A method for preparing a cell-cultured meat biobracket by physical crosslinking

Abstract

The present invention relates to the fields of biomaterials and food, and particularly to a method for physically crosslinking to prepare a cell-cultured meat biocompatible scaffold. The physical crosslinking method enables the internal combination of the biocompatible scaffold material through physical forces to form a biocompatible scaffold. The physical crosslinking method comprises the following steps: 1) shaping and drying the scaffold material; 2) steam crosslinking the dried scaffold material. The present invention uses a physical crosslinking method to prepare an edible protein or polysaccharide scaffold to support animal cell culture. No chemical crosslinking reagents are used during the production process, and no coatings such as ECM proteins are used. At the same time, cells proliferate well on the scaffold. The physical crosslinking step of this method can be combined with the sterilization step, with simple operating steps, low cost, and is applicable to the preparation of scaffolds of various forms.

Description

Technical Field

[0001] The present invention relates to the fields of biomaterials and food, and particularly to a method for physically cross-linking to prepare a cell culture meat biocompatible scaffold. Background Art

[0002] Due to factors such as growing meat consumption, environmental degradation, animal welfare issues, human nutrition, food safety, etc., there is a need to develop alternative proteins. Cultured meat is an emerging technology, also known as in vitro meat, cultured meat, clean meat or lab-grown meat, which is produced by culturing cells in vitro rather than by harvesting animal tissues. A major challenge in the commercialization of cultured meat is large-scale production, which includes: (1) cell line development; (2) serum-free medium development; (3) bioreactor design; (4) scaffold development. Scaffold biomaterials serve as a supporting network, enabling cell attachment and facilitating the transport of oxygen and nutrients to thick tissues. In addition, the scaffold can direct cell proliferation, differentiation, and alignment. Ideally, the scaffold material for cultured meat should be easily digestible, safe to eat, and delicious. In addition, the cell meat scaffold also needs to meet specific taste, texture, thermal stability, and nutritional requirements, and be supplied on a large scale and at low cost.

[0003] Currently, the reported methods for producing scaffolds for cultured meat include preparing a fibrous gelatin scaffold using an immersion rotary jet spinning technique, which reproduces some of the structures and mechanical properties of natural meat products, but this method is only applicable to scaffolds containing a large amount of gelatin components and has limitations in terms of scaffold formulation. Textured soy protein (TSP) scaffolds have recently been used to support the growth and differentiation of bovine skeletal muscle cells, and fibrinogen is used to improve cell adhesion during cell seeding. Scaffolds composed of plant proteins and natural polysaccharides usually need to be coated with extracellular matrix (ECM) proteins (such as collagen, laminin, vitronectin) or positively charged polymers (such as polylysine and polyornithine) to improve cell adhesion. Although animal ECM proteins support cell adhesion and proliferation, they need to be avoided in the production of cultured meat scaffolds because they require livestock to produce, which to some extent negates the benefits of products rich in non-animal proteins.

[0004] In tissue engineering and regenerative medicine, many scaffolds are composed of synthetic polymer materials to ensure mechanical strength. Cross-linking agents are often required to maintain performance during production, and common cross-linking agents include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC / NHS) and glutaraldehyde. However, these chemicals do not have generally recognized food safety, so they cannot be used in food production. For cultured meat production, the production of scaffolds needs to avoid using chemical cross-linking agents or additives because these cross-linking agents will affect the taste, quality, and health. Therefore, the selection of cross-linking agents or cross-linking methods that meet food safety is required for the production of cultured meat scaffolds. Summary of the Invention

[0005] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a method for preparing a cell-cultured meat biologic scaffold by physical crosslinking to solve the problems in the prior art.

[0006] To achieve the above purpose and other related purposes, the present invention provides a method for preparing a cell-cultured meat biologic scaffold, and the preparation method includes using a physical crosslinking method to form a biologic scaffold by physical force binding inside the biologic scaffold material.

[0007] In one embodiment, the physical crosslinking method includes the following steps:

[0008] 1) Shape and dry the scaffold material;

[0009] 2) Steam crosslink the dried scaffold material.

[0010] The physical force binding inside the biologic scaffold material is by hydrogen bond binding.

[0011] The present invention also provides a cell-cultured meat biologic scaffold obtained by the above preparation method.

[0012] The present invention also provides a cell-cultured meat, and the cell-cultured meat includes the cell-cultured meat biologic scaffold and cells attached thereto.

[0013] As described above, the method for preparing a cell-cultured meat biologic scaffold by physical crosslinking of the present invention has the following beneficial effects: A new method is adopted to prepare an edible protein or polysaccharide scaffold to support animal cell culture. Chemical crosslinking reagents are not used in the production process, and coatings such as ECM proteins are not used, but the cells proliferate well on the scaffold. The present method uses a water annealing method to physically crosslink the scaffold, which not only introduces a hydrogen bond crosslinking method under high-temperature steam conditions to stabilize the scaffold, but also combines with the high-temperature and high-pressure steam sterilization step for scaffold preparation. The operation steps are simple, the cost is low, and it is applicable to the preparation of scaffolds of various morphologies. Brief Description of the Drawings

[0014] Figure 1 It shows a schematic diagram of the preparation method of the sponge scaffold of the present invention.

[0015] Figure 2 It shows a schematic diagram of the preparation method of the directional bionic scaffold of the present invention.

[0016] Figure 3 It shows the macroscopic (upper left), microscopic morphology (left) of the gluten protein sponge scaffold in Example 1 and the cell growth situation (right figure, live and dead staining).

[0017] Figure 4Shown are the macroscopic (upper left), microscopic morphology (left), and cell growth (right, live / dead staining) of the gluten biomimetic oriented scaffold in Example 2.

[0018] Figure 5 Shown are the macroscopic (upper left), microscopic morphology (left), and cell growth (right, live / dead staining) of the konjac glucomannan sponge scaffold in Example 3.

[0019] Figure 6 Shown is a schematic diagram of the self-made mold of the present invention.

[0020] Figure 7 Shown is the preparation flow chart of the cultured meat of the present invention. Detailed implementation manners

[0021] The present invention provides a method for preparing a biological scaffold for cultured meat, and the preparation method includes using a physical cross-linking method to form a biological scaffold by physical force binding inside the biological scaffold material.

[0022] In one implementation manner, the physical cross-linking method includes the following steps:

[0023] 1) Shape and dry the scaffold material;

[0024] 2) Steam cross-link the dried scaffold material.

[0025] The scaffold material is a dispersion system. In one implementation manner, the dispersed phase of the dispersion system is selected from proteins and / or polysaccharides. The proteins include but are not limited to one or several of soybean protein, pea protein, wheat protein, gluten protein, potato protein, rice protein, chickpea protein, mung bean protein, peanut protein, seaweed protein, yeast protein, corn protein, almond protein, quinoa protein, etc. The polysaccharide is an edible polysaccharide. The polysaccharides include but are not limited to one or several of curdlan, konjac glucomannan, arabic gum, xanthan gum, hyaluronic acid, locust bean gum, guar gum, sodium alginate, etc. Specifically, the scaffold material is, for example, gluten protein, glutenin, konjac glucomannan.

[0026] In one embodiment, the scaffold material is a solution. The concentration of the scaffold material solution is 1 mg / mL to 200 mg / mL. The concentration of the scaffold material solution can be determined according to different scaffold materials. The concentration of the scaffold material solution is selected from any of the following ranges: 1 to 25 mg / mL, 25 to 50 mg / mL, 50 to 75 mg / mL, 75 to 100 mg / mL, 100 to 125 mg / mL, 125 to 150 mg / mL, 150 to 175 mg / mL, 175 to 200 mg / mL. For example, in one embodiment, when the gluten solution is used as the scaffold material solution, the concentration is 10 mg / mL to 150 mg / mL; in another embodiment, when the gliadin solution is used as the scaffold material solution, the concentration is 1 mg / mL to 150 mg / mL. In another embodiment, when the konjac glucomannan solution is used as the scaffold material solution, the concentration is 5 mg / mL to 50 mg / mL.

[0027] In certain embodiments of the present invention, the pH of the gluten solution and the gliadin solution as scaffold materials is 1 - 5. Specifically, the pH can be 1 to 2, 2 to 3, 3 to 4, or 4 to 5. In some other embodiments, the pH of the konjac glucomannan solution as the scaffold material is 7 - 12.

[0028] In certain embodiments of the present invention, the method of shaping the scaffold material is to freeze the scaffold material solution to solidify it for shaping. In one embodiment of the present invention, the scaffold material solution is transferred to a mold with a preset shape and then frozen.

[0029] The shape of the mold can be made or selected according to the shape that the scaffold is desired to form. In certain embodiments of the present invention, the mold is a well plate. The well plate is selected from a six - well plate, a 12 - well plate, a 24 - well plate, a 48 - well plate, or a 96 - well plate. The shape of the scaffold formed using the well plate as the mold is consistent with the shape of each well on the well plate.

[0030] In certain embodiments of the present invention, the mold is a self - made mold. The self - made mold is an open - top container, and the inner cavity of the open - top container is divided into a freezing part and a scaffold material accommodating part by a temperature - conducting member. In one embodiment, the self - made mold is as Figure 6 shown.

[0031] The material of the self - made mold can withstand a low temperature of - 200 °C. In certain embodiments of the present invention, the material of the open - top container is PDMS. The temperature - conducting member can conduct temperature. The material of the temperature - conducting member is a metal material. The metal material is selected from steel, iron, copper, aluminum, and tin.

[0032] The freezing part is used to hold liquid nitrogen.

[0033] During the shaping in step 1), the temperature for freezing and solidifying only needs to freeze the scaffold material solution. The temperature is, for example, -10°C to -200°C. The temperature is selected from any of the following ranges: -10°C to -40°C, -40°C to -70°C, -70°C to -80°C, -80°C to -85°C, -85°C to -90°C, -90°C to -100°C, -100°C to -150°C, -150°C to -180°C, -180°C to -190°C, or -190°C to -200°C.

[0034] In one embodiment, the shaping is to transfer the mold containing the scaffold material solution to an ultra-low temperature refrigerator for freeze shaping.

[0035] In another embodiment, the step of shaping is to fill the freezing part with liquid nitrogen and use liquid nitrogen for directional freezing to solidify and form. After adding liquid nitrogen and the scaffold material solution to the freezing part and the scaffold material container respectively, the temperature conduction member can conduct the temperature of the liquid nitrogen to the scaffold material solution, and the solution closer to the temperature conduction member will be frozen first, and the solution farther away will be gradually frozen, so as to achieve the effect of directional freezing.

[0036] In one embodiment, the drying in step 1) is freeze-drying; in a preferred embodiment, the temperature of freeze-drying in step 1) is -40°C to -100°C, and / or the time of freeze-drying is 24 - 72 hours. In one embodiment, the temperature of the freeze-drying is selected from any of the following: -40°C to -50°C, -50°C to -60°C, -60°C to -70°C, -70°C to -80°C, -80°C to -90°C, or -90°C to -100°C. The freeze-drying is carried out in a freeze-dryer.

[0037] The freeze-drying can be carried out one or more times at different or the same temperature. The specific freeze-drying steps need to be determined according to the properties of the scaffold material solution.

[0038] In one embodiment, when the scaffold material solution is konjac gum solution, it is freeze-dried once before shaping, soaked in alkali solution, and then shaped and subjected to the second freeze-drying to form a freeze-dried sponge. Soaking in the alkali solution can remove acetyl groups, and the konjac gum after removing acetyl groups can crosslink under heating conditions. The alkali solution is, for example, selected from Na2CO3 solution or Ca(OH)2 solution.

[0039] In one embodiment, the specific steps of steam crosslinking are: after demolding the freeze-dried scaffold material, put it into a heat-resistant container, seal the heat-resistant container with a breathable material, and carry out steam crosslinking. Steam can achieve the purposes of sterilization and crosslinking at the same time.

[0040] In one embodiment, the heat-resistant container is selected from a glass bottle, a metal container or heat-resistant plastics.

[0041] In one embodiment, the breathable material is selected from blotting paper or fabric. The breathable material can limit the condensation of water vapor to ensure that the steam fills the heat-resistant container and the support material.

[0042] The temperature for steam sterilization and crosslinking is 50 - 200 °C, and / or the air pressure is 1 psi - 100 psi.

[0043] The temperature is selected from any of the following ranges: 50 - 70 °C, 70 - 90 °C, 90 - 110 °C, 110 - 130 °C, 130 - 150 °C, 150 - 170 °C, 170 - 190 °C, 190 - 200 °C.

[0044] The air pressure is selected from any of the following ranges: 1 psi - 10 psi, 10 psi - 20 psi, 20 psi - 30 psi, 30 psi - 40 psi, 40 psi - 50 psi, 50 psi - 60 psi, 60 psi - 70 psi, 70 psi - 80 psi, 80 psi - 90 psi or 90 psi - 100 psi.

[0045] The time for steam crosslinking is 1 - 60 minutes. The steam crosslinking time is selected from any of the following ranges: 1 - 10 minutes, 10 - 20 minutes, 20 - 40 minutes, 40 - 60 minutes.

[0046] In one embodiment, after steam crosslinking in step 2), drying is further included.

[0047] Steam crosslinking is a key step in producing a stable scaffold. Steam crosslinking is a water annealing process that introduces hydrogen bonds between macromolecules, promotes crystallization, and stabilizes the three-dimensional structure. Proteins, such as silk fibroin, form a stable three-dimensional structure after water annealing due to the crystallization in regions with an increased β-sheet content. FTIR results show that the content of random coils in the secondary structure of glutenin decreases after water annealing, while the α-helix and β-sheet structures increase, which means the formation of intramolecular hydrogen bonds. In an aqueous solution, protein molecules are dispersed in the solution. As water molecules are removed (drying), the protein molecules become closer in space. Under the action of water annealing, water molecules penetrate into the protein chain network and plasticize the system, forming a protein-water binding network with a higher chain mobility. The protein system absorbs the thermal energy of free water vapor molecules and transfers the thermal energy to the protein-water system in the form of long-distance molecular migration, and finally self-assembles to form stacked β-sheet crystals. High-temperature heating also increases the recombination of disulfide crosslinks in the protein network. The physical crosslinking introduced by water annealing avoids the use of toxic chemical crosslinking agents.

[0048] Inside the biological scaffold material, it is combined by physical force, specifically by hydrogen bonding.

[0049] The steam crosslinking method can also be used for the crosslinking and stabilization of various one-dimensional materials (such as fibers), two-dimensional materials (such as films), and three-dimensional materials (such as the porous sponge or directional freezing scaffold of the present invention).

[0050] The present invention also provides a cell-cultured meat biological scaffold obtained by the preparation method.

[0051] The cell-cultured meat biological scaffold is a porous sponge scaffold and a directionally frozen porous scaffold. The diameter of the cell-cultured meat porous scaffold is 10 - 500 μm.

[0052] The present invention also provides a cell-cultured meat, which includes the cell-cultured meat biological scaffold and cells attached thereto.

[0053] The cells are animal cells. In one embodiment, the cells are mammalian cells.

[0054] The cells are selected from the group consisting of myoblasts, muscle satellite cells, fibroblasts, adipocytes, adipose progenitor cells, adult stem cells, and pluripotent stem cells.

[0055] The cells include, but are not limited to, muscle cells, hepatocytes, osteoblasts, fibroblasts, adipocytes, odontoblasts, adult neural progenitor cells, neural stem cells, multiple multipotent stem cells from the subventricular forebrain region, ependymal neural stem cells, hematopoietic stem cells, hepatic hematopoietic stem cells, bone marrow stem cells, adipose fibroblasts, adipose stem cells, stem cells that produce multiple pancreatic islet cells, pancreatic-derived pluripotent islet-producing stem cells, mesenchymal stem cells, placental cells, bone marrow stromal cells, muscle side population cells, bone marrow-derived recovered cells, blood-derived mesenchymal progenitor cells, bone marrow-derived side population cells, muscle progenitor cells, circulating skeletal stem cells, neural progenitor cells, multipotent adult progenitor cells, mesoderm progenitor cells, spinal cord progenitor cells, and spore-like cells, and any combination thereof.

[0056] The following specific examples illustrate the embodiments of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0057] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments, rather than limiting the protection scope of the present invention; in the specification and claims of the present invention, unless otherwise clearly indicated in the text, the singular forms "a", "an" and "the" include plural forms.

[0058] When an embodiment gives a numerical range, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, devices, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention.

[0059] The present invention adopts a new method for the preparation of a stable biological scaffold to support animal cell culture. Experiments were carried out using C2C12 mouse skeletal muscle myoblasts, freshly isolated bovine satellite cells (BSCs), and mouse embryonic fibroblasts (3T3-L1). Three-dimensional porous sponges and oriented porous scaffolds were prepared and characterized, and the growth of muscle cells on the materials was investigated. No chemical cross-linking reagents were used during the production process, and no coatings such as ECM proteins were used in the preparation of the plant protein scaffold, but the muscle cells proliferated well on the protein scaffold. This method uses a water annealing method to physically cross-link the scaffold, that is, introducing a hydrogen bond cross-linking method under high-temperature steam conditions to stabilize the scaffold. This method can be combined with the high-temperature and high-pressure steam sterilization step in the scaffold preparation. The operation steps are simple and applicable to the preparation of scaffolds of various morphologies.

[0060] Example 1

[0061] Preparation method of sponge-like porous gluten protein scaffold: Dissolve 40 mg / mL of gluten protein in water, stir at room temperature for 24 h, and adjust the pH to 3 with 0.1 N HCl and 0.1 M NaOH. Then place these gluten protein solutions in a 90 °C water bath and stir for 5 - 60 minutes to fully disperse and unfold the gluten protein. Then disperse the solution into a 48-well plate, freeze at -20 °C for 48 hours, and then freeze-dry at -81 °C for 48 hours. After freeze-drying, it becomes a sponge-like shape called freeze-dried sponge. Transfer the freeze-dried sponge to a glass vial, seal the mouth of the glass vial with a water-absorbing paper towel and a rubber band to limit the condensation of water vapor and ensure that the steam fills the glass vial and the porous scaffold. After sealing, sterilize with steam (autoclaving method) at 121 °C for 15 minutes and dry for 10 minutes to cross-link and sterilize it.

[0062] The C2C12 cells were seeded on the obtained scaffolds at a seeding density of five hundred thousand cells per milliliter.

[0063] The macroscopic and microscopic morphologies of the gluten protein sponge scaffolds and the cell growth conditions are as Figure 3 shown. The gluten protein scaffolds after steam crosslinking can remain stable in the buffer (the stable time can be up to 6 months), showing a porous structure with pore diameters ranging from 10 - 500 μm. The live / dead staining results show the situation 7 days after cell seeding on the scaffolds. As shown in the figure, the green represents live cells and the red represents dead cells. The number of red cells is small and almost invisible; the green live cells are spindle-shaped and attached to the scaffolds, indicating that the cells grow well on the scaffolds.

[0064] Example 2

[0065] Preparation of the PDMS mold: A PDMS solution was poured into a rectangular open trough with dimensions of 10 * 6 * 2 cm, spread into a thin layer, placed in an oven, and cured at 60 °C. Then it was taken out. A rectangular module with dimensions of 6 * 3 * 1 cm was placed in the middle of the thin layer, and then the PDMS solution was poured around the module and placed in the oven for curing at 60 °C. Then the module was taken out to obtain a hollow PDMS mold, and a cavity with a rectangular cross-section was separated by a steel plate.

[0066] Preparation method of the biomimetic oriented gluten scaffold: Gluten with a concentration of 30 mg / mL and a pH value of 3 was transferred to a rectangular cavity of the PDMS mold. Liquid nitrogen was added to the other side of the PDMS mold to immediately freeze the gluten solution directionally. After freezing, it was freeze-dried at -81 °C to form a gluten scaffold with directionally arranged pores. Finally, the gluten scaffold with oriented pores was placed in a sterilized sealed bag, autoclaved at 121 °C for 15 minutes, and then dried for 10 minutes to crosslink and sterilize it.

[0067] Bovine primary cells were seeded on the obtained scaffolds at a seeding density of five hundred thousand cells per milliliter.

[0068] The macroscopic and microscopic morphologies of the biomimetic oriented gluten scaffolds and the cell growth conditions are as Figure 4 shown. The gluten scaffolds after steam crosslinking show a directionally porous structure with pore diameters ranging from 10 - 200 μm. The live / dead staining results show the situation 7 days after cell seeding on the scaffolds. As shown in the figure, the green represents live cells and the red represents dead cells. The number of red cells is small and almost invisible; the green live cells are spindle-shaped and attached to the scaffolds, indicating that the cells grow well on the scaffolds.

[0069] Example 3

[0070] Preparation of konjac porous scaffold: Konjac glucomannan at 10 mg / mL was stirred evenly in water, and then the solution was dispersed into a well plate or mold, frozen and shaped at -20 °C, and then freeze-dried at -81 °C for 48 hours. Then it was soaked in 1 M Na2CO3 solution for 2 hours, and freeze-dried at -81 °C for another 48 hours. After freeze-drying, it became sponge-like and was called freeze-dried sponge. The freeze-dried sponge was transferred to a glass vial, and the mouth of the glass vial was sealed with absorbent paper napkins and rubber bands to limit the condensation of water vapor and ensure that the steam filled the glass vial and the porous scaffold. After sealing, it was steam sterilized (autoclaving) at 121 °C for 15 minutes and dried for 10 minutes to crosslink and sterilize it.

[0071] Mouse preadipocytes (3T3-L1) were seeded on the obtained scaffold at a seeding density of five million cells per milliliter.

[0072] The macroscopic, microscopic morphologies and cell growth of the konjac glucomannan sponge-like scaffold are as Figure 5 shown. The konjac scaffold after steam crosslinking was porous, and the pore diameter range was between 20 - 500 μm. The live / dead staining results were for the situation 7 days after cell seeding on the scaffold. As shown in the figure, green represents live cells and red represents dead cells. The number of red cells was small and almost invisible; most of the green live cells were spindle-shaped and attached to the scaffold, indicating that the cells grew well on the scaffold.

[0073] The above embodiments are for illustrating the implementation schemes disclosed by the present invention and should not be construed as limitations on the present invention. In addition, various modifications listed herein and changes in the methods of the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, all obvious modifications to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A method for preparing a cell-cultured meat biological scaffold, characterized in that, The preparation method includes using a physical crosslinking method to form a biological scaffold by physical force binding inside the biological scaffold material, and the physical force binding inside the biological scaffold material is by hydrogen bond binding; the physical crosslinking method includes the following steps: 1) Shape and freeze-dry the scaffold material. The temperature of freeze-drying is -40°C to -100°C, and the time of freeze-drying is 24 - 72 hours; the scaffold material is a dispersion system, and the dispersed phase of the dispersion system is selected from gluten protein, gliadin or konjac gum. The method of shaping the scaffold material is to freeze the scaffold material to make it solidify and thus shape; the temperature of freeze-solidification during shaping is -10°C to -200°C; when the scaffold material is konjac gum, it is freeze-dried once before shaping, soaked in alkali solution, and then shaped and freeze-dried for the second time; 2) Steam crosslink the dried scaffold material. The specific steps of the steam crosslinking are: put the scaffold material treated in step 1) into a heat-resistant container, seal the heat-resistant container with a breathable material and then carry out steam crosslinking; the temperature of the steam crosslinking is 50 - 200°C, the air pressure is 1 psi to 100 psi, and the time is 1 - 60 minutes.

2. The preparation method according to claim 1, characterized in that, The concentration of the dispersed phase in the dispersion system is 1 - 200 mg / mL.

3. The preparation method according to claim 1, wherein, The dispersant of the dispersion system is water.

4. The preparation method according to claim 1, characterized in that, The method of shaping the scaffold material is to transfer the scaffold material to a mold with a preset shape and then freeze it.

5. The preparation method according to claim 1, wherein The heat-resistant container is selected from a glass bottle, a metal container or a heat-resistant plastic, and / or, the breathable material is selected from absorbent paper or fabric.

6. A cell-cultured meat biological scaffold obtained by the preparation method according to any one of claims 1 - 5.

7. A cultured meat, characterized in that, The cell-cultured meat includes the cell-cultured meat biological scaffold according to claim 6 and cells attached thereto.

8. The cell-cultured meat according to claim 7, wherein, The cells attached to the cell-cultured meat fiber scaffold are animal cells.

9. The cultured meat according to claim 7, wherein The cells are selected from myoblasts, muscle satellite cells, adipocytes, fibroblasts, adipose precursor cells.

10. The cultured meat according to claim 7, characterized in that, The cells are adult stem cells.

11. The cultured meat according to claim 7, wherein The cells are pluripotent stem cells.

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