Cultured meat components

Abstract

The present invention relates to a method for producing an edible composition comprising incubating a three-dimensional porous scaffold with multiple cell types including myoblasts or their precursor cells, at least one extracellular (ECM)-secreting cell and endothelial cells or their precursor cells, and inducing differentiation of the myoblasts into myotubes. [Selection diagram] No diagram

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 532,998, filed on 15 July 2017, the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates, in particular, to a cultured meat composition and a method for producing the same. [Background technology]

[0003] Cultured meat, also known as artificial meat or clean meat, is produced from cell cultures using tissue engineering technology and is a significant alternative to conventional meat production that uses live animals. Over the past decade, the concept has gained increasing attention in public opinion, influential media, investors, and the scientific community, particularly after the production of the first cultured beef burger. Producing food using animals is considered inefficient because animals consume large amounts of food throughout their lives. In this production, 80-90% of calories are wasted in the metabolism of the animals and the production of non-edible tissues. While meat has the greatest environmental impact when comparing various industries, generally speaking, all animal-based products have a larger environmental footprint compared to plant-based products in terms of soil and water demand, as well as greenhouse gas (GHG) emissions. According to a report by the United Nations Food and Agriculture Organization, the livestock sector accounts for 18% of GHG emissions, uses 30% of the Earth's landform or 70% of arable land, and uses 8% of the world's freshwater. Furthermore, global meat demand is projected to double by 2050, which means that conventional meat production systems are no longer sustainable. Compared to several meat sources, cultured meat is estimated to reduce energy use by 7–45%, GHG emissions by 78–96%, land use by 99%, and water use by 82–96%.

[0004] Intensive factory farming and inadequate animal welfare conditions contribute to the spread of foodborne diseases such as swine and avian influenza, as well as E. coli, Salmonella, and Campylobacter, which can be found in meat. Producing meat in a sterile environment can help improve food safety. Furthermore, 70% of all antibiotics used in the United States are given to livestock as food additives, which promote the selection of antimicrobial-resistant strains and increase the likelihood of multidrug-resistant bacteria. The overuse of antibiotics is a major cause of the emergence of antibiotic-resistant bacteria, resulting in an economic burden of $55 billion annually in the United States alone, 2 million infections, 250,000 hospitalizations, and at least 23,000 deaths. In recent years, bacteria resistant to colistin, a last resort antibiotic, have emerged in pig farms in China.

[0005] Current cultured meat technology focuses on satellite cell culture. Cells are grown, separated, differentiated, and harvested on microcarriers in two-dimensional (2D) flasks or suspensions. However, tissue is not composed solely of cells. The extracellular matrix (ECM), which consists of macromolecules such as glycoproteins and oligosaccharides and confers biochemical and biomechanical properties to tissue, constitutes the majority of tissue. The ECM regulates cell behavior and influences its composition. Therefore, ECM-producing cells are essential for cultured meat, and processes such as cell harvesting should be avoided. Furthermore, three-dimensional (3D) cell culture mimics the natural cellular environment and is crucial for precise cell behavior that influences the biochemical contents of cells. [Overview of the project] [Problems that the invention aims to solve]

[0006] In some embodiments, the present invention relates to edible compositions containing myotubes and methods for producing edible compositions.

[0007] This invention is partly based on the discovery that cell cultures containing multiple cell types (e.g., satellite cells, ECM secretory cells, and endothelial cells) showed better survival, proliferation, and myotubation compared to controls when grown on a three-dimensional porous scaffold.

[0008] This invention is further partly based on the surprising discovery that satellite cells, such as non-human satellite cells, exhibited better differentiation activity when co-cultured at low endothelial cell (EC) concentrations rather than high endothelial cell (EC) concentrations.

[0009] According to one embodiment, a method for producing an edible composition is provided, the method comprising the following steps: (a) incubating a three-dimensional porous scaffold and a plurality of cell types comprising (i) myoblasts or their progenitor cells; and (ii) at least one extracellular matrix (ECM) secreting cell, or (iii) endothelial cells or their progenitor cells, wherein the myoblasts or their progenitor cells and endothelial cells or their progenitor cells are incubated in a ratio ranging from 10:1 to 1:10; and (b) inducing the differentiation of myoblasts or their progenitor cells into myotubes, thereby producing an edible composition.

[0010] In some embodiments, the multiple cell types include myoblasts or their progenitor cells, at least one extracellular matrix (ECM) secreting cell, and endothelial cells or their progenitor cells.

[0011] In some embodiments, ECM secretory cells are selected from the group consisting of stromal cells, fibroblasts, pericytes, smooth muscle cells, and their precursor cells.

[0012] In some embodiments, the multiple cell types include myoblasts, ECM secretory cells, and endothelial cells.

[0013] In some embodiments, the progenitor cells of myoblasts are satellite cells.

[0014] In some embodiments, the endothelial cells are selected from skeletal microvascular endothelial cells, aortic smooth muscle cells, or combinations thereof.

[0015] In some embodiments, the plurality of cell types includes satellite cells, ECM-secreting cells, and endothelial cells.

[0016] In some embodiments, myoblasts or their progenitor cells and ECM-secreting cells are incubated at a ratio in the range of 10:1 to 1:1.

[0017] In some embodiments, ECM-secreting cells and endothelial cells are incubated at a ratio in the range of 1:10 to 1:1.

[0018] In some embodiments, satellite cells, ECM-secreting cells, and endothelial cells are incubated at a ratio in the range of 10:1:1 to 2:1:10.

[0019] In some embodiments, the ECM-secreting cells are fibroblasts, their progenitor cells, or combinations thereof.

[0020] In some embodiments, the three-dimensional porous scaffold is selected from the group consisting of textured proteins, non-textured proteins, and polysaccharides. In some embodiments, the textured protein is a textured soy protein. In some embodiments, the three-dimensional porous scaffold contains pores having an average diameter in the range of 20 to 1,000 micrometers.

[0021] In some embodiments, the plurality of cell types are non-human cells. In some embodiments, the plurality of cells are derived from domestic mammals.

[0022] In some embodiments, myoblasts or their progenitor cells and the three-dimensional porous scaffold are such that the myoblasts or their progenitor cells are 10 3 ~10 7It is incubated in a ratio within the range of . In some embodiments, the three-dimensional porous scaffold further includes an extracellular matrix.

[0023] In another embodiment, (a) three-dimensional porous scaffolding; (b) three-dimensional porous scaffolding 1 mm 3 A composition is provided comprising (c) myotubes containing 100,000 to 250,000 myotubes per unit; and (d) a plurality of cell types, selected from the group consisting of (i) myoblasts or their progenitor cells; and (ii) at least one type of ECM secretory cell, or (iii) at least one endothelial cell or its progenitor cell constituting less than 15% of the plurality of cells.

[0024] In some embodiments, the composition is edible.

[0025] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as those generally understood by those skilled in the art relating to the present invention. Similar or equivalent methods and materials to those described herein may be used in carrying out or testing embodiments of the present invention, but exemplary methods and / or materials are described below. In case of any inconsistency, the patent specification containing the definitions shall prevail. Furthermore, materials, methods, and examples are illustrative and not necessarily intended to limit the scope.

[0026] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given below. However, please understand that the detailed description and specific examples, while illustrating preferred embodiments of the present invention, are given only as examples, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the modes for carrying out the invention. [Brief explanation of the drawing]

[0027] [Figure 1]Figures 1A-1C. Photographs showing commercially available TSP. (A) shows large, medium, and small chunk products of TSP. (B) and (C) show two other commercially available TSP flake products annotated as TVP (left) and Arcon (right). [Figure 2] Figures 2A-2B. Photographs showing the preparation of the TSP scaffold (A) and the TSP scaffold containing cells (B). [Figure 3] Figures 3A-3B. SEM images at 100x magnification of large TSP (A) and moderate TSP (B). [Figure 4] Figures 4A-4B. SEM images at 2x10⁵ magnification of large TSP (A) and moderate TSP (B). [Figure 5] Figures 5A-5C. Confocal microscope images of TSP scaffolds incorporating fibroblasts (red) and endothelial cells (green) at 8 days (A), 18 days (B), and 21 days (C) after fibroblast seeding. [Figure 6] These are confocal microscope images taken 14 days after seeding 200,000 fibroblasts (red) into three different scaffold samples (Samples 1-3) obtained from different sources, indicated as large, medium, small, and 90° to medium. The scaffold indicated as 90° to medium was cut from the pore side of the TSP scaffold prepared using the TSP scaffold procedure. Scale bar = 1 mm. [Figure 7] These are confocal microscope images of myoblasts cultured on a TSP scaffold for 14 days (blue - DAPI, green - desmin). [Figure 8] These are confocal microscope images of bovine skeletal muscle cells cultured on a TSP scaffold for 14 days (blue - DAPI, green - phalloidin). [Figure 9] Figures 9A-9E. Micrographs of bovine aortic smooth muscle cells (BAOSMCs) (8 passages) seeded in different media. (A) Basal medium, (B) Commercial medium, (C) Fetal bovine serum (15%) supplemented, (D) Non-essential amino acids supplemented, and (E) Pyruvate supplemented. Scale bar = 100 μm. [Figure 10]Figures 10A-10B. These vertical bar graphs show a comparison of the proliferation of bovine aortic smooth muscle cells in different culture media at passage 9(A) and passage 10(B) three days after culture. [Figure 11] Figures 11A-11B. Graphs illustrating the growth rates of bovine dermal fibroblasts (BDF) (A) and bovine aortic smooth muscle cells (BAOSMC; B). [Figure 12] Figures 12A–12L. Images of fluorescently labeled bovine aortic endothelial cells (BAECs) seeded in co-culture with supporting cells on day 2 (A, D, G, and J), day 6 (B, E, H, and K), and day 8 (C, F, I, and L). BAEC cells (green) were seeded with either bovine aortic smooth muscle cells (SMCs; A–F) or bovine dermal fibroblasts (BDFs; G–L) (both red). A–C and G–I are 3x3 tile scans at 5x magnification. D–F and J–L are at 20x magnification. Scale bar = 100 μm. [Figure 13] Figures 13A–13L. Images of fluorescently labeled bovine skeletal muscle microvascular endothelial cells (BSkMVECs) seeded in co-culture with supporting cells on day 2 (A, D, G, and J), day 6 (B, E, H, and K), and day 8 (C, F, I, and L). BSkMVEC cells (green) were seeded with either bovine aortic smooth muscle cells (SMC A–F) or bovine dermal fibroblasts (BDF; G–L) (both red). A–C and G–I are 3x3 tile scans at 5x magnification. D–F and J–L are at 20x magnification. Scale bar = 100 μm. [Figure 14] Figures 14A–14F. Images of co-cultures of fluorescently labeled cells containing BAEC and SMC in different ratios. (A and D) BAEC vs. SMC 5:1; (B and E) BAEC vs. SMC 1:1; (C and F) are higher magnification images of B and E, respectively. A–C were taken on day 2, and D–F were taken on day 6. BAEC is red. Scale bar = 100 μm. [Figure 15]Figures 15A–15E. Images of co-cultures of fluorescently labeled cells containing BAEC and BDF in different ratios. (A and C) BAEC vs. SMC 5:1, (B and D) BAEC vs. SMC 1:1. (E) is a higher magnification of D. A and B are images taken on day 2. C and D are images taken on day 6. BAEC is red. Scale bar = 100 μm. [Figure 16] Figures 16A–16L. Images of co-cultures of fluorescently labeled cells containing BAEC and SMC in different ratios. (A–D) BAEC vs. SMC 1:3; (E–H) BAEC vs. SMC 1:1; and (I–L and F) BAEC vs. SMC 5:1. (B, D, F, H, J, and L) are higher magnifications than (A, C, E, G, I, and K), respectively. (A, B, E, F, I, and J) are images taken on day 2, and (C, D, G, H, K, and L) are images taken on day 6. BAEC is red. Scale bar = 100 μm. [Figure 17] Figures 17A–17G show the differentiation and myotube formation of bovine satellite cells (BSCs). (A–C) are images of pre-differentiation BSC cells. (D–F) are images of post-differentiation BSC cells. (G) is a vertical bar graph showing the fusion index (FI) of the samples. (A and D) light microscope, (B and E) Hoechst, and (C and F) myogenin. There was no difference between automated and manual FI, but the difference between pre- and post-differentiation automated FI was found to be statistically significant (P-value = 0.00003). [Figure 18] Figures 18A-18G. Bright-field micrographs of BSCs on day 0 of differentiation. (A) Control, (B) Bovine fibroblast growth factor (bFGF), (C) Epidermal growth factor (EGF), (D) IGF-1 (insulin-like growth factor 1), (E) Pro-LIF (growth medium without growth factor LIF (leukemia inhibitor)), (F) Weaning, and (G) Dil. Scale bar = 300 μm. [Figure 19]Figures 19A-19G. Bright-field micrographs of BSCs on day 4 of differentiation. (A) Control; (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) Dil. Scale bar = 300 μm. [Figure 20] Figures 20A-20H. These figures demonstrate myotube formation in BSCs on day 7 of differentiation. (A-G) are bright-field micrographs of (A) control, (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) Dil. Scale bar = 300 μm. (H) is a vertical bar graph of quantified myotube area % for each of the different dilation conditions. [Figure 21] This is a vertical bar graph showing the quantitative values ​​of cell coverage on day 7 in a porous scaffold of growth medium or Pro-LIF medium. [Figure 22] Figures 22A-22L. Confocal microscope images of fluorescently labeled BSCs differentiated on a porous scaffold. Diastolic stage: (A-D) Cells cultured in growth medium, (E-H) Cells cultured on Pro-LIF medium, (I-L) Cells cultured in Pro-LIF-bFGF medium. Differentiation stage: (A, E, and I) No growth factor (GF) was added to the differentiation medium. (B, F, and J) IGF-1 was added to the differentiation medium. (C, G, and K) EGF was added to the differentiation medium. (D, H, and L) IGF-1 and EGF were added to the differentiation medium. Dil (red), desmin (green), scale bar = 100 μm. [Figure 23]Figures 23A-23O. Immunofluorescence microscopy images of three cultures grown on PLLA / PLGA scaffolds, taken 14 days after seeding. BSC, SMC, and BAEC were seeded on PLLA / PLGA scaffolds at different cell densities (A-E) 2:1:1 and (F-J) 2:1:5. (K-O) are magnifications of (A-E). Scaffolds were stained with DAPI (A, F, and K), myogenin (B, G, and L), DiI (C, H, and M), and CD31 (D, I, and N). (E, J, and O) are combined images. Scale bar = 100 μm (A-J); 10 μm (K-O). [Figure 24] Figures 24A–24F. Immunofluorescence micrographs of PLLA / PLGA scaffolds 14 days after seeding. BSCs, SMCs, and BECs were seeded on PLLA / PLGA scaffolds at different cell densities: (A–C) 2:1:1 and (D–F) 2:1:5. (A and D) BSCs and SMCs (2:1), (B and E) BSCs and BECs (2:1), and (C and F) BSCs only in single cultures. Scaffolds were stained with DAPI (blue), myogenin (gray), DiI (red), and CD31 (green). Scale bar = 100 μm. [Figure 25] Figures 25A-25H. Immunofluorescence microscopy images of BSCs co-cultured with SMCs on a textured soy protein scaffold 14 days after seeding. Different cell densities (A-D) 2:1:1 and (E-H) 2:1:5 were tested. The scaffolds were stained with DAPI (A and E), myogenin (B and F), and DiI (C and G). (D) and (H) are combined images of (A-C) and (E-G), respectively. Scale bar = 100 μm. [Figure 26] Figures 26A-26E. Immunofluorescence microscope images of BSCs, SMCs, and BECs cultured 14 days after sowing on a textured soy protein scaffold. The scaffold was stained with (A) DAPI, (B) myogenin, (C) DiI, and (D) CD31. (E) is a combined image of (A-D). [Figure 27]Figures 27A-27B. Micrographs of trichrome-stained cultures of BSC, SMC, and BEC seeded 14 days after seeding in a 2:1:1 ratio on PLLA / PLGA scaffolds. (A) 5x magnification tile scan and (B) 10x magnification tile scan. The scaffolds were trichrome-stained. Positive extracellular matrix (ECM) staining is circled. Scale bar = 100 μm. [Figure 28] Figures 28A-28C. These are vertical bar graphs showing the effect of supporting cells on the importance of myogenic differentiation and different cell ratios in culture. Myogenic differentiation is shown by statistical analysis for three cultures (BSC:SMC:BEC) and a control (co-culture or monoculture) with cell densities of (A) 2:1:1, (B) 2:1:5 on PLLA / PLGA scaffolds, and (C) 2:1:1 on textured soy protein. [Figure 29] Figures 29A-29N. These figures describe the differentiation of cells in three cultures on textured protein scaffolds. (A-L) are fluorescence images of the textured soy protein scaffolds after immunostaining following 14 days of seeding. Cells were seeded into three cultures of textured soy protein scaffolds: (A, E, and I) BSC, (B, F, and J) BSC+BEC, (C, G, and K) BSC+SMC, and (D, H, and L) BSC+SMC+BEC. (A-D) x20 magnification, (E-H) x20 tile scans, and (I-L) myogenin staining, x63 magnification. Scaffolds were stained with DAPI (blue), myogenin (gray), DiI (red), and CD31 (green). Scale bar = 100 μm. (M) is a bar graph showing the quantification of myogenin expression on TSP scaffolds in single culture (BSC only), co-culture (BSC + BEC; or BSC + SMC), and triple culture (BSC + SMC + BEC). (N) is a bar graph showing the quantification of BSC coating on TSP scaffolds in single culture and co-culture with SMC. [Figure 30] This is a fluorescence image of immunohistochemically stained BSCs (Best Surfactants) 14 days after seeding, seeded on a Gelfoam scaffold without gel. The scaffold was stained with DAPI (blue), myogenin (gray), DiI (red), and CD31 (green). Scale bar = 50 μm. [Modes for carrying out the invention]

[0028] The present invention relates to compositions, kits, and methods for producing cultured meat for food consumption. In some embodiments, the present invention relates to an edible composition comprising a porous textured protein, myoblasts attached thereto, and a three-dimensional polymorphic cell tissue comprising one or more cell types. The present invention further relates to a method for producing the composition by in-vitro culturing myoblasts and one or more cell types together with a three-dimensional porous scaffold (e.g., a porous textured protein) under specific conditions.

[0029] In some embodiments, the compositions of the present invention may be intended for consumption by humans, non-human animals, or both. In some embodiments, the cultured meat product is a food product for human consumption. In other embodiments, the cultured meat product is used in animal feed, such as livestock feed, aquaculture feed, or household pet feed.

[0030] In some embodiments, the present invention relates to a method for producing an edible composition comprising: a. Incubating a three-dimensional porous scaffold, and at least one ECM-secreting cell type selected from the group consisting of myoblasts, adipocytes, fibroblasts, and their progenitor cells, as well as multiple cell types including endothelial cells or their progenitor cells, b. To enable multiple cell types to expand on a three-dimensional porous scaffold, c. Inducing the differentiation of myoblasts into myotubes, This allows cells to form edible compositions containing muscle cells and three-dimensional polymorphic cell tissues including porous textured proteins.

[0031] As used herein, the term “myotube” refers to a multinucleated fiber formed from the fusion of multiple myoblasts and / or muscle cells. As used herein, the term “muscle cell” refers to any cell that contributes to muscle tissue and may include myoblasts, satellite cells (SCs), myotubes, muscle fibers, and myofibrils.

[0032] In some embodiments, this method includes culturing myoblasts in vitro or ex vivo and differentiating these cells into a specific type of muscle cell, such as skeletal muscle cells or smooth muscle cells.

[0033] In some embodiments, the present invention relates to an edible composition comprising a porous textured protein and a three-dimensional polymorphic cell tissue comprising muscle cells, wherein the three-dimensional polymorphic cell tissue is attached to the porous textured protein, and the three-dimensional polymorphic cell tissue is derived from culturing in vitro with the porous textured protein a plurality of cell types, including myoblasts and one or more cell types selected from adipocytes, fibroblasts, smooth muscle cells, endothelial cells and their precursor cells.

[0034] As illustrated below, the composition may include fibroblasts that secrete extracellular molecules, such as forming an extracellular matrix (ECM) that provides further structural and mechanical support to the cells.

[0035] As further illustrated below, the composition may include endothelial cells (ECs) or endothelial progenitor cells (EPCs) for providing tissue support, providing signaling, and / or forming capillary endothelium.

[0036] In some embodiments, the three-dimensional polymorphic cell tissue includes muscle cells such as skeletal muscle cells, smooth muscle cells, and satellite cells. In some embodiments, the three-dimensional polymorphic cell tissue includes fat cells (e.g., adipocytes). In some embodiments, the three-dimensional polymorphic cell tissue includes an extra cell matrix secreted by specialized cells (e.g., fibroblasts). In some embodiments, the three-dimensional polymorphic cell tissue includes endothelial cells such as aortic endothelial cells and skeletal microvascular endothelial cells, or capillary endothelium formed by endothelial cells, but is not limited to these. In some embodiments, the three-dimensional polymorphic cell tissue further includes an extra cell matrix. In some embodiments, the three-dimensional polymorphic cell tissue further includes adipocytes. In some embodiments, the three-dimensional polymorphic cell tissue further includes capillaries.

[0037] In some embodiments, the present invention relates to compositions suitable for cell proliferation, comprising porous textured proteins and cell culture media. In other embodiments, the compositions suitable for cell proliferation further comprise growth factors, 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.

[0038] In some embodiments, the present invention relates to compositions comprising myoblasts and one or more cell types selected from adipocytes, fibroblast endothelial cells, smooth muscle cells, and their precursor cells, and comprising multiple cell types that adhere to porous textured proteins.

[0039] In some embodiments, the present invention provides a kit comprising a three-dimensional porous scaffold (e.g., a porous textured protein), and one or more cell types selected from myoblasts and adipocytes, fibroblasts, smooth muscle cells, endothelial cells, and their progenitor cells. In some embodiments, the kit is for the production of an edible composition. In some embodiments, the kit further comprises at least one component selected from cell culture media, growth factors, differentiation media, and differentiation inducers. In some embodiments, the kit further comprises cell culture media. In another embodiment, the cell culture media is selected from dry powder media, granular preparations, aqueous liquids, or culture medium concentrates. In some embodiments, the multiple cell types are frozen. In some embodiments, the kit further comprises instructions for use. Multiple cell types

[0040] As those skilled in the art will understand, multiple cell types or cell populations can be cultured with a three-dimensional porous scaffold, thereby enabling the formation of a three-dimensional polymorphic cell tissue architecture. In some embodiments, one or more cell types are selected from myoblasts, ECM secretory cells, and endothelial cells.

[0041] In some embodiments, one or more cell types are myoblast progenitor cells. In some embodiments, one or more cell types are ECM secretory cell progenitor cells. In some embodiments, one or more cell types are endothelial cell progenitor cells.

[0042] As used herein, progenitor cells include mesenchymal stem cells (MSc), embryonic stem cells (ESc), adult stem cells, differentiated ESc, differentiated adult stem cells, and induced pluripotent stem cells (iPSc). As used herein, the term “progenitor cell” refers to cells that can give rise to differentiated cells in multiple lineages, such as myoblasts, fibroblasts, adipocytes, stromal cells, pericytes, smooth muscle cells, and endothelial cells. “Progenitor cells” typically differ from stem cells in that they do not possess extensive self-renewal capabilities.

[0043] In some embodiments, the cells of the plurality of cell types of the present invention are progenitor cells. In some embodiments, the progenitor cells are cultured in single culture. In some embodiments, the progenitor cells are differentiated in single culture. In some embodiments, the progenitor cells are differentiated in single culture and then incubated with the plurality of cells in a three-dimensional porous scaffold according to the method of the present invention. Non-limiting examples, but not limited to these, include culturing mesenchymal stem cells and differentiating them into myoblasts, then seeding the differentiated myoblasts onto a three-dimensional porous scaffold and subsequently incubating them. Methods for culturing progenitor cells and inducing differentiation into mature cells will be apparent to those skilled in the art.

[0044] In some embodiments, the multiple cell types include myoblasts and fibroblasts. In some embodiments, the multiple cell types include myoblasts, fibroblasts and / or fibroblast progenitor cells. In some embodiments, the multiple cell types include myoblasts, fibroblasts and adipocytes. In some embodiments, the multiple cell types include myoblasts, fibroblasts, adipocytes, and / or fibroblast progenitor cells and / or adipocyte progenitor cells. In some embodiments, the multiple cell types include myoblasts, fibroblasts and endothelial cells. In some embodiments, the multiple cell types include myoblasts, fibroblasts and endothelial cells, and / or fibroblast progenitor cells and / or endothelial progenitor cells. In some embodiments, the multiple cell types include myoblasts and smooth muscle cells. In some embodiments, the multiple cell types include myoblasts, smooth muscle cells and endothelial cells. In some embodiments, the multiple cell types include myoblasts, smooth muscle cells, endothelial cells and adipocytes. In some embodiments, the cell types include myoblasts, fibroblasts, endothelial cells, and adipocytes. In some embodiments, the cell types include myoblasts, fibroblasts, endothelial cells, adipocytes and / or fibroblast progenitor cells, and / or adipocyte progenitor cells, and / or endothelial progenitor cells.

[0045] In some embodiments, multiple cell types are obtained from living animals and cultured as primary cell lines. In a non-limiting example, cells may be obtained by biopsy and may also be cultured ex vivo. In another non-limiting example, cells may be obtained from a commercial source.

[0046] 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 is known to those skilled in the art, MSCs can be muscle cells, adipocytes, osteocytes, and chondrocytes. In another embodiment, the cells are induced pluripotent stem cells (iPS or iPSCs). In yet another embodiment, the cells are derived from totipotent embryonic stem cells, such as cells from the blastocyst stage, fertilized egg, placenta, or umbilical cord of these animals.

[0047] In some embodiments, the cell types are derived from non-human cells. In some embodiments, the 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 cell types are derived from mammals. In some embodiments, the cell types are derived from non-human mammals. In some embodiments, the cell types are derived from domesticated mammals. As used herein, “domesticated” includes domesticated mammals, semi-domesticated mammals, or wild mammals in captivity. Non-limiting examples of non-human mammals include antelopes, bears, beavers, bison, wild boars, camels, reindeer, cattle, deer, elephants, elk, foxes, giraffes, goats, rabbits, horses, ibex, kangaroos, lions, llamas, moose, peccaries, pigs, rabbits, seals, sheep, squirrels, tigers, whales, yaks, and zebras, or combinations thereof. In some embodiments, the cell types are derived from bird cells. Non-limiting examples of birds include chickens, ducks, emus, geese, grouse, ostriches, pheasants, pigeons, quail, and turkeys, or combinations thereof. In some embodiments, multiple cell types are derived from fish. Non-limiting examples of fish include bass, catfish, carp, cod, eels, flounder, pufferfish, groupers, haddock, halibut, herring, mackerel, parachute, swordfish, orange roughy, perch, pike, Alaska pollock, salmon, sardines, sharks, snapper, flounder, swordfish, tilapia, trout, tuna, and walleye, or combinations thereof. In some embodiments, multiple cell types are derived from invertebrates. Non-limiting examples of invertebrates include lobsters, crabs, shrimp, clams, oysters, mussels, and sea urchins. In some embodiments, multiple cell types are derived from reptiles. Non-limiting examples of reptiles include snakes, crocodiles, and turtles. In some embodiments, multiple cell types are derived from amphibians. Non-limiting examples of amphibians include frogs. Cell seeding and culture

[0048] As will be understood by those skilled in the art, each cell type used in the compositions and methods described herein may have a preferred or optimal range of cell densities, and a culture medium or growth factor favorable for cell viability may be preferred. In some embodiments, each cell type is seeded at a specific cell density. In some embodiments, the cells are seeded simultaneously or sequentially.

[0049] In some embodiments, myoblasts are given 10 mg of porous textured protein. 3 ~10 7Cells are seeded at a specific cell density. In some embodiments, different cell types are seeded in specific ratios. In some embodiments, the ratio of seeded myoblasts to seeded fibroblasts is in the range of 1:1,000 to 1,000:1. In some embodiments, the ratio of seeded myoblasts and seeded fibroblasts to seeded endothelial cells is in the range of 1:20 to 20:1. In some embodiments, the ratio of seeded myoblasts and seeded fibroblasts to seeded adipocytes is in the range of 1:5000 to 5000:1. In some embodiments, the ratio of seeded satellite cells to seeded smooth muscle cells is in the range of 5:1 to 1:5. In some embodiments, the ratio of seeded satellite cells to seeded skeletal microvascular endothelial cells is in the range of 10:1 to 1:10. In some embodiments, the ratio of seeded smooth muscle cells to seeded skeletal microvascular endothelial cells is in the range of 10:1 to 1:10. In some embodiments, the ratio of seeded satellite cells to seeded smooth muscle cells to seeded skeletal microvascular endothelial cells is in the range of 10:1:1 to 2:1:10. In one embodiment, the ratio of seeded satellite cells to seeded smooth muscle cells to seeded skeletal microvascular endothelial cells is 2:1:1 to 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 2:1:1 to 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 2:1:1 to 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 2:1:1 to 2:1:4, or any ratio in between. Each possibility represents a distinct embodiment of the present invention.

[0050] In some embodiments, the seeded cell density and the incubated cell density are approximately the same.

[0051] In one embodiment, “Coverage %” refers to the area or volume of the porous scaffold in contact with cells or myotubes. In another embodiment, Coverage % refers to the area or volume of the porous scaffold occupied by cells or myotubes. As used herein, cells in contact with the scaffold are located on it, inside it, or a combination thereof.

[0052] In some embodiments, the coverage percentage of multiple cells is at least 10%, at least 20%, 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%. In some embodiments, the coverage percentage of multiple cells 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 distinct embodiment of the present invention.

[0053] In some embodiments, the satellite cell coverage percentage 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, the satellite cell coverage percentage is 25–40%, 35–50%, 45–60%, 55–70%, 65–80%, 75–90%, 85–100%, or any range in between. Each possibility represents a distinct embodiment of the present invention.

[0054] In some embodiments, the endothelial cell coverage percentage is up to 2%, up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, or up to 50%. In some embodiments, the endothelial cell coverage percentage is 5–15%, 10–25%, 20–35%, 30–50%, or any range in between. Each possibility represents a distinct embodiment of the present invention.

[0055] In some embodiments, endothelial cells are used to increase the proliferation of myoblasts or their progenitor cells. In some embodiments, endothelial cells inhibit the differentiation of myoblasts or their progenitor cells into myotubes. In some embodiments, endothelial cells assist in the growth and proliferation of myoblasts or their progenitor cells. In some embodiments, endothelial cells are not required for the differentiation of myoblasts or their progenitor cells. In some embodiments, according to the method of the present invention, endothelial cell activity (e.g., assisting in the secretion of myogenic substances, the growth, survival, or both of myoblasts) is maintained for a defined period. In some embodiments, the defined period of endothelial activity is the period required for myoblasts or their progenitor cells to achieve a coverage rate 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 the three-dimensional porous scaffold. In some embodiments, the defined period of endothelial activity is the period required for myoblasts or their progenitor cells to achieve a coverage rate of at least 30%.

[0056] In some embodiments, the myotube coverage rate % is at least 5%, at least 20%, at least 35%, at least 50%, at least 70%, at least 85%, at least 90%, at least 90%, or at least 99%. In some embodiments, the myotube coverage rate % is 1 - 10%, 5 - 20%, 15 - 35%, 30 - 50%, 40 - 65%, 60 - 85%, 80 - 90%, 90 - 100%, or any range therebetween. Each possibility represents a separate embodiment of the present invention.

[0057] In some embodiments, the myotubes of the composition of the present invention are 1 mm of the three-dimensional porous scaffold 3 per 10,000 - 100,000 nuclei, 1 mm of the three-dimensional porous scaffold 3 per 10,000 - 100,000 nuclei, 1 mm of the three-dimensional porous scaffold 3 per 15,000 - 200,000 nuclei, 1 mm of the three-dimensional porous scaffold 350,000 to 500,000 cores per 1 mm of three-dimensional porous scaffolding 3 5,000 to 1,000,000 cores per 1 mm of three-dimensional porous scaffolding 3 100,000 to 250,000 nuclei per 1 mm of three-dimensional porous scaffolding 3 Each includes 10,000 to 1,500,000 nuclei, or any range in between. Each possibility represents a distinct embodiment of the present invention.

[0058] As will be apparent to those skilled in the art, the extracellular matrix (ECM) influences the differentiation of myoblasts into myotubes. In some embodiments, myoblast differentiation is improved by ECM-secreting cells utilized according to the methods of the present invention. In some embodiments, ECM-secreting cells improved myoblast differentiation by simulating the physical properties of the tissue.

[0059] As defined herein, the terms “improved” and “increased” are synonymous.

[0060] 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 1,000%, at least 2,500%, or at least 5,000%. In some embodiments, the improvement is 5-15%, 10-35%, 25-45%, 40-70%, 65-90%, 85-150%, 100-500%, or 250-1,000%. Each possibility represents a distinct embodiment of the present invention.

[0061] Non-limiting examples of the physical properties of a microstructure include, but are not limited to, robustness, porosity, flexibility, and rigidity. A microstructure can be physically defined according to its Young's modulus, viscosity, or other parameters, all of which will be obvious to those skilled in the art.

[0062] In some embodiments, the method of the present invention further includes a step of sterilizing the porous textured protein. In some embodiments, the porous textured protein is sterilized before seeding or incubating multiple cell types. In some embodiments, sterilization is by gamma irradiation. In other embodiments, sterilization is ethanol-based sterilization. The sterilization procedure will be apparent to those skilled in the art.

[0063] Those skilled in the art will understand that cell seeding and / or culture is performed in the presence of a cell culture medium. In another embodiment, the cell culture medium includes growth factors, cytokines, bioactive agents, nutrients, amino acids, antibiotic compounds, anti-inflammatory compounds, or any combination thereof. Suitable media and compounds that are favorable for cell viability and growth are known to those skilled in the art.

[0064] Growth factors that can be used in the methods and compositions of the present invention include, but are not limited to, platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF-1). PDGF and IGF-1 are known to stimulate mitotic, chemotactic, and proliferative (differentiated) cell responses. Growth factors may include, but are not limited to, one or more of the following: PDGF, e.g., PDGF AA, PDGF BB; IGF, e.g., IGF-I, IGF-II; Fibroblast growth factor (FGF), e.g., acidic FGF, basic FGF, β-endothelial growth factor, FGF 4, FGF 5, FGF 6, FGF 7, FGF 8, and FGF 9; Transforming growth factor (TGF), e.g., TGF-P1, TGF β1, β2, TGF-β2, TGF-β3, TGF-β5; Bone morphogenetic protein (BMP), e.g., BMP 1, BMP 2, BMP 3, BMP 4; Vascular endothelial growth factor (VEGF), e.g., VEGF, placental growth factor; epidermal growth factor (EGF), e.g., EGF, amphiregulin, beta-cerulin, heparin-bound EGF; interleukins, e.g., 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 factor (CSF), e.g., CSF-G, CSF-GM, CSF-M; nerve growth factor (NGF); stem cell factors; hepatocyte growth factor, and ciliary neurotrophic factor.

[0065] As illustrated below, for bovine-derived cells, the optimized satellite cell (SC) expansion medium (i.e., Pro-LIF medium) contains bovine SC (BSC) growth medium (99%), amphotericin B (AB / AM; 1% 1x), ZnCl2 (50 μM), EGF (62 ng / ml), IGF-1 (100 ng / ml), and bFGF (10 ng / ml).

[0066] As illustrated below, for bovine-derived cells, the optimized SC growth medium includes DMEM / HEPES (43.5%), Ham's F-10 nutrient mix (43.5%), fetal bovine serum (10%), MEM NEAA (1% 1x), GlutaMAX (1%), and AB / AM (1% 1x).

[0067] As illustrated below, for bovine-derived cells, the optimized SC differentiation medium contains DMEM / HEPES (97%), donor horse serum (2%), AB / AM (1% 1x), IGF-1 (100 ng / ml), and EGF (62 ng / ml). Porous scaffolding

[0068] In some embodiments, a plurality of cells of the present invention are incubated with a porous scaffold. As used herein, the term “scaffold” refers to a structure comprising material that provides a surface suitable for cell attachment / adhesion, maturation, differentiation, and proliferation. The scaffold may further provide mechanical stability and support. The scaffold may have a specific shape or form to influence or define the three-dimensional shape or morphology assumed by the population of proliferating cells. In some embodiments, the porous scaffold of the present invention is three-dimensional.

[0069] In some embodiments, the average pore size of the porous scaffold is 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 to 700 μm, 50 μm to 600 μm, 50 μm to 500 μm, 50 μm to The ranges are 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 μm, 100 μm to 800 μm, 100 μm to 700 μm, 100 μm to 600 μm, 100 μm to 500 μm, 100 μm to 400 μm, 100 μm to 300 μm, 100 μm to 200 μm, 500 μm to 1000 μm, 500 μm to 900 μm, 500 μm to 800 μm, 500 μm to 700 μm, or 500 μm to 600 μm. Each possibility represents a different embodiment of the present invention. In some embodiments, the average pore size of the porous scaffold is in the range of 20 μm to 1000 μm.

[0070] In some embodiments, the porous scaffold is edible. In some embodiments, the porous scaffold contains a textured protein. In some embodiments, the porous scaffold contains polysaccharides. In some embodiments, the textured protein is a textured plant protein. In some embodiments, the textured protein is a textured soy protein (such as TSP).

[0071] In this specification, the term “texture” is used to refer to a rigid or flexible mass of individual cells that can be easily formed into various sizes, shapes, and configurations and are nondispersible in water.

[0072] Particulate textured protein materials suitable for use herein may consist of 30% to 100% protein and 0% to 70% material related to the protein source or added adjuvant material, on a dry weight basis. Examples of adjuvant materials include carbohydrates, vitamins, flavorings, and colorings. In some embodiments, the protein particles consist of 50% to 100% protein or 50% to 80% protein on a dry weight basis.

[0073] Suitable untextured proteins that can be textured to form textured particulate protein materials are available from a variety of sources. In non-limiting examples, such sources include plant proteins and certain fungal proteins, but animal proteins may also be used. Suitable animal proteins include casein, collagen, and egg white. Suitable plant protein sources include soybeans, safflower seeds, corn, peanuts, wheat, wheat gluten, peas, sunflower seeds, chickpeas, cottonseed, coconut, rapeseed, sesame seeds, leaf proteins, gluten, and single-cell proteins such as yeast.

[0074] Another example of a suitable protein source is mushrooms. In some embodiments, the mushroom protein source contains 15–20% (w / w), 20–30% (w / w), 28–45% (w / w), or 10–40% (w / w) of protein by dry weight.

[0075] Generally, when the protein source is plant-based, the protein is kept in a relatively pure form before use. Therefore, for example, if the protein source is soybeans, the soybeans may be solvent-extracted with hexane or similar solvents to remove the oil. The resulting oil-free soybean meal contains approximately 50% protein.

[0076] Soybean meal can be processed by known methods to remove carbohydrates and obtain products with higher levels of protein, such as soybean protein concentrate containing about 70% protein or soybean protein isolate containing about 90% or more protein. Then, various suitable prior art processes can be used to convert the soybean meal, concentrate, isolate, and other edible protein-carrying materials into suitable textured particulate protein materials.

[0077] A preferred method for converting untextured animal and plant protein carriers into particulate textured proteins is, for example, U.S. Patent No. 2,682,466 granted to Boyer on June 29, 1954, U.S. Patent No. 3,142,571 granted to Kitchel on July 28, 1964, U.S. Patent No. 3,488,770 granted to Atkinson on January 6, 1970, U.S. Patent No. 3,498,794 granted to Calvert et al. on March 3, 1970, and U.S. Patent No. 3,498,794 granted to Loepiktie et al. on September 18, 1973. The patents disclosed in U.S. Patent No. 3,759,715, U.S. Patent No. 3,778,522 granted to Strommer on December 11, 1973, U.S. Patent No. 3,794,731 granted to Dannert et al. on February 26, 1974, U.S. Patent No. 3,814,823 granted to Yang et al. on June 4, 1974, and U.S. Patent Application No. 248,581, filed on April 28, 1972, and jointly assigned, now U.S. Patent No. 3,840,679 granted to Liepa et al. on October 8, 1974, are all incorporated herein by reference.

[0078] In alternative embodiments, the porous textured protein can be replaced with other porous compositions that are both edible and chewable. As used herein, the terms “edible” and “chewable” refer to compositions that can be safely ingested into the body. These compositions include those that are absorbable and those that are not absorbable, as well as those that are digestible and those that are indigestible. As used herein, the term “chewable” refers to a composition that can be broken down into smaller pieces by chewing before swallowing. Those skilled in the art will understand that suitable edible compositions can be selected according to their physical properties (e.g., Young's modulus, viscosity, robustness, etc.) for a desired use (e.g., consumption by adult humans).

[0079] According to the method of the present invention, multiple cell types are seeded themselves onto a three-dimensional porous scaffold. In some embodiments, the multiple cell types seeded onto the three-dimensional porous scaffold do not require any solidifying agent. In some embodiments, the multiple cell types seeded onto the three-dimensional porous scaffold require a solidifying agent. In some embodiments, the solidifying agent increases the adhesion or attachment of the multiple cell types to the three-dimensional porous scaffold. Non-limiting examples of solidifying agents include, but are not limited to, thrombin or fibrin.

[0080] As used herein, the terms "gelling agent" and "solidifying agent" are synonymous.

[0081] Unless otherwise specified, in this discussion, adjectives such as “substantially” and “about” modifying the condition or relational characteristics of the features or features of embodiments of the invention are understood to mean that the condition or characteristic is defined within an acceptable range for the operation of the embodiment for its intended use. Unless otherwise specified, the term “or” in the specification and claims is considered exclusive “or” rather than comprehensive “or” and indicates at least one or any combination of the items to be joined.

[0082] The terms “a” and “an” as used above and elsewhere in this specification should be understood to mean “one or more” of the enumerated components. Unless otherwise specified, it will be obvious to those skilled in the art that the singular form includes the plural form. For this reason, the terms “a,” “an,” and “at least one” are used interchangeably herein.

[0083] For the purpose of better understanding this instruction, and without particularly limiting the scope of the instruction, unless otherwise specified, all numerical terms used in this specification and the claims to represent quantities, percentages or proportions, and other numerical values ​​should in all cases be understood as being modified by the term “about.” Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are approximations that may vary depending on the desired characteristics to be obtained. At a minimum, each numerical parameter should be interpreted taking into account the number of significant figures reported and applying the usual rounding method.

[0084] In the description and claims of this application, the verbs “comprise,” “include,” and “have,” and their conjugations, are used to indicate that the object(s) of the verbs do not necessarily enumerate all components, elements, or parts of the subject(s) of the verbs.

[0085] Other terms used herein are to be defined by their well-known meanings in the art.

[0086] Additional objectives, advantages, and novel features of the present invention are not intended to be limiting and will become apparent to those skilled in the art when considering the following embodiments. In addition, each of the various embodiments and aspects of the present invention detailed above and claimed in the following claims will find experimental support in the following embodiments.

[0087] For clarity, it will be recognized that certain features of the Invention described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the Invention described in the context of a single embodiment for brevity may be provided separately, in any preferred subordinate combination, or as suitable for any other described embodiment of the Invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiments cannot operate without those components. Examples

[0088] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. These techniques are thoroughly described in the literature. For example, "Molecular Cloning: A Laboratory Manual," Sambrook et al., (1989); "Current Protocols in Molecular Biology," Vols. I-III, Ausubel, RM, 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. York (1998); U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057, describing the methods used in "Cell Biology: A Laboratory Handbook," Volumes I-III, Cellis, JE, ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique," Freshney, Wiley-Liss, NY (1994), 3rd edition; "Current Protocols in Immunology," Volumes I-III, Coligan, JESee also: , 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); and "Bacteriophage Methods and Protocols," Volume 1: Isolation, Characterization, and Interactions. All of these are incorporated by reference. Other general references are provided throughout this document. material and method Textured soy protein

[0089] Textured soy protein (TSP) is commercially available as several different products. TSP scaffolds were prepared from five different sources of commercially available TSP—three TSP chunks (Figure 1A) were obtained from a local health food store, and two TSP flakes (Figures 1B-1C) were obtained from ADM. The TSP was sterilized with gamma irradiation at 25-40 kGy for 3.5 hours and stored in a sterile environment, or treated with 70% (v / v) ethanol for 15 minutes. Subsequently, it was washed three times with PBS. Preparation of TSP scaffolding

[0090] TSP flakes (Arcon or TVP) were selected using Graefe tweezers to achieve similar thicknesses of approximately 200–400 μm (per layer). The flakes were incubated overnight at 37°C in 5–10 ml of sterile DDW (in a 50 ml falcon). The following day, the flakes were cut into 6 mm scaffolds using a 6 mm biopsy punch on a non-TC 10 cm plate in a hood. Using a Pasteur pipette, the scaffolds were removed from the biopsy punch and transferred to a falcon containing 5 ml of growth medium. The TSP chunks were incubated overnight at 37°C in DDW. Next, the chunks were cut into cylinders using a biopsy punch (inset in Figure 2A) and then into 1 mm thick discs using a knife (Figure 2A). The scaffolds were then placed in 5 ml of growth medium. Scaffolding seeding

[0091] 2x10 per scaffold 6 The BSC was transferred to an Eppendorf tube and centrifuged at 1,500 rcf for 4 minutes. Meanwhile, each scaffold was placed in the wells of a non-TC 6-well plate with 50 μl of growth medium. After centrifugation, the medium was aspirated from the Eppendorf tube, leaving the cell pellet. Next, for each Eppendorf tube, the following procedure was performed: (1) Using forceps, the medium was completely aspirated from the scaffold, and the scaffold was gently squeezed to aspirate any remaining medium. (2) 7 μl of thrombin (20 MHU / ml sigma in PBS) and 7 μl of fibrinogen (15 mg / ml sigma in PBS) were added. (3) Cells were then seeded onto the scaffold. (4) The scaffold was incubated at 37°C for 30 minutes until dry, and then 2 ml of expanded medium was added to the well. (5) The following day, the scaffold was transferred to a new container (e.g., a 24-non-TC well with coverslip bottoms) using sterile Graefe forceps and 2 ml of expanded medium was added. (6) The scaffolds were incubated at 37°C and 5% CO2. (7) The scaffolds were maintained in expanded medium for 7 days. (8) The medium was changed every 2 days (2 ml). (9) After 7 days, the expanded medium was replaced with BSC differentiation medium. (10) The differentiation medium was changed every 2 days for 5 days. cell culture

[0092] Bovine satellite cells (BSCs) are cultured in BSC growth medium (43.5% DMEM / HEPES (Gibco), 43.5% F-10 Nut Mix (Gibco), 10% FBS (HyClone), 1% NEAA (Gibco), 1% GlutaMAX (Gibco), and 1% penicillin-streptomycin-amphotericin B solution (Biological Industries (Ab / Am,BI)), with the following added: 50 μM ZnCl2 (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) The cells were cultured in (Systems). The BSC differentiation medium consisted of DMEM / HEPES supplemented with 2% FBS and 1% Ab / Am. Red fluorescent protein (RFP) expressing dermal fibroblasts (Lonza, USA) are cultured in Dulbecco's Minimum Essential Medium (DMEM; Gibco Life Technologies) supplemented with 10% FBS (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 (American Type Culture Collection) are cultured in 10% FBS and 2.5% HEPES buffer (Biological Cells were cultured in DMEM supplemented with (Industries) and 1% Pen-strep solution. The myoblast differentiation medium consisted of DMEM supplemented with 2% FBS, 2.5% HEPES buffer, and 1% Pen-strep solution. All incubations were performed in a 5% (v / v) CO2 humidified atmosphere at 37°C. Bovine smooth muscle cells (SMC; Cell Applications) were cultured as described in Table 3. Bovine aortic endothelial cells (BAEC; Cell Applications) were cultured in commercially available medium (Cell Applications).Bovine skeletal muscle microvascular cells (BSkMVEC; AngioProteomie) were cultured in commercially available ECM medium (ScienCell) containing 10% total FBS. Bovine dermal fibroblasts (BDF; Sciencell) were cultured in commercially available medium (FM-2; ScienceCell) or in DMEM high-glucose medium supplemented with 15% FBS. immunohistochemistry

[0093] Rinse the scaffold twice with PBS, then fix it in 4% paraformaldehyde (PFA) on a shaker for 20 minutes. Wash the scaffold three times on a shaker with PBS for 5 minutes each, then permeabilize with 0.3% Triton X-100 (Bio Lab Ltd) for 10 minutes. Next, wash the scaffold three times on a shaker with PBS for 5 minutes each, then immerse it overnight in PBS containing 5% bovine serum albumin (BSA; Millipore) at 4°C. Incubate the scaffold at room temperature for 3 hours with 250 μl of primary antibody (PBS containing 5% BSA). After incubation, wash the scaffold four times on a tin foil-covered plate on a shaker for 3 hours with secondary antibody (in PBS) and 4',6-diamidino-2-phenylindole (DAPI, 1:1000 in PBS; Vector Laboratories). The scaffold is washed with PBS on a shaker for 5 minutes and then imaged with a confocal microscope (LSM 700, Zeiss). Bovine aortic smooth muscle cells (BAMSCs)

[0094] The effects of four different culture medium formulations on cell proliferation were compared with commercially available culture medium (BSM) (Table 3). At the start of the experiment, cells were in 8 passages and at a density of 4,000 cells / cm³ in a 6-well plate. 2 The seeds were sown. On the third day, the cells were treated with trypsin and counted using a hemocytometer, with a count of 4,000 cells / cm³. 2 The cells were then reseeded. The experiment was performed twice using the same cells under each condition. Statistical significance was tested using one-way ANOVA. Cell growth dynamics

[0095] Approximately 4x10 3 cells / cm2 The seeds were sown, and their numbers were counted daily using a hemocytometer for six days. The counted values ​​were plotted. cell staining

[0096] Supporting cells (BDF or BASMC) were stained in 3 ml of fresh medium containing 15 μl of lipophilic tracer DiD (1 mg / ml diluted with anhydrous ethanol; #D7757, Molecular Probes®, USA). Bovine endothelial cells (BEC; BAEC or BSkMVEC) were stained in 3 ml of fresh medium containing 5 μl of lipophilic tracer DiI (3 mg / ml diluted with anhydrous ethanol; #D, Molecular Probes®, USA) and incubated at 37°C for 30 minutes. After washing once with medium and then twice more with PBS, the cells were trypsinized and prepared for seeding as described below. Seeding of cells on a porous scaffold

[0097] Cow EC (1.25x10 5 ) and supporting cells (0.25 x 10 5 The co-culture suspension was resuspended in 5 μl of a 1:1 mixture of 5 mg / ml fibrinogen (Sigma Aldrich) and 20 NIHu / ml thrombin (Sigma Aldrich). The fibrinogen solution was prepared by diluting lyophilized human fibrinogen (Sigma Chemical) in 40 mM glycine-Tris buffer. The thrombin solution was prepared by diluting thrombin (Sigma Aldrich) with 40 mM calcium chloride. Next, the co-culture suspension was seeded onto 4 mm diameter circular PLLA-PLGA porous scaffolds and solidified in a 12-well non-tissue plate in an incubator (37°C, 5% CO2) for 30 minutes. After solidification, 2 ml of endothelial culture medium (ECM, ScienCell) supplemented with additional 5% FBS and 2 nM VEGF was added to each well. The medium was changed daily.

[0098] To further adjust the cell ratios seeded on the porous scaffold, the cells were seeded as described above with the following modifications: The cells were first seeded in 1:1 medium (bEBM, AngioProteomie: commercially available medium for supporting cells) and then 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). Differentiation of bovine satellite cells (BSCs) in 2D

[0099] 5x10 4 BSCs (passage 4) were seeded three times on TC24-well plates with coverslip bottoms. Cells were grown according to the myotubogenesis protocol as follows: 4 days in growth medium and 7 days in differentiation medium (t=0 refers to the start time when differentiation medium was used). Subsequently, cells were stained with Hoechst and myogenin on days 1 and 7. Optimization of BSC differentiation in 2D

[0100] BSCs (4 passages) were grown for 4 days in expanded medium containing different GFs (listed below), and then for 7 days in differentiation medium (DMEM-HEPES + 2% HS). Day 0 was defined as the day the medium was changed to differentiation medium. The expanded medium formulation included: (1) control-growth medium (without GF); (2) bovine fibroblast growth factor (bFGF; 10 ng / ml); (3) epidermal growth factor (EGF; 62 ng / ml); (4) insulin-like growth factor 1 (IGF-1; 100 ng / ml); (5) Pro-LIF; bFGF 10 ng / ml, EGF 62 ng / ml, IGF-1 100 ng / ml, and ZnCl 250 μM); (6) GF weaning: Pro-LIF medium for 3 days, then washed 3 times with growth medium (every 5 minutes), then in growth medium for 1 day. (7) Cells were stained with DiI and grown in growth medium to check whether DiI, a fluorescent color added to track cells, affected cell differentiation.

[0101] The inventors further investigated the effects of GFs (IGF-1, EGF, and combinations thereof) during the differentiation phase. BSCs (4 passages) were grown in growth medium for 4 days, followed by 7 days in differentiation mediums containing different GFs (described below). The differentiation medium formulations included: (1) control – original differentiation medium (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). In all cases, cells were visualized using a bright-field microscope. Optimization of BSC extension on 3D scaffolding

[0102] For this purpose, a dual-element experiment (seeding volume and expanded medium formulation) was set up in overlapping order as follows: (1) Seeding volume and cell number: (a) 10 μl seeding volume and 1 x 10 6 BSC; (b) 10 μl seeding rate and 2 x 10 6 BSC; and (c) 20 μl seeding rate and 2 x 10 6 BSC. (2) Extended media: (a) Growth medium; and (b) Pro-LIF medium (Growth medium + bFGF + EGF + IGF-1 + ZnCl2). BSC (4 passages) were stained with DiI and then seeded on porous scaffolds (Arcon) according to the Arcon seeding protocol. Optimization of BSC differentiation in 3D scaffolding

[0103] For this purpose, a dual-element experiment (week 1 expansion medium and week 2 differentiation medium) was set up as follows: (1) Expansion medium: (a) control (growth medium); (b) Pro-LIF; and (c) Pro-LIF-bFGF. (2) Differentiation medium was either alone or supplemented as follows: (a) control (differentiation medium containing 2% horse serum); (b) + IGF-1; (c) + EGF; and (d) + IGF-1 + EGF. BSCs (passage 4) were stained using DiI. 0.5 x 10⁶ cells were placed in 5 μl of fibrin on a 4 mm porous scaffold. 6BSCs (passage 4) were seeded. Cells were imaged on days 2, 7, and 14 after seeding. Next, the scaffolds were stained with desmin. Staining was performed according to a whole-mount staining protocol using the primary polyclonal antibody 1:100 goat α-desmin (Santa Cruz Cat. No. sc-7559) and the secondary antibody Alexa 1:200 488 donkey α-goat (Invitrogen Cat. No. A11055). Staining of bovine satellite cells (BSCs) before seeding.

[0104] BSCs were stained in 1 ml of fresh medium containing 5 μl of lipophilic tracer DiI (3 mg / ml diluted with anhydrous ethanol, Molecular Probes®, USA) and incubated at 37°C for 30 minutes. The cells were washed once with medium, then twice with PBS, and subsequently trypsinized and prepared for seeding as described. Cell seeding in PLLA-PLGA

[0105] Bovine satellite cells (BSCs) (with and without bovine ECs (BECs), and with and without supporting cells) were resuspended in 6 μl of a 1:1 mixture of 15 mg / ml fibrinogen (Sigma Aldrich) and 20 NIHU / ml thrombin (Sigma Aldrich). The fibrinogen solution was prepared by diluting lyophilized human fibrinogen (Sigma Chemical) in 40 mM glycine-Tris buffer. The thrombin solution was prepared by diluting thrombin (Sigma Aldrich) with 40 mM calcium chloride. The cell suspension was then seeded onto 4 mm diameter circular PLLA-PLGA scaffolds and solidified in a 12-well non-tissue plate in an incubator (37°C, 5% CO2) for 30 minutes. After solidification, 1 ml of a 1:1 mixture of endothelial culture medium and BSC growth medium (Pro-LIF) was added to each well. The medium was changed daily. After one week, the culture medium was replaced with BSC differentiation medium (supplemented with IGF-1 and EGF) for another week. Cell seeding on a textured soy protein scaffold

[0106] Bovine satellite cells (BSCs) (with and without bovine ECs, and with and without supporting 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). The fibrinogen solution was prepared by diluting lyophilized human fibrinogen (Sigma Chemical) in 40 mM glycine-Tris buffer. The thrombin solution was prepared by diluting thrombin (Sigma Aldrich) with 40 mM calcium chloride. The cell suspension was then seeded on both sides of a 6 mm diameter circular textured soy protein scaffold and solidified in a 12-well non-tissue plate in an incubator (37°C, 5% CO2) for 45 minutes. After solidification, 1 ml of a 1:1 mixture of endothelial culture medium and BSC growth medium (Pro-LIF) was added to each well. The medium was changed daily. After one week, the culture medium was replaced with BSC differentiation medium (supplemented with IGF-1 and EGF) for another week.

[0107] As shown in the table below, we tested two seeding ratios and two types of endothelial cells (and appropriate culture media): [Table 1] [Table 2]

[0108] Bovine satellite cells (BSCs) were resuspended in 15 μl of BSC growth medium. The cell suspension was then seeded on both sides of a 6 mm diameter Selineder Gelfoam(c) scaffold and solidified in a 12-well non-tissue plate in an incubator (37°C, 5% CO2) for 30 minutes. After solidification, 1 ml of BSC growth medium (Pro-LIF) was added to each well. The medium was changed daily. After one week, the medium was replaced with BSC differentiation medium (supplemented with IGF-1 and EGF) for another week. Cell growth on a scaffold

[0109] DiI-labeled BSCs seeded on PLLA / PLGA and Arcon scaffolds were imaged using a confocal microscope one and two weeks after seeding to evaluate cell growth. Whole-mount immunofluorescence staining

[0110] Two weeks after seeding, the scaffolds were fixed with 4% paraformaldehyde (PFA; Electron Microscopy Sciences) for 20 minutes, followed by three washes with PBS (Gibco® Life Technologies). Permeabilization was performed by incubating the scaffolds with 0.3% Triton X-100 (Bio Lab Ltd) at room temperature (RT) for 15 minutes. After three washes with PBS, the scaffolds were incubated overnight at 4°C in blocking buffer (10% FBS, 1% (w / v) glycine, and 0.01% Triton in PBS). The scaffolds were then incubated overnight at 4°C with primary antibodies: 1:50 goat anti-CD31 (Santa Cruz), 1:50 mouse anti-MYH (Santa Cruz), diluted with blocking buffer, with or without. After several washes with PBS, samples were incubated for 3 hours at RT with 1:400 donkey anti-goat Alexa Fluor® 488 (Jackson ImmunoResearch), 1:50 Alexa Flour 647-conjugated mouse anti-myogenin (Santa Cruz), and 1:1,000 DAPI (4',6-diamidino-2-phenylindole, Sigma-Aldrich) (with or without 1:300 Alexa Flour 647-conjugated mouse anti-myogenin). After several washes with PBS, scaffolds were immediately imaged using Zen software on a Zeiss LSM700 confocal microscope (Carl Zeiss). Image processing and further analysis were performed using FIJI software. PLLA / PLGA scaffolds were also whole-mount immunofluorescence stained one week post-seeding. Freeze cutting and tricolor staining

[0111] At the end of each experiment, the scaffolds were incubated overnight in a 30% (w / v) sucrose solution at 4°C. The scaffolds were then embedded in an optimal cutting temperature (OCT) compound (Tissue-Tec, USA) and frozen for subsequent freeze-cutting. The OCT-embedded scaffolds were freeze-cut to produce sections 5 μm and 10 μm thick. The 5 μm thick sections were then stained according to the trichrome standard staining protocol. Example 1 Three-dimensional structural analysis of TSP porous scaffolding

[0112] TSP scaffolds were examined using a scanning electron microscope (SEM) to analyze their three-dimensional (3D) structure at both the micron and nanoscale. 100x magnification images showed that the TSPs were porous with pore sizes of 100–1,000 μm and suitable for cell culture. The walls of larger TSPs were shown to be thicker compared to those of medium-sized sources (Figures 3A and 3B). SEM image of a large TSP (magnification 2x10⁻¹⁰) 5 The study showed that TSPs contain 30 nm ball-shaped protein clusters. Analysis of moderate TSPs revealed a more amorphous, adhesive-like substance between these clusters (Figures 4A and 4B). Example 2 Growth and proliferation of fibroblasts cultured in a porous scaffold.

[0113] Fibroblast adhesion and proliferation on TSP scaffolds were investigated. First, 50,000 RFP-labeled dermal fibroblasts were seeded onto TSP scaffolds and cultured. After 14 days of incubation, the TSP scaffolds containing the dermal fibroblasts were further seeded with 200,000 endothelial cells and incubated for another 7 days. Samples of the TSP containing the cells were immunostained and imaged using a confocal microscope 8, 18, and 21 days after fibroblast seeding. These results showed that even with a low cell number of 50,000 cells seeded, fibroblasts proliferated and covered the TSP scaffolds (Figure 5A). After endothelial cell seeding (Figure 5B), the endothelial cells aggregated to form clusters (Figure 5C). Next, scaffolds from each scaffold source and scaffolds cut at a 90° angle to the planar direction of the TSP were examined. Each scaffold was seeded with 200,000 fibroblasts and incubated for 14 days. These results demonstrated that fibroblasts grew and proliferated in both large and medium-sized TSP scaffolds (Figure 6). These results indicate that the cells were able to fill the scaffolds after 14 days of culture. Example 3 Growth of myoblasts cultured in a porous scaffold

[0114] To evaluate the applicability of muscle tissue generation, experiments will be conducted to determine whether muscle cells can grow on a TSP scaffold.

[0115] For this purpose, 0.5x10 6 Myoblasts were seeded on a TSP scaffold and cultured in the presence of a differentiation medium that is low in serum and therefore better suited for the production of cultured meat. Images of myoblasts on day 14 (Figure 7) show that myoblasts proliferated on the TSP scaffold, demonstrating that the cells were able to fill the scaffold after 14 days of culture. Example 4 Proliferation of bovine skeletal muscle cells cultured with TSP

[0116] To evaluate its applicability for human consumption, bovine skeletal muscle cells (bSkMCs) were seeded on a TSP scaffold. Specifically, 0.5 x 10⁻¹⁰ cells were seeded. 6bSkMC cells were seeded on each scaffold and cultured in the presence of differentiation medium. On day 14, the cells were imaged using a confocal microscope. The image (Figure 8) shows that bSkMCs proliferate on the TSP scaffold, indicating that TSP is applicable to the production of cultured meat. Example 5 Proliferation of bovine aortic smooth muscle cells (SMCs)

[0117] The inventors investigated the effects of four different culture medium formulations on the growth of SMCs seeded in the culture medium described below, compared with a commercially available culture medium (BSM; Table 3) (Figures 9A to 9E). [Table 3]

[0118] On day 3, no significant difference in cell count was detected in the five different culture media (Figure 10A). On day 3 of the following experiment (Figure 10B), significant differences were observed in four different treatments (P<0.01), and post-hoc analysis revealed that the number of cells in the pyruvate-supplemented medium was significantly higher compared to the NEAA-supplemented medium and the 15% FBS-supplemented medium (P values ​​<0.05 and <0.01, respectively). The difference between the commercially available medium and the sodium pyruvate-supplemented medium was slightly significant (P=0.052). Therefore, the inventors proceeded with sodium pyruvate-supplemented medium (1 mmol) for BAOSMC culture. Example 6 Growth characteristics of BDF and SMC under defined conditions

[0119] After defining the custom culture medium formulations, the inventors investigated their growth rates under these conditions (Example 5). It was found that the growth rates of BDF (Figure 11A) and BAOSMC were very similar (Figure 11B). Example 7 Effects of EC and supporting cells on angiogenesis

[0120] To investigate whether seeded bovine endothelial cells (EC; BAEC - bovine aortic endothelial cells or BSkMVEC - bovine skeletal muscle microvascular endothelial cells) in co-culture with supporting cells improve angiogenesis and scaffold coverage, BAEC was co-seeded with either BDF or BASMC. Indeed, co-culturing fibroblasts with myoblasts and endothelial cells resulted in increased angiogenesis and improved vascular structure stabilization of the manipulated muscle structure over time. The cells were found to cover the entire scaffold (Figure 12). Furthermore, the inventors noticed a decrease in EC staining over time when BAEC was seeded in co-culture with BASMC (Figure 12 Error! Reference not found. A). Also, in this combination, vascular-like structures were evident on day 2. When BAEC was seeded in co-culture with BDF (Figure 12B), the inventors did not notice any vascular-like structures. When BSkMVEC was seeded in co-culture with BASMC (Figure 13A), the inventors noticed that BASMC was far more abundant than BSkMVEC. Furthermore, throughout the entire experimental period, vascular-like structures were clearly visible when using BASMC (Figure 13A) and BDF (Figure 13B). Example 8 A specific cell ratio improves vascular network formation.

[0121] Furthermore, to improve the vascular network, the inventors seeded bovine BAECs with supporting cells (BDF or BASMC) in different ratios. The inventors investigated three different ratios: EC to supporting cells 1:3, 1:1, and 5:1. The inventors clearly observed the self-assembly of vascularized BAEC cells (Figures 14-16) in the presence of BASMC (Figures 14 and 16) early, such as on day 2. The inventors also noticed that by day 6, the EC levels were lower than on day 2, and that BAECs formed regions of the vascular network with a BAEC-to-BASMC ratio of 1:1. BAECs were not thought to form blood vessels in the presence of BDF (Figure 15). Example 9 Differentiation of bovine satellite cells (BSCs) in cell culture

[0122] Next, the inventors investigated myogenin expression before and after myotube formation from BSCs and further evaluated the ability to use DAPI-myogenin for measuring the fusion index (FI). The inventors found little evidence of myogenin expression in undifferentiated BSCs (Figure 17). Myogenin expression occurred mainly in the nuclei of myotubes and several nuclei around the myotubes. It was shown that the fusion index (FI) could be automatically calculated by the myogenin / Hoechst nucleus ratio, which was found to be comparable to manual FI calculations. This protocol was further used in the quantification of myotube formation. Example 10 Optimization of BSC differentiation in cell culture

[0123] Next, the inventors wanted to optimize BSC differentiation in 2D. They investigated which growth factors (GFs) could be added (1) during the diastole phase without inhibiting myotube formation during differentiation, and (2) at the end of the differentiation phase to increase myotube size and FI.

[0124] The appearance of spontaneous myotubes in samples without bFGF after diastole and before differentiation (Figure 18) suggests that bFGF inhibits myotube formation. EGF, IGF-1, and DiI were not thought to affect myotube formation. Cell density increased with IGF-1, Pro-LIF, and weaning. On day 4 of differentiation, the inventors observed an increase in total myotubes under bFGF and Pro-LIF culture conditions. bFGF maintained the stem cell properties of BSCs, and once the cells were removed, they entered a differentiated state (Figure 19). On day 7 of differentiation, the inventors observed higher myotube area in control, weaning, and DiI compared to other test conditions (Figure 20). The inventors concluded that myotube formation and cell death are separated by a fine line. Therefore, cells should be grown in expanded medium containing bFGF (alone or with Pro-LIF) until they reach a high concentration, and then transferred to differentiation medium within a short period of 2-5 days. Furthermore, the abundance of myotubes was demonstrated when IGF-1 or IGF-1 and EGF were added to the cell culture medium. Example 11 Optimization of BSC extension in porous scaffolding

[0125] Next, the inventors attempted to increase BSC coverage to over 60% on a TSP scaffold, as previous experiments had shown low cell coverage on porous scaffolds. Therefore, GF was added during the diastole phase, increasing the initial cell number at seeding. Quantification of cell coverage area showed that cells cultured in GF-supplemented growth medium (i.e., Pro-LIF) covered 72 ± 15% of the scaffold, while cells cultured in growth medium alone covered approximately 18% (Figure 21). The difference between the two treatments proved statistically significant (P-value = 0.0009). Example 12 Optimization of BSC differentiation in porous scaffolds

[0126] To find the optimal conditions for myotube formation in 3D scaffolds, the inventors scanned multiple combinations of expansion medium and subsequent differentiation medium. Myotubes formed without GF in the differentiation medium (Figures 22A, E, and I) were small and round. Adding IGF-1 to the differentiation medium resulted in myotube formation. This was hardly observed in the growth medium (Figure 22B), common in Pro-LIF-bFGF (Figure 22J), and abundant in Pro-LIF medium (Figure 22F). Adding EGF to the differentiation medium resulted in myotube formation in all expansion mediums (Figures 22C, G, K). Adding IGF-1 + EGF resulted in myotube formation in the growth medium (Figure 22D) and Pro-LIF-bFGF (Figure 22L), with abundant myotubes in Pro-LIF (Figure 22H). Example 13 Increased proliferation, elongation, and coverage of BSCs in porous scaffolds through three culture methods.

[0127] Next, the inventors sought to examine the effect of triculture of BSCs with a fixed amount of smooth muscle cells (SMC) and different amounts of skeletal microvascular endothelial cells (SkMVEC) (2:1:1 and 2:1:5, respectively) on BSC survival, proliferation, and myotube differentiation (positive for the differentiation marker - myogenin). The inventors observed that at a low SkMVEC concentration (2:1:1), BSCs appeared to be more elongated compared to their appearance when incubated with a higher concentration of SkMVEC (2:1:5; Figure 28). The inventors also observed that BSCs increased their coating of the porous scaffold and appeared to be more differentiated compared to either single culture or co - culture (control) containing BSCs. The inventors concluded that triculture better supported BSC growth and differentiation compared to other test groups at both incubation ratios, and summarized the BSC differentiation ability as follows: BSC only < BSC + EC < BSC + SMC < BSC + EC + SMC (Figures 23 - 26 and Figure 28). The inventors also observed extracellular matrix (ECM) secretion in the triculture of BSC + SMC + SkMVEC (2:1:1) (Figures 27A - 27B). Example 14 Triculture increases BSC differentiation on a textured soy protein scaffold.

[0128] Next, the inventors sought to examine the effect of triculture of BSCs with a fixed amount of smooth muscle cells (SMC) and skeletal microvascular endothelial cells (SkMVEC) (2:1:1) on BSC survival, proliferation, and myotube differentiation (positive for the differentiation marker - myogenin). The inventors observed that when seeded either in co - culture with SMC or in triculture with SMC and BEC, BSCs not only increased their coating of the TSP scaffold, but also appeared to be more differentiated compared to either single culture (BSC only) or co - culture (BSC and BEC; Figure 29). Example 15 Seed BSCs without adding solidified gel

[0129] Next, the inventors decided to investigate the effect of seeding BSCs onto an FDA-approved collagen-based scaffold (Gelfoam®) without adding a solidifying agent (e.g., fibrin). The inventors observed successful BSC differentiation, as demonstrated by myotube formation (Figure 30).

[0130] While this specification has illustrated and described certain features of the present invention, many modifications, substitutions, alterations, and equivalents will arise for those skilled in the art. Therefore, it should be understood that the appended claims are intended to cover all such modifications and alterations that fall within the true spirit of the invention.

Claims

1. A method for producing an edible composition, a. An edible three-dimensional porous scaffold containing protein, wherein the protein is selected from the group consisting of textured proteins and non-textured proteins, and (i) Myoblasts or their precursor cells, (ii) at least one extracellular matrix (ECM) secreting cell selected from the group consisting of fibroblasts, smooth muscle cells, stromal cells, pericytes, and their precursor cells, and (iii) Endothelial cells or their progenitor cells Incubating multiple non-human cell types, including The ratio of myoblasts or their progenitor cells, ECM-secreting cells, and endothelial cells or their progenitor cells is 2:1:1, and incubation is performed. b. Inducing myoblasts or their precursor cells to differentiate into myotubes, A method comprising, by means of, inducing the production of the aforementioned edible composition.

2. The method according to claim 1, wherein the myoblast progenitor cell is a satellite cell.

3. The method according to claim 1, characterized in that the myoblasts or their progenitor cells and the endothelial cells or their progenitor cells are incubated for a period of time necessary for the myoblasts or their progenitor cells to achieve a coverage rate of at least 30% of the edible three-dimensional porous scaffold, and then the differentiation of the myoblasts or their progenitor cells into myotubes is induced.

4. The method according to any one of claims 1 to 3, wherein the endothelial cells are skeletal microvascular endothelial cells.

5. The method according to any one of claims 1 to 4, wherein the plurality of cell types include satellite cells, fibroblasts, and endothelial cells.

6. The method according to any one of claims 1 to 4, wherein the plurality of cell types include satellite cells, smooth muscle cells, and endothelial cells.

7. The method according to claim 1, wherein the protein is a textured protein.

8. The method according to claim 7, wherein the textured protein is textured soy protein.

9. The method according to claim 7 or 8, wherein the three-dimensional porous scaffold includes pores having an average diameter in the range of 20 to 1,000 micrometers.

10. The method according to any one of claims 1 to 9, wherein the plurality of cells are derived from livestock mammals.

11. The method according to claim 10, wherein the domesticated mammal is a cattle.

12. The myoblasts or their progenitor cells and the three-dimensional porous scaffold are such that the myoblasts or their progenitor cells are present in 10 mg of the three-dimensional porous scaffold. ３ ~10 ７ The method according to any one of claims 1 to 11, wherein the cells are incubated in a range ratio.

13. The method according to any one of claims 1 to 12, wherein the three-dimensional porous scaffold further comprises an extracellular matrix.

14. a. An edible three-dimensional porous scaffold containing protein, wherein the protein is selected from the group consisting of textured proteins and untextured proteins, b. The three-dimensional porous scaffolding 1 mm ３ Each myotube contains 10,000 to 250,000 myotubular nuclei, c. Multiple cells including multiple cell types, (i) Myoblasts or their precursor cells, (ii) at least one extracellular matrix (ECM) secreting cell selected from the group consisting of fibroblasts, smooth muscle cells, stromal cells, pericytes, and their precursor cells, and (iii) A plurality of cells comprising a plurality of cell types, which constitute less than 15% of the plurality of cells, and which are endothelial cells or their precursor cells, The aforementioned plurality of cell types are cultured in a ratio of (i):(ii):(iii) of 2:1:1 in this edible composition.

15. The composition according to claim 14, wherein the myoblast precursor cells are satellite cells.

16. The composition according to claim 14 or 15, wherein the plurality of cell types include satellite cells, fibroblasts, and endothelial cells.

17. The composition according to claim 14 or 15, wherein the plurality of cell types include satellite cells, smooth muscle cells, and endothelial cells.

18. The composition according to claim 15, wherein the endothelial cells are skeletal microvascular endothelial cells.

19. The composition according to any one of claims 14 to 18, wherein the protein is a textured protein.

20. The composition according to claim 19, wherein the textured protein is textured soy protein.

21. The composition according to claim 14 or 20, wherein the three-dimensional porous scaffold comprises pores having an average diameter in the range of 20 to 1,000 micrometers.

22. The composition according to any one of claims 14 to 21, wherein the plurality of cells are derived from livestock mammals.

23. The composition according to claim 22, wherein the livestock mammal is a cattle.

24. The composition according to any one of claims 14 to 23, wherein the three-dimensional porous scaffold further comprises an extracellular matrix.

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