A personalized customization of cell cultured meat based on porous scaffold material and its production method

Abstract

The present invention discloses a personalized customization of cell-cultured meat based on a porous scaffold material and a production method thereof. The method adopts a material suitable for cell growth and safe and edible, prepares a porous scaffold material with a suitable pore structure based on freeze-drying, and realizes personalized customization of cell-cultured meat by adjusting the cell inoculation parameters. The porous scaffold material constructed by the present invention has good biocompatibility and mechanical properties. Its high porosity, connectivity and suitable pore size structure are suitable for the intercellular information transmission and sufficient exchange of nutrients during cell culture, which is beneficial to the fate processes such as cell growth, proliferation, differentiation and maturation. In addition, the edible qualities such as texture, taste and nutritional value of cell-cultured meat can be improved by inoculating different types of cells and changing the cell inoculation ratio, thereby realizing personalized customization of cell-cultured meat. The porous scaffold material provided by the present invention has important guiding significance for the personalized development of the cell-cultured meat production industry.

Description

Technical Field

[0001] The present invention belongs to the field of cell cultured meat and relates to a method for producing personalized cell cultured meat, and specifically relates to personalized customization of cell cultured meat based on porous scaffold materials and a method for producing the same. Background Art

[0002] Cell cultured meat is a type of meat that is formed by extracting stem cells or adult cells from animals through in vitro proliferation, differentiation and culture based on animal cell scale culture technology and tissue engineering technology. As a new type of cell agriculture product, it does not rely on animal husbandry, but directly obtains real meat through cell culture, which greatly reduces the utilization rate of environmental resources and solves the environmental pollution problem caused by livestock and poultry farming. In the face of the continued increase in demand for meat, animal epidemics such as swine fever and avian influenza also pose severe challenges to meat safety. Therefore, efficient and green cell cultured meat urgently needs to be developed and produced.

[0003] Large-scale induction of differentiation in vitro to form mature muscle tissue is one of the most critical links in cell culture meat technology. At present, the main method used is to inoculate the livestock and poultry seed cells obtained by expansion culture onto three-dimensional materials, and the cells adhere to the materials to proliferate and differentiate to maturity. Among the three-dimensional materials used for cell culture meat production, porous scaffold materials have broad application potential in the field of cell culture meat due to their low preparation cost, simple and environmentally friendly methods, and convenient and efficient processes. As a three-dimensional carrier for cell culture in vitro, the structural properties of porous scaffold materials are crucial to the fate of cells. Among them, the pore structure is the primary reference property of porous scaffold materials. The porous scaffold uses the pore structure to provide a place for three-dimensional cultured cells to obtain nutrients, exchange gases, excrete waste, and grow metabolism. In addition, based on the anisotropic muscle tissue structure, the porous scaffold material provides important support for the three-dimensional culture of muscle stem cells. Its porous structure can guide the functional performance of stimulated cells, including cytoskeleton arrangement, striated myotube formation, and expression of myogenic proteins. Therefore, porous scaffold materials with suitable pore size, high porosity and connectivity can provide cells with a comfortable growth environment, appropriate contact and inhibition space through the pore structure, and further facilitate the fate of cell growth, proliferation, differentiation and maturation. However, most of the existing porous scaffold materials used to construct cell cultured meat often ignore the pore structure requirements, which will be detrimental to the full exchange of nutrients, intercellular information transmission and three-dimensional cell culture fate during cell culture. Therefore, it is worth looking forward to constructing porous scaffold materials with suitable pore structures for cell culture meat production.

[0004] The taste, texture and appearance of meat are important factors in measuring the quality of meat. The taste and texture come not only from the chewiness provided by muscle fibers, but also from the toughness of connective tissue connections. Muscle tissue, connective tissue and capillary tissue can be constructed by culturing stem cells or adult cells such as muscle stem cells, smooth muscle cells, fibroblasts and endothelial cells. Therefore, choosing different types of cell culture can be used to improve the edible quality of cell-cultured meat. In addition, the inoculation ratio of different types of cells has an important influence on the quality of meat through cell-to-cell interactions. However, most of the current cell-cultured meat still remains in the construction method of single muscle stem cell culture, ignoring the role of cell type and inoculation ratio on the taste, texture and appearance of cell-cultured meat, which will affect the overall edible quality and sensory evaluation of cell-cultured meat.

[0005] Purpose of the Invention

[0006] Invention content: In view of the problems existing in the prior art, the present invention provides a personalized customization of cell cultured meat based on porous scaffold materials and a production method thereof, which effectively solves the problem that the pore structure of existing porous scaffold materials is not suitable for life activities such as proliferation and differentiation of muscle stem cells and the edible quality of cell cultured meat is too single. The present invention adopts different pore-forming methods and inoculates different cell types and cell ratios to achieve personalized customization of cell cultured meat.

[0007] Technical solution: In order to achieve the above-mentioned purpose, the personalized customization of cell cultured meat based on porous scaffold materials and its production method described in the present invention include the following steps:

[0008] (1) Preparation of porous scaffold materials: a uniformly dispersed solution is prepared from a material suitable for cell growth and safe and edible, and a hydrogel is prefabricated by physical, chemical or enzymatic crosslinking, and a porous scaffold material is constructed by adding a porogen and / or repeatedly freezing and thawing at low temperature, and then freeze-drying; specifically, a porogen is added before forming a hydrogel and then freeze-dried to construct a porous scaffold material, or a porogen is directly repeatedly frozen and thawed at low temperature and then freeze-dried after forming a hydrogel to construct a porous scaffold material, or a porogen is added before forming a hydrogel, and then a porogen is repeatedly frozen and thawed at low temperature after forming a hydrogel and then freeze-dried to construct a porous scaffold material;

[0009] (2) Cell inoculation and culture: sterilize, clean, and soak the porous scaffold material, then inoculate the cell suspension, wait for the cells to adhere to the scaffold, and slowly add proliferation culture fluid to perform in vitro three-dimensional proliferation culture on the cells; after the cell proliferation culture, induce cell differentiation culture to form cultured meat tissue; the in vitro three-dimensional proliferation culture of the cells is to select a single cell type or different cell types at different cell inoculation ratios of livestock, poultry, or aquatic animal stem cells or adult cells for three-dimensional culture to meet the needs of later personalized customization of cell cultured meat;

[0010] (3) Personalized customization: After the differentiation process is completed, the cell cultured meat is collected, processed into a food product, and cooked according to the cooking method required to obtain the cultured meat product. The safe and edible materials suitable for the adherent growth of muscle stem cells include safe and edible proteins, polysaccharides or protein-polysaccharide complexes and other safe and edible materials.

[0011] Preferably, the safe and edible protein includes natural proteins such as collagen, soy protein, proteolytic peptides, or in vitro recombinant proteins expressed and secreted by Escherichia coli, yeast, mammalian cell lines, etc.; the polysaccharide includes sodium alginate, agar, chitosan, etc.

[0012] Furthermore, in step (1), the material suitable for cell growth and safe and edible is dispersed in water to form a suspension, and the insoluble components are removed by filtration after dissolution; a porogen is added after filtration, and the pH is adjusted to an alkaline environment by adding NaOH or glutaminase is added to the filtrate, and a hydrogel is formed at room temperature; and then vacuum freeze-dried to obtain a porous scaffold material;

[0013] Alternatively, in step (1), a material suitable for cell growth and safe and edible is dispersed in water to form a suspension, and the insoluble components are removed by filtration after dissolution; after filtration, NaOH is added to adjust the pH to an alkaline environment or glutamine transferase is added to the filtrate to form a hydrogel at room temperature; the hydrogel is subjected to low-temperature repeated freezing and thawing to construct a porous structure, and then vacuum freeze-dried to obtain a porous scaffold material;

[0014] Alternatively, in step (1), a material suitable for cell growth and safe and edible is dispersed in water to form a suspension, and the insoluble components are removed by filtering after dissolution; after filtration, a porogen is added, and the pH of the solution is adjusted to an alkaline environment or glutaminase is added to the solution to form a hydrogel at room temperature; the hydrogel is subjected to a low-temperature repeated freeze-thaw method to construct a porous structure, and then vacuum freeze-dried to obtain a porous scaffold material.

[0015] The porogen includes one or more of glycerol, polyethylene glycol (PEG), polyethylene oxide (PEO), polyvinyl alcohol (PVA), sucrose, sodium chloride, and calcium carbonate particles. Other non-toxic porogens may also be used.

[0016] The low-temperature repeated freezing and thawing is a repeated process of ice crystal formation, melting, and re-formation of ice crystals.

[0017] Preferably, the low-temperature repeated freezing and thawing is to freeze at a temperature within the range of -200 to -20°C to form ice crystals, melt at 20 to 40°C, and then freeze at a temperature within the range of -200 to -20°C to complete the repeated freezing and thawing process.

[0018] The porous scaffold material that meets the pore structure requirements of the present invention can be obtained by pore-forming methods such as repeated freezing and thawing and adding porogens, wherein the pore structure is obtained through a repeated process of ice crystal formation-melting-reformation, and the pore size is adjusted by adjusting the proportion of porogen addition.

[0019] The cells in step (2) include muscle stem cells, their progenitor cells, nerve cells, smooth muscle cells, endothelial cells, fibroblasts, mesenchymal stem cells, or stem cells or adult cells of other animals.

[0020] Furthermore, the inoculated cells of the porous scaffold material may be derived from livestock and poultry such as pigs, cows, chickens, ducks, rabbits, or aquatic animals such as fish and shrimps.

[0021] Among them, the personalized customization of the cell-cultured meat can be achieved by inoculating different cell types and different cell inoculation ratios.

[0022] The culture medium for cell proliferation and differentiation culture in step (2) is selected according to the type of cells to be inoculated, and includes F10, DMEM / F12, DMEM or MEM basal culture medium prepared as needed.

[0023] Furthermore, the porous scaffold material sterilization method includes sterilization methods such as 75% ethanol, ultraviolet radiation and ozone penetration.

[0024] Furthermore, the cell cultured meat tissue is processed into food, such as marinating with seasonings, coating with flour, coloring with red yeast rice, and other food additives, and then cooked in different ways, including steaming, frying, pan-frying, and other cooking methods.

[0025] Specifically, the personalized customization of cell cultured meat based on porous scaffold materials and the production method thereof of the present invention include:

[0026] (1) Preparation of porous scaffold materials: Safe and edible materials suitable for the adherent growth of muscle stem cells are used. A uniformly dispersed solution is prepared according to the solubility characteristics and prefabricated to form a hydrogel. A porous scaffold material with high porosity, connectivity, suitable pore structure and good mechanical properties is constructed based on freeze-drying technology.

[0027] (2) Cell inoculation and culture: After the porous scaffold material has been sterilized, cleaned, soaked, and other treatments, the cell type is selected as needed and a cell suspension of appropriate volume and density is inoculated. After the cells are attached to the scaffold, the proliferation culture medium is slowly added to perform in vitro three-dimensional proliferation culture of the cells, wherein the cell number is increased during the proliferation stage, and then the cells are induced to differentiate to form cultured meat tissue; a single type or different cell types and different cell inoculation ratios of livestock and poultry stem cells or adult cells are selected for three-dimensional culture to meet the personalized customization of cell-cultured meat;

[0028] (3) Personalized customization: The cell-cultured meat is collected after the differentiation process is completed, and after being processed into food by different methods, the cooking method is selected as required to obtain the cultured meat product.

[0029] The present invention provides personalized customization of cell-cultured meat based on porous scaffold materials and a production method thereof. The porous scaffold material has a suitable pore structure suitable for three-dimensional cell culture in vitro, can promote the proliferation, differentiation and maturation of muscle stem cells, and by selecting and inoculating different types of cells and changing the cell inoculation ratio, can meet the production of cell-cultured meat with different textures, tastes and nutritional values, thereby realizing personalized customization of cell-cultured meat.

[0030] The materials used in the present invention select appropriate physical, chemical or enzymatic cross-linking methods as needed to construct a three-dimensional network structure and a hydrogel system with a stable gel state. The constructed porous scaffold material has good biocompatibility and mechanical properties, and its high porosity, connectivity and appropriate pore size structure are suitable for the intercellular information transmission and sufficient exchange of nutrients during cell culture, and further, it is beneficial to the fate processes such as cell growth, proliferation, differentiation and maturation. In addition, by inoculating different types of cells and changing the cell inoculation ratio, the edible qualities such as the texture, taste and nutritional value of cell cultured meat can be improved, thereby realizing personalized customization of cell cultured meat. The porous scaffold material provided by the present invention has important guiding significance for the personalized development of the cell cultured meat production industry. The present invention can adopt different pore-forming methods according to different taste textures and nutritional requirements, select different types of cells to be inoculated as needed, and change the cell inoculation ratio for in vitro three-dimensional culture, which can realize personalized customization of cell cultured meat and effectively solve the problem of too single edible quality of cell cultured meat.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0032] (1) The materials used to construct the porous scaffolds of the present invention are low-cost, safe and edible, the preparation method is simple and environmentally friendly, the preparation process is convenient and efficient, the reaction conditions are mild, and no expensive instruments and experimental equipment are required, which greatly reduces the time and financial cost of the research and development and production of cell-cultured meat;

[0033] (2) The present invention can construct a porous scaffold material with a suitable pore structure by adjusting the porosity and pore size by selecting different pore-forming methods (such as adding pore-forming agents, repeated freezing and thawing, or using them in combination), which is beneficial to the intercellular information transmission, sufficient exchange of nutrients, and cell growth, proliferation, differentiation, maturation and other fate processes during cell culture;

[0034] (3) The porous scaffold material constructed by the present invention can improve the edible qualities of cell-cultured meat, such as texture, taste, and nutritional value, by inoculating different types of cells and changing the cell inoculation ratio, so as to achieve personalized customization of cell-cultured meat and meet the development needs of diversification and nutrition of cell-cultured meat. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The process flow chart of the preparation of the porous scaffold material and production of cell cultured meat of the present invention;

[0036] Figure 2 The scanning electron microscope images of the porous scaffold material prepared by adding different porogens of the present invention, wherein (a) is without adding porogen, (b) is with adding PEO porogen, (c) is with adding PEG porogen, (d) is with adding PVA porogen, and the scale bar is 100 μm;

[0037] Figure 3 The scanning electron microscope images of the porous scaffold material of the present invention, wherein (a) is a cross-sectional scanning electron microscope image, and (b) is a longitudinal cross-sectional scanning electron microscope image, and the scale bar is 100 μm;

[0038] Figure 4 This is an immunofluorescence image of the porous scaffold material of the present invention inoculated with muscle stem cells and cultured to the end of differentiation, with a scale bar of 100 μm;

[0039] Figure 5 This is a picture of the finished product of the cell cultured meat constructed by the present invention. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0041] The raw materials and reagents used in the examples are all commercially available.

[0042] In the present invention, collagen, transglutaminase, muscle stem cells, smooth muscle cells and fibroblasts are all commercially available. Among them, food-grade collagen is purchased from Ten Kate Protein Technologies; transglutaminase is purchased from Solarbio; muscle stem cells are porcine skeletal muscle stem cells, and smooth muscle cells and fibroblasts are also porcine cells, and other poultry and aquatic animals can also be used.

[0043] Example 1

[0044] Preparation of food-grade collagen porous scaffold materials

[0045] A method for preparing a porous scaffold material for producing cell cultured meat, wherein the preparation process is as follows: Figure 1 As shown, the following steps are included:

[0046] First, food-grade collagen was dispersed in ultrapure water to form a 4% suspension, fully dissolved in a 50°C warm water bath for 0.5h, and then the insoluble components were filtered out; then a 1% porogen aqueous solution (PEO (MW300000), PEG (MW 400), or PVA (MW 85000)) was mixed with the filtered collagen solution at a volume ratio of 1:2; a 10% glutamine transferase (100u / g) aqueous solution and the filtered collagen solution were uniformly dispersed in the protein-porogen mixture at a volume ratio of 1:10, and a hydrogel was formed at room temperature for 40min. For samples with stable gel state, a porous structure was constructed by repeated freezing and thawing at -196°C liquid nitrogen for 5min, thawing at 25°C for 8min, and then freezing at -80°C for 5h, and then vacuum-freezing for 24h to obtain a porous scaffold material.

[0047] The porous scaffold material prepared in this embodiment has a spongy texture and is fluffy and porous. The pore structure of the porous scaffold material is observed by scanning electron microscopy. Figure 2 As shown, the interconnected porous structure of the scaffold material can be observed. Without adding a porogen group, the pore size distribution is 10-60 μm. Adding PEG can obtain a relatively wide pore size distribution range (10-100 μm); and adding PVA can obtain a pore structure with a more uniform size. In addition, the pore structure varies depending on the type of porogen added. With PEO as a porogen, the constructed porous scaffold material presents a highly interconnected network structure.

[0048] Example 2

[0049] Preparation of food-grade collagen porous scaffold materials

[0050] First, food-grade collagen was dispersed in water to form a suspension with a mass concentration of 4%. After being fully dissolved in a 50°C warm water bath for 0.5 hours, the insoluble components were filtered out. Glutamine transferase (100u / g) was dispersed in water to form a solution with a mass fraction of 10%. It was mixed evenly with the filtered collagen solution at a volume ratio of 10:1, and a hydrogel with a stable gel state was formed at room temperature for 30 minutes. The porous structure was constructed by freezing with liquid nitrogen at -196°C for 5 minutes to form ice crystals, then thawing at 25°C for 8 minutes, and then freezing at -80°C for 5 hours. Finally, the hydrogel in the frozen state was vacuum-dried for 24 hours to obtain a porous scaffold material. The pore structure of the porous scaffold material was observed by scanning electron microscopy, such as Figure 3 As shown, both the horizontal and vertical sections show that the pores are connected, the pore size distribution is uniform, and the channel diameter is mostly around 10-60 μm.

[0051] Example 3

[0052] In vitro cell culture using porous scaffolds

[0053] The method for producing cell cultured meat based on porous scaffold material comprises the following steps:

[0054] The porous scaffold material prepared in Example 2 was sterilized with 75% ethanol, then rinsed with PBS three times, and finally irradiated with ultraviolet light for 40 min under closed conditions. After irradiation sterilization, 100 ul of 1×10 8 / ml porcine skeletal muscle muscle stem cells are inoculated on a porous scaffold material cut with a surgical blade (size is about 10mm×10mm×1mm); the muscle stem cells are inoculated on the porous scaffold material for about 1 hour, and 2ml of F10 proliferation culture medium including 20% ​​fetal bovine serum, 1% penicillin-streptomycin double antibody solution and 5ng / ml fibroblast growth factor is added to the well plate to slowly adhere to the wall, and the culture plate is placed in a saturated humidity, 37°C, 5% CO2 incubator for 3 days, and the proliferation culture medium is replaced every two days; when the muscle stem cell proliferation reaches the pre-differentiation density, the differentiation culture medium is replaced, the differentiation culture medium contains 2% horse serum, 97% DMEM cell culture medium, and 1% penicillin-streptomycin double antibody solution, and 1 / 2 of the differentiation culture medium is replaced every two days, and the cells enter the differentiation process and continue to differentiate for 14 days.

[0055] The cell cultured tissue at the end of differentiation (differentiation day 14) was subjected to immunofluorescence detection, and images were collected under a confocal microscope. Figure 4 The immunofluorescence staining results of F-actin secreted by muscle stem cells loaded with porous scaffold materials are shown in Figure 2. Figure 4 As shown, the food-grade collagen porous scaffold material has good biocompatibility, supports the proliferation and differentiation of muscle stem cells, and expresses a large amount of F-actin at the end of differentiation, indicating that the scaffold material is suitable for the growth, proliferation and differentiation of muscle stem cells, and has marker protein expression.

[0056] In addition, in order to achieve the diversified construction of cell-cultured meat, porous scaffold materials are also used to co-culture pig muscle stem cells, smooth muscle cells and fibroblasts in vitro. The cell type and quantity ratio is muscle stem cells: smooth muscle cells: fibroblasts = 2:1:1. The use of porous scaffold materials for multi-cell co-culture can not only promote the expression of muscle stem cell marker proteins and the production of related extracellular matrix, but also improve the sensory properties and texture of cell-cultured meat, making it closer to the eating experience of natural muscle tissue.

[0057] Example 4

[0058] Cell culture meat processing:

[0059] The method for processing cell cultured meat into food comprises the following steps:

[0060] The porous scaffold material prepared in Example 2 was cut into cylinders with a diameter of about 2.2 cm and a height of 1.5 mm using a surgical blade. The porous scaffold material was sterilized with 75% ethanol, then rinsed with PBS three times, and finally irradiated with ultraviolet light for 40 min under closed conditions. After irradiation sterilization, 100 ul of 1×10 9 / ml porcine skeletal muscle muscle stem cells are inoculated on the treated porous scaffold material; the muscle stem cells are inoculated on the porous scaffold material for about 1 hour, and 2ml of F10 proliferation culture medium including 20% ​​fetal bovine serum, 1% penicillin-streptomycin double antibody solution and 5ng / ml fibroblast growth factor is added to the well plate to slowly adhere to the wall, and the culture plate is placed in a saturated humidity, 37°C, 5% CO2 incubator for 3 days, and the proliferation culture medium is replaced every two days; when the muscle stem cell proliferation reaches the pre-differentiation density, the differentiation culture medium is replaced, the differentiation culture medium contains 2% horse serum, 97% DMEM cell culture medium, and 1% penicillin-streptomycin double antibody solution, and 1 / 2 of the differentiation culture medium is replaced every two days, and the cells enter the differentiation process and differentiate for 14 days.

[0061] After 14 days of differentiation, the porous scaffold material structure remains intact, and the texture of the cell cultured meat tissue is elastic. The cultured meat tissue is harvested and slowly washed several times to remove the culture fluid components; after coloring with red yeast rice, the cultured meat is cooked by frying. The cultured meat is placed in a frying pan and fried on both sides until it turns dark yellow. Figure 5 As shown, after frying, the surface of the cell cultured meat is glossy and its appearance is no different from that of normal muscle tissue. Its taste is similar to fried ham. However, due to its smaller size, the meat flavor is lighter and its aroma is slightly lower.

[0062] According to the above method, the cells are replaced with porcine skeletal muscle stem cells, porcine smooth muscle cells and porcine fibroblasts in a ratio of 2:1:1. After the 14-day differentiation process, the porous scaffold material structure remains intact, the cultured meat tissue is harvested, and slowly washed several times to remove the culture fluid components; after the red yeast red coloring treatment, the cultured meat is cooked by frying, and the cultured meat is placed in a frying pan and fried on both sides until it is dark yellow; the cell cultured meat obtained based on multi-cell co-culture not only has mechanical properties similar to natural muscle tissue, but also the secreted extracellular matrix gives it a distinct glossy appearance, showing sensory characteristics similar to natural muscle tissue, and the cooked cell cultured meat has a typical meaty taste and a pleasant meaty flavor.

[0063] Example 5

[0064] Disperse food-grade collagen or gelatin in water to form a suspension with a mass concentration of 6%. After fully dissolving in a warm water bath at 50°C for 0.5h, filter out the insoluble components; take a 2% polysaccharide (sodium alginate, agar or chitosan) aqueous solution, and mix the protein and polysaccharide at a volume ratio of 2:1. Use 10% glutaminase to induce protein molecule cross-linking, and the volume ratio of glutaminase to the above mixed liquid is 1:10. Let it stand at room temperature for 2h, and form a hydrogel with a stable gel state through the electrostatic interaction between protein and polysaccharide; freeze at -80°C for 5h, then thaw at 25°C for 8min, and then freeze at -80°C for 5h to construct a porous structure. Finally, the hydrogel in the frozen state is vacuum-dried for 24h to obtain a porous scaffold material.

[0065] Example 6

[0066] Disperse food-grade collagen or gelatin in ultrapure water to form a 6% suspension, fully dissolve in a 50°C warm water bath for 0.5h, and filter to remove insoluble components; mix the porogen (glycerol or calcium carbonate) with the filtered collagen solution at a mass ratio of 1:3; disperse 10% glutamine transferase aqueous solution and the filtered collagen solution at a volume ratio of 1:10 in the protein-porogen mixture, and form a hydrogel at room temperature for 40min. Freeze the sample with a stable gel state at -80°C overnight, and then vacuum freeze-dry for 24h to obtain a porous scaffold material.

Claims

1. A personalized customization of cell cultured meat based on porous scaffold materials and a production method thereof, characterized in that: The following steps are involved: (1) Preparation of porous scaffold materials: Food-grade collagen was dispersed in water to form a suspension with a mass concentration of 4%, and the suspension was fully dissolved in a warm water bath at 50°C for 0.5 h, and then the insoluble components were filtered out. 100 u / g glutamyl transferase was dispersed in water to form a solution with a mass fraction of 10%, and the solution was mixed evenly with the filtered collagen solution at a volume ratio of 10:

1. The solution was then incubated at room temperature for 30 min to form a hydrogel with a stable gel state. The porous structure was constructed by freezing at -196°C liquid nitrogen for 5 min to form ice crystals, followed by thawing at 25°C for 8 min, and then freezing at -80°C for 5 h. Finally, the hydrogel in the frozen state was vacuum-freeze-dried for 24 h to obtain a porous scaffold material. The pores of the porous scaffold material were interconnected, the pore size distribution was uniform, and the channel diameter was mostly 10-60 μm. (2) Cell inoculation and culture: The prepared porous scaffold material was sterilized with 75% ethanol, then washed with PBS three times, and finally irradiated with ultraviolet light for 40 min under closed conditions. After irradiation sterilization, 100 μl of 1×10 8 / ml porcine skeletal muscle stem cells or 1×10 8 pcs / ml porcine skeletal muscle stem cells, porcine smooth muscle cells and porcine fibroblasts, with a quantity ratio of 2:1:1, were inoculated on a porous scaffold material cut with a surgical blade, with a size of about 10 mm×10 mm×1 mm; the muscle stem cells were inoculated on the porous scaffold material for 1 hour, and 2 ml of F10 proliferation culture medium including 20% ​​fetal bovine serum, 1% penicillin-streptomycin double antibody solution and 5ng / ml fibroblast growth factor was added to the well plate to slowly adhere to the wall, and the culture plate was placed in a saturated humidity, 37°C, 5% CO2 incubator for 3 days, and the proliferation culture medium was replaced every two days; when the muscle stem cell proliferation reached the pre-differentiation density, the differentiation culture medium was replaced, and the differentiation culture medium contained 2% horse serum, 97% DMEM cell culture medium, and 1% penicillin-streptomycin double antibody solution. 1 / 2 of the differentiation culture medium was replaced every two days, and the cells entered the differentiation process and continued to differentiate for 14 days; or: The prepared porous scaffold material was cut into cylinders with a diameter of about 2.2 cm and a height of 1.5 mm using a surgical blade. The porous scaffold material was sterilized with 75% ethanol, then rinsed with PBS three times, and finally irradiated with ultraviolet light for 40 min under closed conditions. After irradiation sterilization, 100 μl of 1×10 9 / ml porcine skeletal muscle stem cells or 1×10 9 100 cells / ml of porcine skeletal muscle stem cells, porcine smooth muscle cells and porcine fibroblasts were inoculated on the treated porous scaffold material in a ratio of 2:1:1; the muscle stem cells were inoculated on the porous scaffold material for 1 hour, and 2 ml of F10 proliferation culture medium including 20% ​​fetal bovine serum, 1% penicillin-streptomycin double antibody solution and 5 ng / ml fibroblast growth factor was added to the well plate to slowly adhere to the wall, and the culture plate was placed in a saturated humidity, 37°C, 5% CO2 incubator for 3 days, and the proliferation culture medium was replaced every two days; when the muscle stem cell proliferation reached the pre-differentiation density, the differentiation culture medium was replaced, and the differentiation culture medium contained 2% horse serum, 97% DMEM cell culture medium, and 1% penicillin-streptomycin double antibody solution. 1 / 2 of the differentiation culture medium was replaced every two days, and the cells entered the differentiation process and differentiated for 14 days; (3) Personalized customization: Collect the cell-cultured meat after the differentiation process is completed, process it into food, and then choose the cooking method as needed to obtain the cultured meat product.

2. The personalized customization of cell cultured meat based on porous scaffold materials and the production method thereof according to claim 1, characterized in that: The food processing of the cultured meat in step (3) includes marinating with seasonings, coating with flour or coloring with red yeast rice; and then cooking the cultured meat in different ways, including steaming, frying or pan-frying.

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