,000 fibroblasts (red) on three different matrix samples (samples 1-3) obtained from different sources labeled as Large, Medium, Small and Medium from 90°, with matrices, designated as Average of 90°, were cut from the porous side of the TSB during the procedure for obtaining matrices from TSB. Scale bar = 1 mm.

[033] In FIG. Figure 7 shows a confocal microscopy image of myoblasts cultured on a TSB matrix for 14 days (Blue - DAPI (4,6-diamidino-2-phenylindole dihydrochloride), Green - desmin).

[034] In FIG. 8 shows confocal microscopy images of bovine skeletal muscle cells cultured on a TSB matrix for 14 days (Blue - DAPI, Green - phalloidin).

[035] In FIG. 9A-9E are micrographs of bovine aortic smooth muscle cells (SMCC) (passage 8) seeded in different media. ( A ) Basal medium; ( B ) Commercial environment; ( C ) enrichment with fetal bovine serum (15%); ( D ) enrichment in non-essential amino acids; and ( E ) pyruvate enrichment. Scale bar = 100 µm.

[036] In FIG. Fig. 10A-10B are vertical histograms comparing different media during bovine aortic smooth muscle cell proliferation at passage 9 ( A ) and passage 10 ( B ) after 3 days of culture.

[037] In FIG. 11A-11B is a graph showing the growth kinetics of bovine dermal fibroblasts (DBF) ( A ) and bovine aortic smooth muscle cells (BSMAB; B ).

[038] In FIG. 12A-12L are images of fluorescently labeled bovine aortic endothelial cells (ABEC) seeded in coculture with support cells at 2 (A, D, G, and J ), 6 ( B, E, H, and K ), and 8 ( C, F ). , I and L ) days. ABEC cells (green) were seeded with either bovine aortic smooth muscle cells (SMC; AF ) or bovine dermal fibroblasts (DFB; GL ), both red. AC and GI are 3×3 fragment scans with ×5 magnification. DF and JL represent x20 magnification. Scale bar = 100 µm.

[039] In FIG. 13A-13L are images of fluorescently labeled bovine skeletal muscle microvascular endothelial cells (MBECs) seeded in coculture with support cells at 2 ( A, D, G, and J ), 6 ( B, E, H, and K ), and 8 ( C, F, I and L ) days. MEXcMB cells (green) were seeded with either bovine aortic smooth muscle cells (SMC; AF ) or bovine dermal fibroblasts (DFB; GL ), both red. AC and GI are 3×3 fragment scans with ×5 magnification. DF and JL represent ×20 magnification. Scale bar = 100 µm.

[040] In FIG. 14A-14F are images of fluorescently labeled cocultures of cells containing ABEC and SMC in various ratios. ( A and D ) 5:1 ABEC to SMC; ( B and E ) 1:1 ABEC to GMC. ( C and F ) represent increases in B and E , respectively. A-C represent images taken on day 2 and DF on day 6. Scale bar = 100 µm

[041] In FIG. 15A-15E are images of fluorescently labeled cocultures of cells containing ABEC and DPB in various ratios. ( A and C ) 5:1 ABEC to SMC; ( B and D ) 1:1 ABEC to GMC. ( E ) Higher resolution D. A and B represent image taken on day 2, while C and D are image taken on day 6. EKAB - red; Scale bar = 100 µm.

[042] In FIG. 16A-16L are images of fluorescently labeled cell cocultures containing ABEC and SMC in various ratios. ( AD ) 1:3 ABEC to SMC; ( EH ) 1:1 ABEC to MMC; and ( IL and F ) 5:1 ABEC to MMC. ( B, D, F, H, J and L ) represent the magnification of ( A, C, E, G, I and K ), respectively. ( A, B, E, F, I and J ) represent the image taken on day 2 and ( C, D, G, H, K and L ) the image taken on day 6. EKAB - red; Scale bar = 100 µm.

[043] FIG. 17A-17G demonstrate bovine satellite cell (BSC) differentiation and myotube formation. ( AC ) represent images of SBC cells before differentiation; ( DF ) - images of SBC cells after differentiation. ( G ) is a vertical histogram showing the confluence index (CI) of the samples. (A and D ) light microscopy; ( B and E ) Hoechst; and ( C and F ) Myogenin. The difference in automatic IS before and after differentiation was statistically significant (P-value=0.00003), while the difference between automatic and manual IS was not.

[044] In FIG. 18A-18G are bright-field micrographs of CPS on day 0 of differentiation. ( A ) Control group; ( B ) bovine fibroblast growth factor (bFGF); ( C ) epidermal growth factor (EGF); ( D ) IGF-1 (insulin-like growth factor 1); ( E ) Pro-LIF (proliferation medium without growth factor LIF (leukemia inhibitory factor)); ( F ) weaning; and ( G ) DiI. Scale bar = 300 µm.

[045] In FIG. 19A-19G are bright-field micrographs of CPS on day 4 of differentiation. ( A ) Control group; ( B ) bovine fibroblast growth factor (bFGF); ( C ) epidermal growth factor (EGF); ( D ) IGF-1 (insulin-like growth factor 1); ( E ) Pro-LIF; ( F ) weaning; and ( G ) DiI. Scale bar = 300 µm.

[046] FIG. 20A-20H show the formation of the muscular tubule of the SKB after 7 days of differentiation. (AG ) - brightfield micrographs ( A ) Control group; ( B ) bovine fibroblast growth factor (bFGF); ( C ) epidermal growth factor (EGF); ( D ) IGF-1 (insulin-like growth factor 1); ( E ) Pro-LIF; ( F ) weaning; and ( G ) DiI. Scale bar = 300 µm. ( H ) - vertical histogram in % area of myotubes, quantified for each of the various settling conditions.

[047] In FIG. 21 is a vertical bar graph showing the quantification of cell coverage of porous matrices on day 7 with culture medium or Pro-LIF medium.

[048] In FIG. 22A-22L are confocal microscopy images of fluorescently labeled CBP differentiated on a porous matrix. The expansion phase included: ( AD ) cells cultured on a nutrient medium; ( EH ) cells cultured on Pro-LIF medium; ( IL ) Cells cultured on Pro-LIF-bFGF medium. The differentiation phase included: ( A, E and I ) no growth factors (GF) were added to the differentiation medium; ( B, F and J ) IGF-1 was added to differentiation medium; ( C, G and K ) EGF was added to differentiation medium; and ( D, H and L ) IGF-1 and EGF were added to differentiation medium. DiI (red); desmin (green); Scale bar = 100 µm.

[049] In FIG. 23A-23O are immunofluorescence micrographs of tri-cultures grown on L-PLA/PLG arrays taken 14 days after inoculation. SKB, SMC, and ABEC were seeded on L-PLA/PLG matrices with different cell population densities ( AE ) 2:1:1 and ( FJ ) 2:1:5. ( KO ) higher resolution ( AE ). Matrices were stained ( A, F and K ) for DAPI (4,6-diamidino-2-phenylindole dihydrochloride); ( B, G and L ) Myogenin; ( C, H and M ) DiI; ( D, I and N ) CD31. ( E, J and O ) are the merged image. Scale bar = 100 µm ( AJ ); 10 µm ( K-O ).

[050] In FIG. 24A-24F are immunofluorescence micrographs of L-PLA/PLG arrays 14 days after seeding. SKB, SMC, and ECB were seeded on L-PLA/PLG matrices with different cell population densities ( AC ) 2:1:1 and ( DF ) 2:1:5. ( A and D ) SKB and MMC (2:1); (B and E ) SKB and EKB (2:1); and ( C and F ) monoculture of SKB only. Matrices were stained for DAPI (blue); myogenin (gray); DiI (red); and CD31 (green). Scale bar = 100 µm.

[051] In FIG. 25A-25H are immunofluorescence micrographs of SBP co-cultured with MMC on structured soy protein matrices 14 days after inoculation. Different cell densities were tested ( AD ) 2:1:1 and ( EH ) 2:1:5. Templates were stained for ( A and E ) DAPI; ( B and F ) myogenin; ( C and G ) DiI; ( D ) and ( H ) are the combined images of ( AC ) and ( EG ), respectively. Scale bar = 100 µm.

[052] In FIG. 26A-26E are immunofluorescent micrographs of CBP, SMC, and ECP co-cultured on structured soy protein matrices 14 days after inoculation. Matrices were stained for ( A ) DAPI; ( B ) myogenin; ( C ) DiI; and ( D ) CD31. ( E ) is the merged image ( AD ).

[053] In FIG. 27A-27B are micrographs of trichrome staining of three cultures of CBP, MMC, and ECP seeded in L-PLA/PLG at a ratio of 2:1:1, 14 days after inoculation. ( A ) Scan at ×5 magnification and ( B ) ×10 magnification. Matrices are painted in three colors. Positive extracellular matrix (ECM) spots are grouped in circles. Scale bar = 100 µm.

[054] In FIG. 28A-28C are vertical bar graphs showing the effect of support cells on myogenic differentiation and the importance of the ratio of different cells in culture. Myogenic differentiation is shown for tri-cultures (SKB: MMC: ECB) and controls (coculture or monoculture) at ( A ) 2:1:1 and ( B ) 2:1:5 cell population densities on an L-PLA/PLG matrix , ( C ) 2:1:1 on structured soy protein with statistical analysis.

[055] FIG. 29A-29N describe the differentiation of tri-cultures on structured protein matrices. ( AL ) are fluorescence images of cross-linked soy protein immunostained arrays 14 days post seeding. Cells were seeded on matrixes of structured soy protein ( A, E and I ) SBP, ( B, F and J ) SBP + ECP; ( C, G and K ) SKB + MMC; and ( D, H and L ) tri-culture of SKB + SMC + ECB. ( AD ) x20x magnification, ( EH ) scan of fragments x20 and ( IL ) myogenin staining, x63x magnification. Arrays were stained with DAPI (blue), myogenin (grey), DiI (red) and CD31 (green). Scale bar = 100 µm. ( M ) is a vertical histogram of myogenin expression quantification on TBP matrices in monoculture (SBP only), cocultures (SBP + ECP; or SBP + SMC) and tri-culture (SCP + SMC + ECP). ( N ) - Vertical histogram of the quantification of SKB coverage on TSB matrices in monoculture and co-culture with MMC.

[056] In FIG. 30 shows fluorescence images of immunostained CBP seeded without gel on Gelfoam arrays 14 days after seeding. Matrices were stained with DAPI (blue), myogenin (grey), DiI (red); and CD31 (green). Scale bar = 50 µm.

DETAILED DESCRIPTION OF THE INVENTION

[057] The present invention relates to compositions, kits and methods for producing cultured meat for human consumption. The present invention in some embodiments relates to an edible composition comprising a porous structured protein and a three-dimensional multicellular tissue containing myoblasts and one or more cell types attached thereto. The invention further relates to methods for preparing a composition by in vitro culturing myoblasts and one or more cell types with a three-dimensional porous matrix (eg, a porous structured protein) under certain conditions.

[058] In some embodiments, the composition of the invention may be for consumption by humans, non-human animals, or both. In some embodiments, cultured meat products are food products for human consumption. In other embodiments, cultured meat products are used as animal feed, such as livestock feed, aquafeed, or pet food.

[059] In some embodiments, the invention relates to a method for preparing an edible composition comprising the steps of:

a. incubating a three-dimensional porous matrix and a plurality of cell types containing myoblasts and at least one ECM-secreting cell type selected from the group consisting of: adipocytes, fibroblasts, and their precursor cells; and an endothelial cell or its progenitor cell

b. allowing a plurality of cell types to propagate on a three-dimensional porous matrix; and

With. induction of differentiation of myoblasts into muscle tubules;

thereby allowing the cells to form an edible composition containing a three-dimensional multicellular tissue containing muscle cells and a porous structured protein.

[060] As used herein, the term "muscular tube" refers to a multinucleated fiber that results from the fusion of multiple myoblasts and/or myocytes. As used herein, the term “muscle cell” refers to any cell that refers to muscle tissue and may include: myoblasts, satellite cells (SCs), myotubes, myofibrils, and myofibril tissues.

[061] In some embodiments, the method includes culturing myoblasts in vitro or ex vivo and allowing these cells to differentiate into certain types of muscle cells, such as skeletal muscle cells or smooth muscle cells.

[062] In some embodiments, the invention relates to an edible composition comprising: a porous structured protein; and a three-dimensional multicellular tissue containing muscle cells, wherein the three-dimensional multicellular tissue is attached to a porous structured protein, and wherein the three-dimensional multicellular tissue is derived from in vitro cultivation of a plurality of cell types containing: myoblasts and one or more cell types selected from: adipocytes, fibroblasts, smooth muscle cells, endothelial cells and their precursor cells with a porous structured protein.

[063] As illustrated below, the composition may contain fibroblasts that secrete extracellular molecules so as to form an extracellular matrix (ECM), providing additional structural and mechanical support to the cells.

[064] As further illustrated below, the composition may contain endothelial cells (ECs) or endothelial progenitors (ECCs) to provide tissue scaffolding, signaling, and/or capillary endothelial formation.

[065] In some embodiments, the three-dimensional multicellular tissue contains muscle cells, including skeletal muscle cells, smooth muscle cells, and satellite cells. In some embodiments, the three-dimensional multicellular tissue contains fat cells (eg, adipocytes). In some embodiments, the three-dimensional multicellular tissue contains an extracellular matrix secreted by specialized cells (eg, fibroblasts). In some embodiments, the three-dimensional multicellular tissue comprises endothelial cells or capillary endothelium formed by endothelial cells, including, but not limited to, aortic endothelial cells and skeletal microvascular endothelial cells. In some embodiments, the three-dimensional multicellular tissue further comprises an extracellular matrix. In some embodiments, the three-dimensional multicellular tissue further comprises adipocytes. In some embodiments, the three-dimensional multicellular tissue further comprises capillaries.

[066] In some embodiments, the invention relates to a composition suitable for growing cells, containing a porous structured protein and a cell culture medium. In another embodiment, the composition suitable for growing cells further comprises a growth factor, cytokines, bioactive agents, nutrients, amino acids, antibiotic compounds, anti-inflammatory compounds, or any combination thereof. Suitable media and compounds suitable for cell viability and growth are known to those skilled in the art.

[067] In some embodiments, the invention relates to a composition comprising: a plurality of cell types, including myoblasts and one or more cell types selected from: adipocytes, fibroblast endothelial cells, smooth muscle cells, and their precursor cells, attached to a porous structured protein.

[068] In some embodiments, the invention provides a kit containing: a three-dimensional porous matrix (eg, a porous structured protein); and a plurality of cell types comprising: myoblasts and one or more cell types selected from: adipocytes, fibroblasts, smooth muscle cells, endothelial cells, and progenitor cells thereof. In some embodiments, the kit is designed to provide an edible composition. In some embodiments, the kit further comprises at least one component selected from: cell culture medium, growth factor, differentiation medium, differentiation promoters. In some embodiments, the kit further comprises a cell culture medium. In another embodiment, the cell culture medium is selected from a dry powder medium, a granular preparation, an aqueous liquid, or a medium concentrate. In some embodiments, multiple cell types are frozen. In some embodiments, the kit further contains instructions for use.

Many types of cells

[069] As will be appreciated by one of skill in the art, multiple cell types or populations of cells can be cultured with a three-dimensional porous matrix to form a three-dimensional multicellular tissue architecture. In some embodiments, one or more cell types are selected from: myoblasts, ECM-secreting cells, endothelial cells.

[070] In some embodiments, one or more cell types are myoblast precursor cells. In some embodiments, one or more cell types are progenitors of an ECM-secreting cell. In some embodiments, one or more cell types are endothelial cell progenitors.

[071] As used herein, a progenitor cell comprises a mesenchymal stem cell (MSc), an embryonic stem cell (ESC), an adult stem cell, a differentiated ESC, a differentiated adult stem cell, and an induced pluripotent stem cell (iPSC). As used herein, the term "progenitor cell" refers to a cell capable of producing differentiated cells in multiple lineages such as myoblasts, fibroblasts, adipocytes, stromal cells, fibroblasts, pericytes, smooth muscle cells, and endothelial cells. "Progenitor cells" differ from stem cells in that they generally do not have extensive self-renewal capacity.

[072] In some embodiments, a cell from a plurality of cell types according to the present invention is a progenitor cell. In some embodiments, the precursor cell is cultured in a monoculture. In some embodiments, the progenitor cell differentiates into a monoculture. In some embodiments, the progenitor cell is differentiated into a monoculture and then incubated on a multi-cell three-dimensional porous matrix in accordance with the method of the present invention. A non-limiting example includes, but is not limited to, culturing and differentiating a mesenchymal stem cell into a myoblast cell, and then seeding and then incubating the differentiated myoblast on a three-dimensional porous matrix. Methods for culturing and inducing differentiation of progenitor cells into mature cells will be apparent to those skilled in the art.

[073] In some embodiments, the plurality of cell types comprise myoblasts and fibroblasts. In some embodiments, the plurality of cell types comprise myoblasts, fibroblasts, and/or fibroblast precursor cells. In some embodiments, the plurality of cell types comprise myoblasts, fibroblasts, and adipocytes. In some embodiments, the plurality of cell types comprise myoblasts, fibroblasts, adipocytes, and/or fibroblast progenitors and/or adipocyte progenitors. In some embodiments, the plurality of cell types comprise myoblasts, fibroblasts, and endothelial cells. In some embodiments, the plurality of cell types comprises myoblasts, fibroblasts, and endothelial cells, and/or fibroblast progenitor cells and/or endothelial progenitor cells. In some embodiments, the plurality of cell types comprise myoblasts and smooth muscle cells. In some embodiments, the plurality of cell types comprise myoblasts, smooth muscle cells, and endothelial cells. In some embodiments, the plurality of cell types comprises myoblasts, smooth muscle cells, endothelial cells, and adipocytes. In some embodiments, the plurality of cell types comprise myoblasts, fibroblasts, endothelial cells, and adipocytes. In some embodiments, the plurality of cell types comprise myoblasts, fibroblasts, endothelial cells, adipocytes, and/or fibroblast progenitors and/or adipocyte progenitors and/or endothelial progenitors.

[074] In some embodiments, multiple cell types are obtained from live animals and cultured in a primary cell line. As a non-limiting example, cells can be biopsied and cultured ex vivo. As another non-limiting example, cells can be obtained from commercial sources.

[075] In some embodiments, multiple cell types are derived from stem cells, such as pluripotent embryonic stem cells. In another embodiment, mesenchymal stem cells (MSCs) are used. As known to the person skilled in the art, MSCs can serve as a source of muscle cells, fat cells, bone cells and cartilage cells. In another embodiment, the cells are induced pluripotent stem cells (iPSCs). In another embodiment, the cells are derived from totipotent embryonic stem cells, such as cells from the blastocyst stage, fertilized egg, placenta, and umbilical cord from these animals.

[076] In some embodiments, a plurality of cell types are derived from cells other than human cells. In some embodiments, the plurality of cell types are derived from non-human cells selected from the group consisting of: mammals, birds, fish, invertebrates, reptiles, amphibians, and combinations thereof. In some embodiments, the plurality of cell types are mammalian. In some embodiments, the plurality of cell types are non-human mammalian. In some embodiments, the plurality of cell types are derived from mammalian livestock. As used herein, the term "livestock" includes any domestic mammal, semi-domestic mammal, or captive wild mammal. Non-limiting examples of non-human mammals include: antelope, bear, beaver, bison, wild 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, the plurality of cell types are derived from avian cells. Non-limiting examples of birds include: chicken, duck, emu, goose, hazel grouse, ostrich, pheasant, dove, quail, and turkey, or combinations thereof. In some embodiments, multiple cell types are derived from fish. Non-limiting examples of fish include: perch, catfish, carp, cod, eel, flounder, pufferfish, grouper, haddock, halibut, herring, mackerel, mahi, marlin, hoplostetus, tautogolyabrus, pike, pollock, salmon, sardine, shark, pagrus , sole, swordfish, tilapia, trout, tuna and walleye, or combinations thereof. In some embodiments, a plurality of cell types are derived from invertebrates. Non-limiting examples of invertebrates include lobster, crab, shrimp, clams, oysters, mussels, and sea urchins. In some embodiments, a plurality of cell types are derived from reptiles. Non-limiting examples of reptiles include: snake, alligator and turtle. In some embodiments, multiple cell types are derived from amphibians. A non-limiting example of amphibians includes frogs.

Seeding and culturing cells

[077] As one of skill in the art will appreciate, each cell type used in the composition and method described herein may have a preferred or optimal cell density range and preferred media or growth factors appropriate for cell viability. In some embodiments, each cell type is seeded at a specific cell density. In some embodiments, cells are seeded simultaneously or sequentially.

[078] In some embodiments, myoblasts are seeded at a cell density of 10 ³ to 10 ⁷ cells per 10 mg of porous structured protein. In some embodiments, different cell types are seeded in a specific ratio. In some embodiments, the ratio of seeded myoblasts to seeded fibroblasts is between 1:1000 and 1000:1. In some embodiments, the ratio of seeded myoblasts and seeded fibroblasts to seeded endothelial cells is between 1:20 and 20:1. In some embodiments, the ratio of seeded myoblasts and seeded fibroblasts to seeded adipocytes is between 1:5000 and 5000:1. In some embodiments, the ratio of seeded satellite cells to seeded smooth muscle cells is between 5:1 and 1:5. In some embodiments, the ratio of seeded satellite cells to seeded skeletal microvascular endothelial cells is between 10:1 and 1:10. In some embodiments, the ratio of seeded smooth muscle cells to seeded skeletal microvascular endothelial cells is between 10:1 and 1:10. In some embodiments, the ratio of seeded satellite cells to seeded smooth muscle cells to seeded skeletal microvascular endothelial cells is between 10:1:1 and 2:1:10. In one embodiment, the ratio of seeded satellite cells to seeded smooth muscle cells to seeded skeletal microvascular endothelial cells is between 2:1:1 and 2:1:5, or any ratio in between. In one embodiment, the ratio of seeded satellite cells to seeded smooth muscle cells to seeded skeletal microvascular endothelial cells is between 2:1:1 and 2:1:2, or any ratio in between. In one embodiment, the ratio of seeded satellite cells to seeded smooth muscle cells to seeded skeletal microvascular endothelial cells is between 2:1:1 and 2:1:3, or any ratio in between. In one embodiment, the ratio of seeded satellite cells to seeded smooth muscle cells to seeded skeletal microvascular endothelial cells is between 2:1:1 and 2:1:4, or any ratio in between. Each possibility represents a separate embodiment of the present invention.

[079] In some embodiments, seeded cell density and incubated cell density are relatively the same.

[080] In one embodiment, "% coverage" refers to the area or volume of the porous matrix that is in contact with cells or myotubes. In another embodiment, % coverage refers to the area or volume of the porous matrix that is occupied by cells or myotubes. In the context of this document, the cells in contact with the matrix are on or within it, or a combination thereof.

[081] In some embodiments, the % coverage of a plurality of cells is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, by at least 70%, at least 80%, at least 90%, or at least 99%. In some embodiments, % cell coverage is 5-20%, 15-30%, 25-40%, 35-50%, 45-60%, 55-70%, 65-80%, 75-90%, 85 -100% or any range in between. Each possibility represents a separate embodiment of the present invention.

[082] In some embodiments, the % satellite cell coverage is at least 35%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90, or at least 99%. In some embodiments, % satellite cell coverage is 25-40%, 35-50%, 45-60%, 55-70%, 65-80%, 75-90%, 85-100%, or any range in between. Each possibility represents a separate embodiment of the present invention.

[083] In some embodiments, the coverage of % of endothelial cells is not more than 2%, not more than 5%, not more than 10%, not more than 15%, not more than 20%, not more than 25%, not more than 30%, not more than 35 %, not more than 40%, not more than 45% or not more than 50%. In some embodiments, the % endothelial cell coverage is 5-15%, 10-25%, 20-35%, 30-50%, or any range in between. Each possibility represents a separate embodiment of the present invention.

[084] In some embodiments, endothelial cells are used to increase the proliferation of myoblasts or their precursor cells. In some embodiments, the endothelial cells inhibit the differentiation of myoblasts or their progenitor cells into myotubes. In some embodiments, endothelial cells support the growth and proliferation of myoblasts or their precursor cells. In some embodiments, endothelial cells are not required for differentiation of myoblasts or their progenitor cells. In some embodiments, in accordance with the method of the present invention, endothelial cell activity (eg, secretion of myogenic agents, stabilization of myoblast growth, survival, or both) is maintained for a certain period. In some embodiments, the defined period for endothelial activity is the period required for the myoblasts or their progenitor cells to achieve coverage of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of a three-dimensional porous matrix. In some embodiments, the defined period of endothelial activity is the period required for myoblasts or their progenitor cells to achieve coverage of at least 30%.

[085] In some embodiments, the % coverage of myotubes is at least 5%, at least 20%, at least 35%, at least 50%, at least 70%, at least 85%, at least at least 90%, at least 90% or at least 99%. In some embodiments, the % coverage of myotubes is 1-10%, 5-20%, 15-35%, 30-50%, 40-65%, 60-85%, 80-90%, 90-100%, or any range in between. Each possibility represents a separate embodiment of the present invention.

[086] In some embodiments, the muscle tubules in the composition of the present invention comprise 10,000-100,000 cores per mm ³ 3D porous matrix, 10,000-100,000 cores per mm ³ 3D porous matrix, 15,000-200,000 cores per mm ³ 3D porous matrix, 50,000- 500000 cores/mm3 ^3D porous matrix, 5000-1000000 cores/mm3 ^3D porous matrix, 10000-250000 cores/mm3 ^3D porous matrix, 10000-1500000 cores/mm3 ^3D porous matrix, or any range in between. Each possibility represents a separate embodiment of the present invention.

[087] As will be apparent to those skilled in the art, ECM affects the differentiation of myoblasts into myotubes. In some embodiments, ECM-secreting cells used in accordance with the methods of the present invention improve myoblast differentiation. In some embodiments, ECM-secreting cells improved myoblast differentiation by modeling the physical properties of the tissue.

[088] As defined herein, the terms "enhanced" and "enhanced" are used interchangeably.

[089] In some embodiments, the improvement is at least 5%, at least 20%, at least 35%, at least 50%, at least 75%, at least 90% , at least 100%, at least 250%, at least 500%, at least 750%, at least 1000%, at least 2500%, or at least 5000%. In some embodiments, the improvement is 5-15%, 10-35%, 25-45%, 40-70%, 65-90%, 85-150%, 100-500%, or 250-1000%. Each possibility represents a separate embodiment of the present invention.

[090] Non-limiting examples of the physical properties of a fabric include, but are not limited to, strength, porosity, flexibility, stiffness, etc.). The physical properties of a fabric can be determined in accordance with its Young's modulus, viscosity modulus, or other parameters that will be apparent to a person skilled in the art.

[091] In some embodiments, the method of the present invention further includes the step of sterilizing the porous structured protein. In some embodiments, the porous structured protein is sterilized prior to seeding or incubation of multiple cell types. In some embodiments, sterilization is performed by gamma radiation. In another implementation, sterilization is carried out using ethanol. Sterilization procedures are obvious to a person skilled in the art.

[092] One skilled in the art will appreciate that seeding and/or cell culture is carried out in the presence of a cell culture medium. In another embodiment, the cell culture medium contains a growth factor, cytokines, bioactive agents, nutrients, amino acids, antibiotic compounds, anti-inflammatory compounds, or any combination thereof. Suitable media and compounds suitable for cell viability and growth are known to those skilled in the art.

[093] Growth factors that can be used in the methods and compositions of the invention include, but are not limited to, platelet-derived growth factor (TPGF), insulin-like growth factor (IGF-1). It is known that TCFR and IGF-1 induce mitogenic, chemotactic and proliferative (differentiating) cellular responses. The growth factor may be, but is not limited to, one or more of the following: TCFR, eg platelet-derived growth factor AA, platelet-derived growth factor BB; IGF, for example, IGF-I, IGF-II; fibroblast growth factors (FGF), eg, acidic FGF, basic FGF, β-endothelial growth factor, FGF 4, FGF 5, FGF 6, FGF 7, FGF 8, and FGF 9; transforming growth factors (TGF), eg TGF-P1, TGF-β1,2, TGF-β2, TGF-β3, TGF-β5; bone morphogenetic proteins (BMPs), eg BMP 1, BMP 2, BMP 3, BMP 4; vascular endothelial growth factors (VEGF), eg VEGF, placental growth factor; epidermal growth factors (EGF), eg EGF, amphiregulin, betacellulin, heparin-binding EGF; interleukins, for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL- 12, IL-13, IL-14; colony stimulating factors (CSF), eg G-CSF, GM-CSF, M-CSF; nerve growth factor (NGF); stem cell factor; hepatocyte growth factor,

[094] As illustrated below, in the case of bovine-derived cells, the optimal satellite cell (SC) expansion medium (i.e. Pro-LIF medium) contains Bovine SC Growth Medium (BSC) (99%), Amphotericin B (AB/ AM; 1% 1×), ZnCl ₂ (50 μM), EGF (62 ng/ml), IGF-1 (100 ng/ml) and bFGF (10 ng/ml).

[095] As illustrated below, in the case of bovine-derived cells, the optimal CK medium contains DMEM/HEPES (Dulbecco's Modified Eagle's Minimal Medium/4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) (43, 5%), Ham F-10 Formula (43.5%), Fetal Bovine Serum (10%), MEM NEAA (Minimum Culture Medium, Non-Essential Amino Acids) (1% 1×), GlutaMAX (1%) and AB/ AM (1% 1×).

[096] As illustrated below, in the case of bovine-derived cells, the optimal media for SC differentiation contains DMEM/HEPES (97%), donor horse serum (2%), AB/AM (1% 1×), IGF-1 (100 ng/ml) and EGF (62 ng/ml).

porous matrix

[002] In some embodiments, a plurality of cells of the invention are incubated with a porous matrix. In the context of this document, the term "matrix" refers to a structure containing a material that provides a surface suitable for adhesion/attachment, maturation, differentiation and proliferation of cells. The matrix may additionally provide mechanical stability and support. The matrix may have a particular geometry or shape in order to influence or restrict the three-dimensional geometry, or a shape intended by the population of proliferating cells. In some embodiments, the implementation of the porous matrix according to the invention is three-dimensional.

[097] In some embodiments, the average pore diameter of the porous matrix is in the range of 20 micrometers (µm) to 1000 µm, 20 µm to 900 µm, 20 µm to 800 µm, 20 µm to 700 µm, 20 µm to 600 µm, 20 µm to 500 µm, 20 µm to 400 µm, 20 µm to 300 µm, 20 µm to 200 µm, 20 µm to 100 µm, 50 µm to 1000 µm, 50 µm to 900 µm, 50 µm to 800 µm, 50 µm. up to 700 µm, 50 µm to 600 µm, 50 µm to 500 µm, 50 µm to 400 µm, 50 µm to 300 µm, 50 µm to 200 µm, 50 µm to 100 µm, 100 µm to 1000 µm, 100 µm to 900 100 to 800 µm, 100 to 700 µm, 100 to 600 µm, 100 to 500 µm, 100 to 400 µm, 100 to 300 µm, 100 to 200 µm, 500 to 1000 µm, 500 to 900 µm, 500 to 800 µm, 500 to 700 µm, or 500 to 600 µm. Each possibility represents a separate embodiment of the present invention. In some embodiments, the average pore diameter of the porous matrix is in the range of 20 to 1000 microns.

[098] In some embodiments, the porous matrix is edible. In some embodiments, the porous matrix contains a structured protein. In some embodiments, the porous matrix contains a polysaccharide. In some embodiments, the structured protein is a structured plant protein. In some embodiments, the structured protein is a structured soy protein (eg, TSB).

[099] The term "structure" is used herein to refer to a rigid mass or flexible mass, individual cells that can easily form various sizes, geometries and configurations, and which are not dispersed in water.

[0100] Suitable particulate structured protein materials for use herein can be comprised of 30% to 100% protein on a dry weight basis and 0% to 70% materials associated with the original protein material or added auxiliary materials. Examples of auxiliary materials are carbohydrates, vitamins, flavors, colorants, or others. In some embodiments, the protein particles are composed of 50%-100% protein, or 50%-80% protein based on dry matter weight.

[0101] Suitable unstructured proteins that can be structured to form structured particulate protein materials are available from a variety of sources. As a non-limiting example, the source of such proteins is vegetable protein and some fungal proteins; however, animal protein may be used. Examples of suitable animal proteins are casein, collagen and egg white. Examples of suitable vegetable protein sources are soybeans, safflower seeds, corn, peanuts, wheat, wheat gluten, peas, sunflower seeds, chickpeas, cottonseed, coconut, canola, sesame seeds, leaf proteins, gluten, single cell proteins such as yeast, etc.

[0102] Mushrooms are another example of a suitable protein source. In some embodiments, the mushroom protein source contains an amount of protein 15-20% (w/w), 20-30% (w/w), 28-45% (w/w), or 10-40% (w/w) based on dry weight. substances.

[0103] In general, if the protein source is a vegetable protein, the protein is placed in a relatively pure form before use. For example, if the protein source is soybeans, the soybeans can be extracted with a solvent, such as hexane, to remove oil from them. The resulting defatted soybean meal contains about 50% protein.

[0104] Soybean meal can be processed in a known manner to remove carbohydrates and produce higher protein products, such as soy protein concentrates containing about 70% protein or soy protein isolates containing about 90% or more protein. In turn, a variety of suitable prior art methods can be used to convert soybean meal, concentrate, isolate, and other edible protein-containing materials into suitable particulate structured protein materials.

[0105] Suitable methods for converting unstructured animal and plant protein-containing materials into particulate structured proteins are disclosed, for example, in the following US Pat. No. 2,682,466, issued June 29, 1954 to Boyer; No. 3142571 issued July 28, 1964 to Kitchel; No. 3488770, issued January 6, 1970 to Atkinson; No. 3498794 issued March 3, 1970 to Calvert et al.; No. 3759715 issued September 18, 1973 to Loepikte et al.; No. 3778222, issued December 11, 1973 to Strommer; No. 3794731 issued Feb. 26, 1974 to Dannert et al.; No. 3814823 issued June 4, 1974 to Young et al.; and filed application for US patent No. 248581, filed April 28, 1972, currently Pat. No. 3840679 issued on October 8, 1974 to Liepa et al.; which are incorporated herein by reference.

[0106] In alternative embodiments, the porous structured protein may be replaced with other porous compositions that are ingestible and edible, as well as chewable. As used herein, the terms "ingestible" and "edible" refer to compositions that can be safely administered to the body. These compositions include absorbable and non-absorbable, and digestible and non-absorbable. In the context of this document, the term "chewable" refers to a composition that can be broken/chopped into smaller pieces by chewing before swallowing. One skilled in the art will appreciate that a suitable comestible composition may be selected according to the physical properties (eg, Young's modulus, viscosity modulus, hardness, etc.) for the desired use (eg, adult consumption).

[0107] In accordance with the method according to the present invention, many types of cells are seeded on a three-dimensional porous matrix as such. In some embodiments, the plurality of cell types seeded on the 3D porous matrix do not require hardeners. In some embodiments, the plurality of cell types seeded on the 3D porous matrix require hardeners. In some embodiments, hardeners increase adhesion or attachment of multiple cell types to the three-dimensional porous matrix. Non-limiting examples of hardeners include, but are not limited to, thrombin or fibrin.

[0108] In the context of this document, the terms "gelling agent" and "curing agent" are used interchangeably.

[0109] In the discussion, unless otherwise indicated, adjectives such as "substantially" and "about" a feature-modifying condition or relationship of a feature or features of an embodiment of the invention are understood to mean that the condition or feature is defined within acceptable tolerances. to implement the implementation option in the application for which they are intended. Unless otherwise indicated, the word "or" in the specification and claims is considered to be inclusive "or" and not exclusive or, and indicates at least one of or any combination of the elements to which it is attached.

[0110] It should be understood that the terms in the singular used above and elsewhere in this document refer to "one or more" of the listed components. One skilled in the art will appreciate that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms singular and "at least one" are used interchangeably in this application.

[0111] For the purposes of a better understanding of the present ideas, and in no way to limit the scope of these ideas, unless otherwise indicated, all numbers expressing quantities, percentages or proportions and other numerical values used in the description and claims, mean modified by the term "near". Accordingly, unless otherwise indicated, the numerical parameters set forth in the following description and the appended claims are approximate values that may vary depending on the desired properties to be obtained. As a minimum, each numerical parameter must at least be interpreted in terms of the number of significant digits declared and using normal rounding techniques.

[0112] In the description and claims of the present application, each of the verbs "comprise", "include" and "have" and their conjugations is used to indicate that the object or objects following the verb are not necessarily a complete list of components, elements or parts of the subject or subject of the verb.

[0113] Other terms in the context of this document are defined by their well-known meanings in the art.

[0114] Additional objects, advantages and new features of the present invention will become apparent to a person skilled in the art when considering the following examples, which are not intended to be limiting. In addition, each of the various embodiments and aspects of the present invention described above and claimed in the claims section below finds experimental support in the following examples.

[0115] It is understood that some features of the invention, which are described in the context of separate embodiments for clarity, may also be provided in combination in one embodiment. Conversely, various features of the invention, which for brevity are described in the context of one embodiment, may also be provided separately or in any suitable combination, or as suitable in any other described embodiment of the invention. Certain features described in the context of various implementations should not be considered essential features of those implementations unless the implementation is feasible without those elements.

EXAMPLES

[0116] In general, the nomenclature used herein and the laboratory procedures used in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such methods are described in detail in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); the methodology is set forth in US Pat. No. 4,666,828; No. 4,683,202; No. 4,801,531; #5,192,659 and #5,272,057; "Cell Biology: A Laboratory Handbook," Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); "Bacteriophage Methods and Protocols", Volume 1: Isolation, Characterization, and Interactions, which are incorporated herein by reference. Other general references are provided in this document.

Materials and methods

structured soy protein

[0117] Structured soy protein (TSP) is commercially available in several different products. TBP matrices were obtained from 5 different sources of commercial TBP - 3 pieces of TBP ( Figure 1A ) purchased from a local health food store and 2 samples of TBP flakes ( Figures 1B-1C ) purchased from ADM. TSB was sterilized in 25-40 kGy gamma irradiation for 3.5 hours and stored in a sterile environment or using 70% (v/v) ethanol for 15 minutes. This was followed by 3 washes with PBS (phosphate-buffered saline).

Preparation of the TSB matrix

[0118] TSB flakes (from Arcon or TVP) were selected using Graefe's tweezers to obtain a uniform thickness of about 200-400 microns (per 1 layer). The flakes were incubated in 5-10 ml sterile DFA (Deuterium Depleted Water) (at 37°C overnight (in a 50 ml vial). The following day, the flakes were cut into 6 mm matrices using a 6 mm biopsy needle inside the The matrix was removed from the biopsy needle using a Pasteur pipette and transferred to a vial containing 5 ml of culture medium. .2A ) using a biopsy syringe and cut into 1 mm thick discs using a knife ( Fig. 2A ) The matrices were then placed in 5 ml of culture medium.

Matrix seeding

[0119] For one matrix 2×10 ⁶ SKB was transferred to Eppendorf and centrifuged for 4 minutes at 1500 RCF (relative centrifugal acceleration). Meanwhile, each template was placed in the well of a 6-well plate, untreated with cell culture, with 50 μl of growth medium. After centrifugation, the medium was aspirated from the Eppendorf tube, leaving the tray with cells. The following steps were then performed with each Eppendorf: (1) the medium was carefully aspirated from the matrix using tweezers to gently compress the matrix, and the remaining medium was evacuated; (2) 7 μl of thrombin (20 MGE/ml sigma in PBS) and 7 μl of fibrinogen (15 mg/ml sigma in PBS) were added; (3) the cells were then seeded onto a matrix; (4) matrices were incubated at 37° C. for 30 minutes until dry and then 2 ml of propagation medium was added to the well; (5) the day after the matrix was transferred to a new container (eg, 24 wells, uncultured, coverslip bottom) using sterile Graefe forceps and 2 ml of propagation medium was added to it; (6) the matrix was incubated at 37°C and 5% CO ₂ ; (7) the matrix was stored in propagation medium for 7 days; (8) medium changed every 2 days (2 ml); (9) after 7 days, the propagation medium was replaced with the medium for differentiating CPS; and (10) the differentiation medium was changed every 2 days for 5 days.

Cell culture

[0120] Bovine Satellite Cells (BSCs) were cultured in BBS Proliferation Medium 43.5% DMEM/HEPES (Gibco), 43.5% Nut Mix F-10 (Gibco), 10% PBS (HyClone), 1% non-essential amino acids (Gibco), 1% GlutaMAX (Gibco), and 1% penicillin-streptomycin-amphotericin solution B (Biological Industries (Ab/Am, BI) supplemented with 50 μM ZnCl ₂ (Millipore), 62 ng/ml EGF (R&D Systems), 100 ng/mL IGF-1 (R&D Systems), 10 ng/mL LIF (R&D Systems) and 10 ng/mL bFGF (R&D Systems) SCD differentiation medium consisted of DMEM/HEPES supplemented with 2% FBS and 1% Ab /Am Red fluorescent protein (RFP) expressing dermal fibroblasts (Lonza, USA) was cultured in Dulbecco's minimal nutrient medium (DMEM; Gibco LIFe Technologies) supplemented with 10% PBS (HyClone; Thermo Fisher Scientific), 1% non-essential amino acids ( Biological Industries), 0.2% β-mercaptoethanol (Biological Industries) and 100 units/mL penicillin and 0.1 mg/mL streptomycin (Pen-Strep Solution, Biological Industries, Israel) Myoblasts (America n Type Culture Collection) were cultured in DMEM supplemented with 10% FBS, 2.5% HEPES buffer (Biological Industries) and 1% penicillin/streptomycin solution. Myoblast differentiation medium consists of DMEM supplemented with 2% FBS, 2.5% HEPES buffer, and 1% penicillin/streptomycin solution. Incubations were carried out in a 5% (v/v) humidified CO ₂ atmosphere at 37°C. Bovine smooth muscle cells (SMC; CellApplications) were cultured as described in Table 3. Bovine aortic endothelial cells (ABEC; CellApplications) were cultured in commercial media (CellApplications). Bovine skeletal muscle microvascular ECs (MEKScMB; AngioProteomie) were cultured in commercial ECM medium (ScienCell) with 10% of the total amount of PBS. Bovine dermal fibroblasts (DBF; ScienCell) were cultured either in commercial medium (FM-2; ScienCell) or in high glucose DMEM supplemented with 15% PBS.

Immunohistochemistry

[0121] Matrices were washed twice in PBS, then fixed in 4% paraformaldehyde (PFA) for 20 minutes on a shaker. The matrices were washed three times with PBS for 5 minutes on a shaker and then permeabilized with 0.3% Triton X-100 (Bio Lab Ltd) for 10 minutes. The matrices were then washed three times with PBS for 5 minutes and then immersed in 5% bovine serum albumin (BSA; Millipore) in PBS overnight at 4°C. Matrices were incubated with 250 μl of primary antibody (in 5% BSA in PBS) for 3 hours at room temperature. After incubation, the matrices were washed 4 times with PBS for 5 minutes. Cells were then incubated at room temperature for 3 hours with secondary antibody (in PBS) and 4',6-diamidino-2-phenylindole (DAPI, 1:1000 in PBS; Vector Laboratories) in a tin foil plate on a shaker. The matrices were washed with PBS for 5 minutes on a shaker and imaged using a confocal microscope (LSM 700, Zeiss).

Bovine aortic smooth muscle cells (BSC)

[0122] The effect on cell proliferation of four different medium formulations was compared to commercial medium (BSM) ( Table 3 ). At the beginning of the experiment, the cells were in passage 8 and seeded at 4000 cells/cm ² in a 6-well plate. On day 3, cells were trypsinized, counted with a hemocytometer and reseeded at 4000 cells/cm ² . The experiment was carried out twice, using the same cells for each condition. One-way analysis of variance was used to test for statistical significance.

Cell Growth Kinetics

[0123] Approximately 4×10 ³ cells/cm ^{2 were} seeded and counted every day using a hemocytometer for six days. The calculated values were plotted on a graph.

Cell staining

[0124] Support cells (DPB or GMAB) were stained with 3 ml of fresh medium with 15 μl of DiD lipophilic labeling substances (1 mg/ml, dissolved in absolute ethanol; #D7757, Molecular Probes®, USA). Bovine endothelial cells (ECB; ECAB or MEXcMB) were stained with 3 ml of fresh medium with 5 μl of DiI lipophilic labels (3 mg/ml, dissolved in absolute ethanol; #D, Molecular Probes®, USA) and incubated for 30 min at 37 °C. After one wash with medium and then two more washes with PBS, the cells were trypsinized and ready for seeding as described below.

Seeding cells on a porous matrix

[0125] Bovine EC (1.25×10 ⁵ ) and support cells (0.25×10 ⁵ ) were resuspended in 5 µl of a 1:1 mixture of 15 mg/ml fibrinogen (Sigma Aldrich) and 20 NIH units/ml thrombin (Sigma Aldrich). Fibrinogen solution was prepared by diluting lyophilized human fibrinogen (Sigma Chemical) in 40 mM glycine tris buffer. Thrombin solution was prepared by diluting thrombin (Sigma Aldrich) in 40 mM calcium chloride. The coculture slurry was then seeded onto 4 mm round porous L-PLA-PLG matrices and allowed to solidify in uncultured 12-well plates for 30 minutes in an incubator (37° C., 5% CO ₂ ). After curing, 2 ml of endothelial culture medium (ECM, ScienCell) was added to each well with an additional 5% FBS and 2 nM VEGF. The medium was changed every other day.

[0126] To further customize the ratio of cells seeded on porous arrays, cells were seeded as described above with the following modifications: cells were first seeded in 1:1 medium (bEBM epithelial basal medium, AngioProteomie: commercial cell maintenance medium) and cells were resuspended in 5 µl of a 1:1 mixture of 15 mg/ml fibrinogen (Johnson and Johnson) and 2 U/ml thrombin (Johnson and Johnson).

2D Bovine Satellite Cell (BSC) Differentiation

[0127] 5×10 ⁴ CBP (passage 4) were plated on a 24-well plate, treated with cell culture, with a coverslip bottom in triplicate. Cells were grown according to the myotube formation protocol as follows: 4 days in growth medium and 7 days in differentiation medium (t = 0 refers to the point when differentiation medium was used). Cells were then stained with Hoechst and Myogenin on days -1 and 7.

Optimization of SBC differentiation in 2D

[0128] SKB (passage 4) were grown for 4 days in propagation medium with various RFs (as described below) and grown for 7 days in differentiation medium (DMEM-HEPES + 2% HS). Day 0 was defined as the day the medium was placed on the differentiation medium.

The compositions of the media for reproduction included: (1) Control group - Nutrient medium (without the addition of FR); (2) bovine fibroblast growth factor (bFGF; 10 ng/ml); (3) epidermal growth factor (EGF; 62 ng/ml); (3) insulin-like growth factor 1 (IGF-1; 100 ng/ml); (4) Pro-LIF; bFGF 10 ng/ml, EGF 62 ng/ml, IGF-1 100 ng/ml and ZnCl ₂ 50 μM); (6) Weaning RF: pro-LIF medium for 3 days, then 3 culture medium washes (every 5 minutes), then 1 day of culture medium; (7) Cells were stained with media-grown DiI to test whether DiI, a fluorescent dye added for cell tracking, affected cell differentiation.

[0129] The inventors further tested the effect of FR: IGF-1, EGF and combinations thereof during the differentiation phase. CBP (passage 4) were grown for 4 days in growth medium and then grown for 7 days in differentiation medium with various RFs (as shown below). Differentiation media formulations included: (1) Control group - stock differentiation media (DMEM-HEPES + 2% HS); (2) IGF-1 (100 ng/ml); (3) EGF (62 ng/ml); and (4) IGF-1 (100 ng/ml) + EGF (62 ng/ml). Cells were visualized using a bright field microscopy system for all cases described above.

Optimization of SKB reproduction on 3D matrices

[0130] For this purpose, a double two-factor experiment (seed volume and propagation medium composition) was set up in duplicate as follows: (1) seed volume and cell number: (a) seed volume 10 μl and 1×10 ⁶ CBP; (b) 10 µl seed volume and 2 x 10 ⁶ CBP; and (c) 20 µl of seed volume and 2 x 10 ⁶ CBP. (2) Propagation medium: (a) Nutrient medium; and (b) pro-LIF medium (nutrient medium + bFGF + EGF + IGF-1 + ZnCl ₂ ). SBC (passage 4) were stained with DiI and then seeded onto porous arrays (Arcon) according to the Arcon seeding protocol.

Optimization of SKB differentiation on 3D matrices

[0131] For this purpose, a two-factor experiment was set up (propagation medium in the first week and differentiation medium in the second week): (1) propagation medium: (a) Control group (Nutrient medium); (b) Pro-LIF; and (c) Pro-LIF - bFGF. (2) Differentiation medium either alone or enriched as follows: (a) Control group (differentiation medium with 2% horse serum); (b) + IGF-1; (c) + EGF; and (d) + IGF-1 + EGF. SBC (passage 4) were stained using DiI. Four (4) mm porous matrices were seeded with 0.5×10 ⁶ CBP (passage 4) in 5 µl of fibrin. Received the image of the cells at 2, 7 and 14 days after seeding. The matrices were stained with desmin. Staining was performed according to the staining protocol for total slide staining using a 1:00 goat anti-desmin primary polyclonal antibody (Santa Cruz Cat. No. SK-7559) and a 1:200,488 donkey anti-goat IgG secondary antibody (Invitrogen Cat. No. A11055). ).

Bovine satellite cell (BSC) staining before seeding

[0132] BSC were stained with 1 ml of fresh medium with 5 μl of DiI lipophilic labels (3 mg/ml, diluted in absolute ethanol, Molecular Probes®, USA) and incubated for 30 minutes at 37°C. After 1 wash with medium and then 2 washes with PBS, the cells were trypsinized and ready for seeding as described above.

Seeding cells on L-PLA-PLG

[0133] Bovine satellite cells (BSC) with or without bovine EC (BEC), and with or without support cells were resuspended in 6 µl of a 1:1 mixture of 15 mg/ml fibrinogen (Sigma Aldrich) and 20 NIH U/ml thrombin ( Sigma Aldrich). Fibrinogen solution was prepared by diluting lyophilized human fibrinogen (Sigma Chemical) in 40 mM glycine tris buffer. Thrombin solution was prepared by diluting thrombin (Sigma Aldrich) in 40 mM calcium chloride. The cell suspension was then seeded onto 4 mm round L-PLA-PLG arrays and allowed to solidify in uncultured 12-well plates for 30 minutes inside an incubator (37° C., 5% CO ₂ ). After curing, 1 ml of a 1:1 mixture of endothelial culture medium with SKB nutrient medium (Pro-LIF) was added to each well. The medium was changed every other day. One week later, the medium was replaced with CCD differentiation medium (enriched with IGF-1 and EGF) for an additional week.

Seeding cells on matrixes of structured soy protein

[0134] Bovine satellite cells (BSC) with or without bovine EC and with or without support cells were resuspended in 15 µl of a 1:1 mixture of 15 mg/ml fibrinogen (Sigma Aldrich) and 20 NIHU/ml thrombin (Sigma Aldrich). Fibrinogen solution was prepared by diluting lyophilized human fibrinogen (Sigma Chemical) in 40 mM glycine tris buffer. Thrombin solution was prepared by diluting thrombin (Sigma Aldrich) in 40 mM calcium chloride. The cell suspension was then seeded on both sides of 6 mm cross-linked soy protein circular arrays and allowed to solidify in uncultured 12-well plates for 45 minutes inside an incubator (37° C., 5% CO ₂ ). After curing, 1 ml of a 1:1 mixture of endothelial culture medium with SKB nutrient medium (Pro-LIF) was added to each well. The medium was changed every other day. One week later, the medium was replaced with CPS differentiation medium (enriched with IGF-1 and EGF) for an additional week.

[0135] Two ratios of seeding and 2 types of endothelial cells (and their appropriate medium) were tested as described in the tables below:

Table 1 - Seeding on matrices L-PLA\PLG

culture SKB MMC EKB GENERAL Tri-culture 2:1:1 250000 125000 125000 500000 Tri-culture 2:1:5 125000 62500 312500 500000 Coculture 2:1 250000 125000 375000 Coculture 2:1 250000 125000 375000 Control group monoculture for 2:1:1 250000 250000 Coculture 2:1 125000 62500 187500 Coculture 2:5 125000 312500 437500 Monoculture of the control group 2:1:5 125000 125000

Table 2 - Seeding on structured soy protein matrices

culture SKB MMC EKB GENERAL Tri-culture 2:1:1 750000 375000 375000 1500000 Tri-culture 2:1:5 375000 187500 937500 1500000 Control group monoculture for 2:1:1 750000 750000 Coculture 2:1 750000 375000 112500 Coculture 2:1 750000 375000 112500 Control group monoculture for 2:1:5 375000 375000 Coculture 2:1 375000 187500 562500 Coculture 2:5 375000 937500 1312500

Seeding cells on Gelfoam (gelatin hemostatic sponge)

[0136] Bovine cells (BBS) were resuspended in 15 µl of BBS culture medium. The cell suspension was then seeded on both sides of 6 mm diameter Gelfoam © arrays and allowed to solidify in uncultured 12-well plates for 30 min inside an incubator (37° C., 5% CO ₂ ). After curing, 1 ml of SKB nutrient medium (Pro-LIF) was added to each well. The medium was changed every other day. One week later, the medium was replaced with SCD differentiation medium (supplemented with IGF-1 and EGF) for an additional week.

Growing cells on matrices

[0137] One and two weeks after seeding, DiI-labeled CBP seeded on L-PLA/PLG and Arcon arrays were imaged using confocal microscopy to assess cell growth.

Immunofluorescent staining by the method of total preparation

[0138] Two weeks after seeding, matrices were fixed with 4% paraformaldehyde (PFA; Electron Microscopy Sciences) for 20 minutes, followed by three washes with PBS (Gibco® Life Technologies). Permeabilization was achieved by incubating the matrices for 15 minutes at room temperature (RT) in 0.3% Triton X-100 (Bio Lab Ltd). After three washes with PBS, the matrices were incubated overnight in blocking buffer (10% FBS, 1% (w/v) glycine and 0.01% Triton in PBS) at 4°C. Matrices were then incubated overnight at 4° C. with primary antibodies: 1:50 goat anti-CD31 (Santa Cruz), with or without 1:50 mouse anti-MYH (Santa Cruz), diluted in blocking buffer. After several PBS washes, samples were incubated with 1:400 donkey anti-goat Alexa Fluor®488 (Jackson ImmunoResearch), 1:50 Alexa Flour 647-conjugated mouse anti-myogenin (Santa Cruz), and 1:1000 DAPI (4', 6-diamidino-2-phenylindole, Sigma-Aldrich) with or without 1:300 vs. Alexa Flour 647 mouse, for 3 hours at RT. After several washes with PBS, arrays were immediately imaged on a Zeiss LSM700 confocal microscope (Carl Zeiss) using Zen software. Image processing and additional analysis was performed using FIJI software. L-PLA/PLG arrays were also immunofluorescently stained by the total preparation method one week after seeding.

Cryo-separation and trichrome staining

[0139] At the end of each experiment, the matrices were incubated overnight in 30% (w/v) sucrose solution at 4°C. Then the matrices were embedded in an optimum cutting temperature (OTC) compound (Tissue-Tec, USA) and frozen for subsequent cryo-separation. The matrices enclosed in OTP were subjected to cryo-separation into sections with a thickness of 5 and 10 μm. The 5 µm sections were then stained according to the standard trichrome staining protocol.

Example 1

3D Structural Analysis of a Porous TSB Matrix

[0140] TSB matrices were examined using scanning electron microscopy (SEM) to analyze their three-dimensional (3D) structure at both micron and nanoscale scales. The 100× images showed that TSB had a porosity with a pore size of 100-1000 µm, which can be used for cell culture. It was shown that the walls of the large TSB were thicker than those of the medium TSB ( Fig. 3A and 3B ). SEM images (2×10 ⁵ magnification) of a large TSB showed that the TSB contained 30 nm beaded protein clusters. Analysis of the average TSB showed a more amorphous glue-like material between these clusters ( Fig. 4A and 4B ).

Example 2

Growth and Proliferation of Fibroblasts Cultivated on a Porous Matrix

[0141] Investigated the attachment and proliferation of fibroblasts on the matrix of TSB. First, 50,000 CPB-labeled dermal fibroblasts were plated on a TSB matrix and cultured. After 14 days of incubation, the TSB matrix colonized with dermal fibroblasts was additionally seeded with 200,000 endothelial cells and incubated for an additional 7 days. Immunostaining of TSB samples populated with cells was carried out, images were obtained using confocal microscopy 8, 18 and 21 days after seeding of fibroblasts. The results demonstrated that fibroblasts proliferated and covered the TSB matrix even when seeded at a low number of 50,000 cells ( Fig. 5A ). After seeding the endothelial cells ( Fig. 5B ), endothelial cells were collected in clusters ( Fig. 5C ). Then matrices were examined from each source of matrices, cut at an angle of 90° to the direction of the TSB surface. Each of the matrices were seeded with 200,000 fibroblast cells and incubated for 14 days. The results demonstrated that fibroblast cells grew and proliferated on the large and medium TSB matrix (Figure 6 ). These results indicated that the cells could fill the matrix after 14 days of culture.

Example 3

Growing myoblasts cultured on a porous matrix

[0142] To evaluate the applicability of generating muscle tissue, experiments were performed to study the cultivation of muscle cells on a matrix of TSB.

[0143] For this purpose, 0.5x10 ⁶ myoblasts were plated on TSB arrays and cultured in the presence of differentiation medium, which was low in serum and therefore better suited for cultured meat. Images of myoblasts at day 14 ( Figure 7 ) showed that the myoblasts proliferated on the TSB matrix, hence the cells could populate the matrix after 14 days of culture.

Example 4

Proliferation of bovine skeletal muscle cells cultured on TSB

[0144] To assess applicability for human consumption, bovine skeletal muscle cells (bSKM) were seeded onto a TBP matrix. In particular, 0.5×10 ⁶ bKskM cells were seeded on each matrix and cultured in the presence of a differentiation medium. On day 14, cells were imaged by confocal microscopy. Images ( Fig. 8 ) showed that bKsM proliferated on the TSB matrix, indicating that TSB is useful for producing cultured meat.

Example 5

Proliferation of bovine aortic smooth muscle cells (SMCs)

[0145] The inventors examined the effect of four different media formulations compared to commercial media (BSM; Table 3) on the proliferation of MMC seeded ( FIGS. 9A-9E ) in the media described below.

Table 3. Compositions of various media tested for MMC

one 2 (basal) 3 four 5 BSM + - - - - Dulbecco's Modified Eagle's Medium (DMEM) - + + + + Fetal bovine serum (FBS) - ten% fifteen% ten% ten% Penicillin-streptomycin (PS) - one% one% one% one% Non-essential amino acids (NAC) - - - one% - sodium pyruvate - - - - 1 mM

[0146] On day 3, there was no significant difference in cell numbers in the five different media ( FIG. 10A ). On day 3 of the next experiment ( Fig. 10B ), there was a significant difference between the four different treatments (P < 0.01), and in retrospective analysis, it was found that the number of cells exposed to pyruvate-enriched medium was significantly higher compared to with medium enriched in non-essential amino acids and medium supplemented with 15% PBS (with P values<0.05 and <0.01, respectively). The difference between commercial medium and sodium pyruvate enriched medium was not significant (P=0.052). Therefore, the inventors used a medium enriched with sodium pyruvate (1 mm) for culturing HMBAB.

Example 6

Characteristics of the cultivation of DPB and MMC under certain conditions

[0147] After determining consumer media compositions, the inventors investigated the kinetics of their growth under these conditions (Example 5). It was found that the growth rates of DPB ( Fig. 11A ) and GMCAB are very similar ( Fig. 11B ).

Example 7

Effects of ECs and Supporting Cells on Vascularization

[0148] In order to test whether bovine endothelial cells (EC; BEAC - bovine aortic endothelial cells, or MEKScMB - bovine skeletal muscle microvascular endothelial cells) inoculated in co-culture with maintenance cells enhance vascularization and matrix coverage, ABEC was seeded together with DPB or GMKAB. Indeed, co-cultivation of fibroblasts along with myoblasts and endothelial cells has been shown to increase the vascularization of artificial muscle constructs and contribute to the stabilization of the vessel structure over time. The cells were found to cover the entire matrix ( Fig. 12 ). In addition, when ABEC was seeded in co-culture with GMAB ( Fig. 12A ), the inventors noticed that EC staining decreased over time. Also in this combination, a vessel-like structure was visible on day 2. When ABEC was seeded in co-culture with DPB ( Fig. 12B ), the inventors did not notice the vascular structures. When MEXxMB was seeded in co-culture with GMAB ( FIG. 13A ), the inventors noticed much more GMAB than MEXxMB. In addition, vessel-like structures were visible with GMAB ( Fig. 13A ) and with DPB ( Fig. 13B ), during the entire period of the experiment.

Example 8

A certain ratio of cells improves the formation of a network of blood vessels

[0149] To further improve the vascular network, the inventors seeded bovine ABEC with supporting cells (DPB or GMAB) in various ratios. The inventors investigated 3 different ratios of EC: 1:3, 1:1 and 5:1 to support cells. The inventors clearly observed self-assembly of ABEC cells (FIGS . 14-16 ), which created vessels, as early as day 2 in the presence of HMBAB (FIGS . 14 and 16 ). The inventors also noticed that by day 6 there were fewer ECs than by day 2 and that ABEC created a grid area of ships at a ratio of 1:1 ABEC to GMAB. ABEC in the presence of DPB did not appear to create vessels ( Fig. 15 ).

Example 9

Differentiation of bovine satellite cells (BSC) into cell culture

[0150] The inventors then examined myogenin expression before and after muscle tube formation from CBP and further assessed the ability to use DAPI-myogenin to measure fusion index (IC). The inventors found little evidence of myogenin expression in undifferentiated SCD ( Fig. 17 ). The expression of myogenin occurred mainly in the nuclei of the myotubes and in some nuclei around the myotubes. It was shown that the fusion index (FI) can be calculated automatically from the ratio of the number of myogenin/Hoechst nuclei, which turned out to be comparable to the manual calculation of FI. This protocol was additionally used to quantify myotube formation.

Example 10

Optimization of SBC differentiation in cell culture

[0151] Next, the inventors wanted to optimize 2D differentiation of SBCs. The inventors tested which growth factors (GFs) can be added during (1) the breeding phase, which will not interfere with the formation of the muscular tube during the differentiation stage, and (2) at the end of the differentiation phase, which will increase the size of the muscular tube and IS.

[0152] After the breeding phase and before the differentiation phase, samples that did not contain bFGF ( Fig. 18) appeared spontaneous myotubes, indicating that bFGF inhibits the formation of myotubes. EGF, IGF-1 and DiI do not appear to affect muscle tubule formation. An increase in cell population density occurred in IGF-1, Pro-LIF and weaning. On day 4 of differentiation, the inventors observed an increase in the total number of myotubes under bFGF and Pro-LIF culture conditions. bFGF maintained the stemness of the CBP, and after cell removal, differentiation suddenly began ( Fig. 19 ). On day 7 of differentiation, the inventors observed a greater area of the muscular tube in the control group, weaning and DiI compared to other test conditions ( Fig. 20 ). The inventors concluded that there is a fine line between muscle tubule formation and cell death. Accordingly, cells should be grown in growth medium containing bFGF (alone or with Pro-LIF) until population is reached, and then transferred to differentiation medium for a short period of 2-5 days. In addition, the overspreading of myotubes is shown when IGF-1 or IGF-1 and EGF are added to the cell culture medium.

Example 11

Optimization of SKB Reproduction on a Porous Matrix

[0153] The inventors then attempted to increase the CBP coverage on the TSB matrix above 60%, as previous experiments showed low coverage of cells on a porous matrix. Thus, FR was added to the expansion phase, and the initial number of cells increased upon seeding. Quantification of cell coverage showed that cells cultured with FR-enriched medium (i.e., Pro-LIF) covered 72 ± 15% of the matrix, while cells cultured solely on the medium covered approximately 18% of the matrix ( Fig. 21 ). Difference that was found to be statistically significant between the two treatments (P value = 0.0009).

Example 12

Optimization of SBC Differentiation on a Porous Matrix

[0154] In order to find the optimal conditions for the formation of a muscular tube on 3D matrices, the inventors scanned many combinations of media for reproduction and sequential media for differentiation. Muscle tubules formed without RF in differentiation media ( Fig. 22A, E and I ) were small and rounded. The addition of IGF-1 to differentiation media resulted in the appearance of myotubes, which were rarely observed in nutrient media ( Fig. 22B ), usually in Pro-LIF-bFGF ( Fig. 22J ), and abundant in Pro-LIF media ( Fig. 22F ). The addition of EGF to differentiation media resulted in the appearance of myotubes in all growth media ( Fig. 22C, G and K ). The addition of IGF-1 + EGF resulted in the appearance of myotubes in the nutrient medium ( Fig. 22D ) and Pro-LIF-bFGF ( Fig. 22L ), as well as the abundance of myotubes in Pro-LIF ( Fig. 22H ).

Example 13

Tri-Culture Increases Proliferation, Elongation, and Coating of SKB on a Porous Matrix

[0155] Next, the inventors wanted to investigate the effect of tri-culture SCD with a constant number of smooth muscle cells (SMC) and varying amounts of skeletal microvascular endothelium (ScMVEC) (2:1:1 and 2:1:5, respectively) on SCD survival, proliferation and differentiation of myotubes (positive for the marker of differentiation, myogenin). The inventors observed that at a low concentration of CMBEK (2:1:1), the CBP appeared to be more elongated compared to their appearance when incubated with a higher concentration of CMBEK (2:1:5; FIG. 28 ). The inventors also observed an increase in CBP coverage on the porous matrix and, apparently, an increase in differentiation compared to monoculture or co-cultures containing CBP (control groups). The inventors concluded that the tri-cultures better supported SBP proliferation and differentiation compared to other groups tested in both incubation ratios and deduced the potential for SBP differentiation as follows: SBP only <SBP+EC<SCP+GMC<SCP+EC+ MMC ( Fig. 23-26 and 28 ). The inventors also observed secretion of extracellular matrix (ECM) in tri-cultures of SKB+SMC+ScMWEC (2:1:1) ( FIGS. 27A-27B ).

Example 14

Tri-Culture Enhances CBP Differentiation in Structured Soy Protein Matrix

[0156] Next, the inventors wanted to investigate the effect of culturing three cultures of SBCs with a constant number of smooth muscle cells (SMCs) and skeletal microvascular endothelium (SCMEC) (2:1:1, respectively) on SCS survival, proliferation and differentiation of myotubes (positive for the marker differentiation - myogenin). The inventors observed not only an increase in SBP coverage on the TBP matrix when seeded either in co-culture with SMA or in a tri-culture with SMA and ECP, but also an increase in the differentiation of SBP compared to monoculture (SBP only) or co-culture (SBP with ECP; Fig. 29 ).

Example 15

Seeding SKB without adding hardener gel

[0157] Next, the inventors wanted to study the effect of seeding of SBCs on a US-approved collagen-based matrix (Gelfoam ©) without the addition of hardeners (e.g., fibrin). The inventors observed successful differentiation of SBCs, which was demonstrated by the formation of a muscular tube ( Fig. 30 ).

[0158] While some features of the invention have been illustrated and described herein, many modifications, substitutions, alterations, and equivalents will now be apparent to those skilled in the art. Therefore, it is to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims (29)

1. A method for producing an edible composition, comprising the steps of: a. incubating an edible three-dimensional porous matrix and a plurality of non-human animal cell types containing: (i) myoblasts or progenitor cells thereof; and at least one of the following: (ii) at least one cell type that secretes extracellular matrix (ECM); or (iii) at least one type of ECM-secreting cells and endothelial cells or progenitors thereof, wherein said endothelial cells or progenitors thereof comprise less than 15% of said plurality of cells; and b. inducing differentiation of myoblasts or their progenitor cells into myotubes, resulting in an edible composition. 2. The method of claim 1, wherein said plurality of cell types comprises myoblasts or progenitor cells thereof, at least one extracellular matrix (ECM) secreting cell type, and endothelial cells or progenitor cells thereof. 3. The method according to any one of claims 1 or 2, wherein said ECM-secreting cell is selected from the group consisting of stromal cells, fibroblasts, pericytes, smooth muscle cells, and progenitor cells thereof. 4. The method according to any one of claims 1 to 3, wherein said plurality of cell types comprises myoblasts, ECM-secreting cells, and endothelial cells. 5. The method according to any one of claims 1 to 4, wherein said myoblast progenitor cell is a satellite cell. 6. The method of any one of claims 1 to 5, wherein said endothelial cells are selected from skeletal microvascular endothelial cells, aortic smooth muscle cells, or combinations thereof. 7. The method of any one of claims 1 to 6, wherein said plurality of cell types comprises satellite cells, ECM-secreting cells, and endothelial cells. 8. The method of claim 7, wherein said ECM-secreting cell is a fibroblast, a progenitor cell thereof, or a combination thereof. 9. The method of claim 1, wherein said three-dimensional porous matrix is selected from the group consisting of a structured protein, an unstructured protein, and a polysaccharide. 10. The method of claim 9 wherein said structured protein is structured soy protein. 11. The method according to any one of claims 9 or 10, wherein said three-dimensional porous matrix contains pores with an average diameter in the range of 20 to 1000 micrometers. 12. The method according to any one of claims 1 to 11, wherein said plurality of cells originate from a livestock mammal. 13. The method according to any one of claims 1 to 12, wherein said myoblasts or progenitor cells thereof and said three-dimensional porous matrix are incubated at a ratio in the range of 103 to 107 ^of said myoblasts or progenitor cells thereof to 10 mg ^of said three-dimensional porous matrices. 14. The method according to any one of claims 1 to 13, wherein said three-dimensional porous matrix further comprises an extracellular matrix. 15. An edible composition containing: a. an edible three-dimensional porous matrix; b. muscle tubules of a non-human animal containing 10,000-250,000 myotube nuclei per mm ^{3 of} said three-dimensional porous matrix; and With. a plurality of non-human animal cell types selected from the group consisting of: (i) myoblasts or progenitor cells thereof; and at least one of: (ii) at least one type of ECM-secreting cell; or (iii) endothelial cells or their precursor cells, wherein said endothelial cells or their precursor cells comprise less than 15% of said plurality of cells. 16. The composition of claim 15 wherein said plurality of cell types comprises myoblasts or progenitor cells thereof, at least one extracellular matrix (ECM) secreting cell type, and endothelial cells or progenitor cells thereof. 17. A composition according to claim 15 or 16, wherein said myoblast progenitor cell is a satellite cell. 18. A composition according to any one of claims 15-17, wherein said plurality of cell types comprises satellite cells, ECM-secreting cells, and endothelial cells. 19. A composition according to any one of claims 15-18, wherein said ECM-secreting cell is a fibroblast, its progenitor cell, or a combination thereof. 20. A composition according to any one of claims 15-19, wherein said three-dimensional porous matrix is selected from the group consisting of a structured protein, an unstructured protein, and a polysaccharide. 21. The composition of claim 20 wherein said structured protein is a structured soy protein. 22. A composition according to any one of claims 15 or 21, wherein said three-dimensional porous matrix contains pores with an average diameter in the range of 20 to 1000 micrometers. 23. A composition according to any one of claims 15 to 22, wherein said plurality of cells are derived from a livestock mammal. 24. A composition according to any one of claims 15-23, wherein said three-dimensional porous matrix further comprises an extracellular matrix.

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