A chunky cell culture meat and a method of preparing the same

Abstract

The present application provides a kind of block cell culture meat and its preparation method.The present application meets the demand of seed cell large-scale preparation of block cell culture meat by suspension cell scale culture, and realizes the adhesion, proliferation and integration of seed cell on edible scaffold material by specific culture medium induction of suspension cell adhering to wall.The method can be in stirred tank cell bioreactor by sterile scaffold material and cooperate with specific induction culture solution and stirring process, so that the adhesion rate of cell and scaffold material reaches 96.8%.The method uses suspension cell as seed cell, and combines corresponding process and culture medium, effectively solves the problems of seed cell difficult to be large-scale prepared and difficult to be effectively integrated with scaffold material in the culture process of block cell culture meat, and provides scalable scheme for industrialized production of block cell culture meat.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and in particular to a block-shaped cultured meat and its preparation method. Background Technology

[0002] With the increasing global demand for protein, the traditional livestock industry is rapidly expanding, leading to a series of environmental problems and resource pressures. Cell-cultured meat, which produces protein using animal cells in a controlled environment, is an environmentally friendly and sustainable protein production method. Currently, most cell-cultured meat production methods still focus on culturing single cells or cell composite materials into microparticle tissue forms. The harvested cell-cultured meat is often loose, textureless mince, making it difficult to replicate the texture and grain of animal meat. It needs to be mixed with plant-based ingredients to be processed into various meat substitutes. Existing culture technologies struggle to reproduce the complex structure of natural muscle, resulting in cultured products that are mostly in the form of minced meat, failing to meet market demands for the shape and texture of cut meats (such as steak and chicken breast).

[0003] Block-shaped cultured meat is produced by seeding cells onto a scaffold material, where the cells adhere, proliferate, and undergo matrix remodeling (integration), ultimately forming block-shaped meat with the texture and structure of animal meat. This represents the next trend and direction for the development of cell-cultured meat. However, there are still significant technical bottlenecks in the current process for preparing block-shaped cultured meat: (1) Large-scale preparation of seed cells is difficult. Currently, the seed cells for block-shaped cultured meat are mostly adherent cells, and their production relies on plate culture and microcarrier culture. Plate culture faces drawbacks such as large footprint, complex operation, high labor costs, and difficulty in scaling up. In microcarrier culture, the separation of cells from microcarriers and centrifugation processes increase the complexity of the process and challenges in quality consistency. (2) Seed cells and scaffold materials are difficult to integrate effectively. Currently, the main methods for integrating cells and materials based on tissue engineering construction strategies are static seeding and cell 3D printing. Static seeding faces constraints such as uneven cell distribution and difficulty in scale-up; cell 3D printing faces problems such as low efficiency and high equipment costs.

[0004] Therefore, solving the problems of large-scale preparation of seed cells and effective integration with scaffold materials in the process of cultured meat from bulk cells is the key to promoting the establishment of industrial production technology for cultured meat from bulk cells. Summary of the Invention

[0005] This invention provides a method for preparing block-shaped cultured meat. The method involves adjusting the culture medium composition and optimizing the culture process when suspension cells reach peak density, controlling cell interactions and signal transduction to induce cell adhesion to a scaffold material (gelatin-modified plant textured protein scaffold). Subsequently, cells proliferate and migrate, secreting extracellular matrix (ECM), thereby cultivating block-shaped cultured meat with a structure and texture close to real meat. This method uses suspension cells as seed cells, making it suitable for large-scale industrial proliferation culture, effectively improving production efficiency and scale, and meeting the demand for large-scale seed cell preparation in cultured meat. Furthermore, this method achieves efficient integration of seed cells and scaffold material, with a cell adhesion rate as high as 96.8%, effectively solving the problems of large-scale seed cell preparation and effective integration with scaffold material in the culture of block-shaped cultured meat, providing a new pathway for the large-scale production of block-shaped cultured meat.

[0006] This invention also provides a block-shaped cultured meat, prepared by the above-described method for preparing block-shaped cultured meat. The inventors' research shows that this block-shaped cultured meat can maintain a good morphological structure.

[0007] The first aspect of the present invention provides a method for preparing block-shaped cultured meat, comprising the following steps:

[0008] Suspension cells were inoculated into medium No. 1 for proliferation culture. When the cell density reached its peak, the proliferated cells were obtained.

[0009] After proliferation culture, the cells were centrifuged, the cell pellet was collected, and the cell pellet was resuspended in medium No. 2 to obtain a cell suspension.

[0010] Cell suspensions were inoculated onto gelatin-modified plant textured protein scaffolds, and after adding culture medium No. 2, variable-speed culture and constant-speed culture were performed sequentially to obtain block-shaped cultured meat.

[0011] The formulation of culture medium No. 1 includes: basal culture medium, fetal bovine serum 8-12% v / v, anti-cell aggregation agent 23-25ug / mL, and poloxamer 188 0.8-1mg / mL;

[0012] The formulation of culture medium No. 2 includes: basal culture medium, fetal bovine serum 8-12% v / v, astragalus polysaccharide 95-100ug / mL, inulin 2-2.5mg / mL, vitamin C 166.12-176.12ug / mL, selenium-enriched yeast 0.2-0.5ug / mL, and insulin 0.0106-0.0116ug / mL.

[0013] In the method for preparing block-shaped cultured meat as described above, the cell suspension density is 1E6-5E6 cells / mL.

[0014] The method for preparing block-shaped cell cultured meat as described above, wherein the suspended cells include chicken embryo fibroblast cell lines;

[0015] And / or, in the centrifugation process, the rotation speed is 800-1000 rpm and the time is 5-8 min;

[0016] And / or, the basal culture medium includes any one of DMEM medium, DMEM / F12 medium, and MEM medium.

[0017] The method for preparing block-shaped cultured meat as described above, wherein the gelatin-modified plant textured fibroin scaffold is obtained by a preparation method including the following steps:

[0018] Plant textured protein was cross-linked with gelatin and sterilized to obtain the initial gelatin-modified plant textured protein.

[0019] The initial gelatin-modified plant textured protein was soaked in sterile water and swelled after fully absorbing water to obtain swelled gelatin-modified plant textured protein.

[0020] The expanded gelatin-modified plant textured protein was soaked in culture medium No. 2 to obtain a gelatin-modified plant textured protein scaffold.

[0021] Culture medium No. 2 is the culture medium No. 2 mentioned above.

[0022] In the above-described method for preparing block-shaped cultured meat, the soaking treatment is carried out at a temperature of 38-39°C for 20-30 minutes.

[0023] And / or, the plant textured protein is any one of soybean textured protein, peanut textured protein, and wheat textured protein.

[0024] In the method for preparing block-shaped cultured meat as described above, the inoculation amount of the suspended cells is 2E5-3E5 cells / mL.

[0025] The method for preparing block-shaped cultured meat as described above, wherein the suspension cells are inoculated into culture medium No. 1 for proliferation culture, and when the cell density reaches its peak, the proliferated cells are obtained, specifically including:

[0026] Suspension cells were inoculated into medium No. 1 and cultured for 4-6 days at 110-120 rpm, 38-39℃, pH 7.2-7.4, and 50%-60% dissolved oxygen. Medium No. 1 was replaced every 1-2 days during the culture period. When the cell density reached its peak, the cultured cells were obtained.

[0027] The method for preparing block-shaped cultured meat as described above, wherein the variable-speed culture program is: 70-80 rpm, 5-6 min; 0 rpm, 1-1.2 h, 24 cycles;

[0028] The variable-speed culture was carried out at 38-39℃, pH=7.2-7.4, and 50%-60% dissolved oxygen for 20-24 hours.

[0029] The method for preparing block-shaped cultured meat as described above, wherein the cells are adhered to the surface of a gelatin-modified plant textured fibroin scaffold and then cultured at a constant rate to obtain block-shaped cultured meat, specifically includes:

[0030] After the cells adhered to the surface of a gelatin-modified plant textured protein scaffold, they were cultured at a constant rate for 7-10 days at 90-110 rpm, 38-39℃, pH 7.2-7.4, and 50%-60% dissolved oxygen. During the constant rate culture, the culture medium No. 2 was replaced every 1-2 days to obtain block-shaped cultured meat.

[0031] A second aspect of the present invention provides a cultured meat, which is prepared by the method for preparing block-shaped cultured meat.

[0032] The solution of the present invention has at least the following effects:

[0033] (1) This invention addresses the need for large-scale seed cell preparation in block-type cultured meat by using large-scale suspension cell culture. Furthermore, it induces re-adhesion of suspension cells onto the scaffold material using Culture Medium No. 2, achieving cell adhesion, proliferation, and matrix remodeling. This method uses suspension cells as seed cells, passing them through a scaffold material and employing a specific culture medium (Culture Medium No. 2) and agitation techniques (variable-rate and constant-rate culture). This satisfies the need for large-scale seed cell preparation in cultured meat and achieves efficient integration of seed cells (suspension cells) with the scaffold material, with a cell adhesion rate reaching 96.8%. This effectively solves the problems of large-scale seed cell preparation and effective integration with the scaffold material during block-type cultured meat cultivation, providing a new pathway for the large-scale production of block-type cultured meat.

[0034] (2) The method for preparing block-shaped cultured meat of the present invention uses culture medium No. 2 as the medium for inducing the adhesion, proliferation and migration of suspension cells. It induces the re-adhesion of suspension cells through the synergistic effect of multiple targets, taking into account energy supply, matrix remodeling, signal activation and antioxidant protection. It is more innovative and stable than traditional single factors (such as collagen or serum).

[0035] (a) Energy metabolism (inulin + insulin), matrix synthesis (vitamin C), signaling pathway (astragalus polysaccharide), and antioxidation (selenium-enriched yeast) work together in four dimensions to cover the entire adhesion process;

[0036] (b) Inulin and selenium-enriched yeast reduce oxidative damage and interference from inflammatory factors during culture by regulating the immune microenvironment. Studies have shown that using the No. 2 culture medium provided by this invention, the cell adhesion rate can reach 96.8%, and the cell morphology is well maintained, achieving a stable adhesion effect. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 The growth states of the chicken embryo fibroblast cell line in Example 1 in different culture media are shown; wherein, Figure 1 Figure A shows the results after culturing the CEF cell line in medium 1 for 2 hours. Figure 1 Figure B shows the results after culturing the CEF cell line in medium 2 for 2 hours.

[0039] Figure 2 The block-shaped cultured meat obtained by culturing CEF cell lines of different densities with gelatin-modified soybean textured fibrous scaffolds as described in Example 2; wherein, Figure 2 A is a block of cell cultured meat obtained by culturing a CEF cell line of 1E6 cells / mL with a gelatin-modified soybean textured scaffold. Figure 2 B is a block of cell cultured meat obtained by culturing a CEF cell line of 3E6 cells / mL with a gelatin-modified soybean textured protein scaffold. Figure 2 The block-shaped cultured meat was obtained by culturing a CEF cell line with 5E6 cells / mL and a gelatin-modified soybean textured scaffold.

[0040] Figure 3 Cell adhesion rates of CEF cell lines at different densities on gelatin-modified soybean fibrous scaffolds;

[0041] Figure 4 This is a flowchart illustrating the preparation of block-shaped cultured meat in Example 3 of the present invention;

[0042] Figure 5 This is an image showing the HE staining results of the block-shaped cell cultured meat in Example 3;

[0043] Figure 6 This is a diagram showing the DAPI staining results of the block-shaped cell cultured meat in Example 3; where, Figure 6 A represents the cell nuclear staining results on the surface of a gelatin-modified plant fibroin scaffold; Figure 6 B represents the staining result of the cell nuclei inside the gelatin-modified plant fibroin scaffold;

[0044] Figure 7 This is a diagram showing the Dil staining results of the block-shaped cell cultured meat in Example 3; where, Figure 7 A represents the result of cell nuclear staining; Figure 7 B represents the result of Dil staining; Figure 7 A combined image of C cell nuclear staining results and Dil staining results. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this art or in accordance with the product instructions. Reagents or instruments used without specified manufacturers are all conventional products that can be obtained commercially.

[0046] It should be noted that in this invention, "and / or" means at least one of the connected objects, and the character " / " generally indicates that the preceding and following objects are in an "or" relationship.

[0047] Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains.

[0048] The first aspect of the present invention provides a method for preparing block-shaped cultured meat, comprising the following steps:

[0049] Suspension cells were inoculated into medium No. 1 for proliferation culture. When the cell density reached its peak, the proliferated cells were obtained.

[0050] After proliferation culture, the cells were centrifuged, the cell pellet was collected, and the cell pellet was resuspended in medium No. 2 to obtain a cell suspension.

[0051] Cell suspensions were inoculated onto gelatin-modified plant textured protein scaffolds, and after adding culture medium No. 2, variable-speed culture and constant-speed culture were performed sequentially to obtain block-shaped cultured meat.

[0052] The formulation of culture medium No. 1 includes: basal culture medium, fetal bovine serum 8-12% v / v, anti-cell clumping agent 23-25ug / mL, and poloxamer 188 0.8-1mg / mL;

[0053] The formula for culture medium No. 2 includes: basal culture medium, fetal bovine serum 8-12% v / v, astragalus polysaccharide 95-100ug / mL, inulin 2-2.5mg / mL, vitamin C 166.12-176.12ug / mL, selenium-enriched yeast 0.2-0.5ug / mL, and insulin 0.0106-0.0116ug / mL.

[0054] I understand that in culture medium 1, 8-12% v / v fetal bovine serum (FBS) means the volume of FBS accounts for 8-12% of the total volume of culture medium 1; 23-25 ​​μg / mL anti-cell aggregation agent means the concentration of anti-cell aggregation agent in culture medium 1 is 23-25 ​​μg / mL; and 0.8-1 mg / mL poloxamer 188 means the concentration of poloxamer 188 in culture medium 1 is 0.8-1 mg / mL. In culture medium 2, 8-12% v / v FBS means the volume of FBS accounts for 8-12% of the total volume of culture medium 2; 95-100 μg / mL astragalus polysaccharide means the concentration of astragalus polysaccharide in culture medium 2 is 95-100 μg / mL; and 2-2.5 mg / mL inulin means the concentration of inulin in culture medium 2 is 2-2.5 mg / mL. Vitamin C... 166.12-176.12 ug / mL refers to the concentration of vitamin C in culture medium No. 2, 0.2-0.5 ug / mL refers to the concentration of selenium-enriched yeast in culture medium No. 2, and 0.0106-0.0116 ug / mL refers to the concentration of insulin in culture medium No. 2.

[0055] In this invention, the components in culture medium 1 work together to effectively promote cell proliferation; the components in culture medium 2 work together to effectively promote cell adhesion, achieving efficient integration of cells and scaffold materials.

[0056] The concentrations of each component in culture medium No. 2 are within the reasonable range selected in this invention. If the concentrations exceed or fall below the ranges defined in this application, they will not be able to promote cell adhesion and growth.

[0057] Specifically, the present invention first inoculates suspended cells into culture medium No. 1 for proliferation culture. When the cell density reaches its peak, proliferated cells are obtained. The proliferated cells are centrifuged, and the cell pellet is collected. The cell pellet is resuspended in culture medium No. 2 to obtain a cell suspension. The cell suspension is inoculated onto a gelatin-modified plant textured protein scaffold, and culture medium No. 2 is added to cover the gelatin-modified plant textured protein scaffold. Then, variable-speed culture is performed to allow the cells to adhere to the surface of the gelatin-modified plant textured protein scaffold. Then, constant-speed culture is performed to allow the adhered cells to proliferate and migrate and secrete extracellular matrix (ECM). After maturation, block-shaped cell cultured meat is obtained.

[0058] This invention addresses the need for large-scale seed cell preparation in block-type cultured meat by scaling up suspension cell culture. It utilizes a No. 2 culture medium to induce re-adhesion of suspension cells, achieving adhesion, proliferation, and integration of suspension cells onto a scaffold material. This method uses suspension cells as seed cells, passing them through a scaffold material and employing a specific culture medium (No. 2 medium) and agitation techniques (variable-rate and constant-rate culture). This satisfies the need for large-scale seed cell preparation in cultured meat and achieves efficient integration of seed cells (suspension cells) with the scaffold material, with a cell adhesion rate reaching 96.8%. It effectively solves the challenges of large-scale seed cell preparation and effective integration with scaffold materials in block-type cultured meat cultivation, providing a new pathway for the large-scale production of block-type cultured meat.

[0059] In some embodiments, the density of the cell suspension is 1E6-5E6 cells / mL.

[0060] When the cell suspension density is 1E6-5E6 cells / mL, it exhibits a high cell adhesion rate.

[0061] Furthermore, the density of the cell suspension is preferably 3E6 cells / mL.

[0062] A higher cell adhesion rate was achieved when the cell suspension density was 3E6 cells / mL.

[0063] In some embodiments, the suspended cells include chicken embryo fibroblast cell lines.

[0064] In this invention, the chicken embryo fibroblast cell line is a suspension cell line that has undergone suspension domestication. This invention does not impose any particular limitation on the specific method for preparing the chicken embryo fibroblast cell line; methods known in the art can be used.

[0065] This invention uses chicken embryo fibroblast cell lines (suspension cells) as seed cells, which can be used for large-scale cell expansion, effectively improving production efficiency and scale, meeting the demand for large-scale preparation of seed cells in cell-cultured meat, and achieving efficient integration of suspension cells and scaffold materials, reducing the process complexity of seed cells in the large-scale industrial production of cell-cultured meat.

[0066] In some embodiments, the centrifugation process described above is performed at a speed of 800-1000 rpm for 5-8 minutes.

[0067] In some embodiments, the basal culture medium includes any one of DMEM medium, DMEM / F12 medium, and MEM medium, preferably DMEM medium.

[0068] The present invention does not impose any particular limitation on the size of the gelatin-modified plant textured protein scaffold. In some embodiments, the size of the gelatin-modified plant textured protein scaffold may be 1cm x 1cm x 1cm.

[0069] In some embodiments, the gelatin-modified plant fibroin scaffold described above is obtained by a preparation method comprising the following steps:

[0070] Plant textured protein was cross-linked with gelatin and sterilized to obtain the initial gelatin-modified plant textured protein.

[0071] The initial gelatin-modified plant textured protein was soaked in sterile water and swelled after fully absorbing water to obtain swelled gelatin-modified plant textured protein.

[0072] The expanded gelatin-modified plant textured protein was soaked in culture medium No. 2 to obtain a gelatin-modified plant textured protein scaffold.

[0073] Culture medium No. 2 is the aforementioned culture medium No. 2.

[0074] In this invention, soaking the expanded gelatin-modified plant textured protein in culture medium No. 2 allows the gelatin-modified plant textured protein scaffold to absorb nutrients from culture medium No. 2, providing a nutrient-rich microenvironment for the cells, which is beneficial for cell adhesion and growth.

[0075] The present invention does not specifically limit the sterilization method described above. In some embodiments, sterilization can be performed using cobalt-60 gamma ray irradiation.

[0076] In this invention, the gelatin is a gelatin aqueous solution with a mass fraction of 5%.

[0077] In some embodiments, the soaking treatment described above is performed at a temperature of 38-39°C for a time of 20-30 minutes.

[0078] When the temperature and time parameters during the soaking treatment are within the above range, the gelatin-modified plant fibroin scaffold can fully absorb the nutrients in culture medium No. 2.

[0079] In some embodiments, the aforementioned plant-based textured protein is soybean textured protein, peanut textured protein, or wheat textured protein, preferably soybean textured protein.

[0080] In some embodiments, the seeding amount of the above-mentioned suspended cells is 2E5-3E5 cells / mL.

[0081] In some embodiments, the suspension cells are seeded in culture medium No. 1 for proliferation culture, and when the cell density reaches its peak, proliferated cells are obtained, specifically including:

[0082] Seed cells were inoculated into medium No. 1 and cultured for 4-6 days at 110-120 rpm, 38-39℃, pH 7.2-7.4, and 50%-60% dissolved oxygen. Medium No. 1 was replaced every 1-2 days during the proliferation culture. When the cell density reached its peak, the proliferated cells were obtained.

[0083] In some implementations, the above-mentioned variable-speed culture program is as follows: 70-80 rpm, 5-6 min; 0 rpm, 1-1.2 h, 24 cycles;

[0084] The variable-speed culture was carried out at 38-39℃, pH=7.2-7.4, and 50%-60% dissolved oxygen for 20-24 hours.

[0085] In some embodiments, cells are attached to a gelatin-modified plant textured fibroin scaffold and then cultured at a constant rate to obtain block-shaped cultured meat, specifically including:

[0086] After the cells adhered to the surface of a gelatin-modified plant textured protein scaffold, they were cultured at a constant rate for 7-10 days at 90-110 rpm, 38-39℃, pH 7.2-7.4, and 50%-60% dissolved oxygen. During the constant rate culture, the culture medium No. 2 was replaced every 1-2 days to obtain block-shaped cultured meat.

[0087] A second aspect of the present invention provides a block-shaped cultured meat, which is prepared by the above-described method for preparing block-shaped cultured meat.

[0088] The present invention will be further described below through specific embodiments.

[0089] Experimental materials:

[0090] The chicken embryo fibroblast cell line, abbreviated as CEF cell line, is derived from our laboratory's cell bank. Cells extracted from chicken embryos undergo spontaneous immortalization and suspension domestication to obtain suspension cells, thus obtaining the CEF cell line. For details of the process, please refer to the literature [Pasitka, L., et al. "Spontaneous immortalization of chicken fibroblasts generates stable, high-yield cell lines for serum-free production of cultured meat." Nature Food 4.1(2023):35-50.].

[0091] DMEM medium (Gibco, 31600083); fetal bovine serum (Tianhang Biotechnology, 11011-8611, abbreviated as FBS); sodium dextran sulfate (Maclean, D808268-50g); poloxamer 188 (Solepro, S7070100g, also known as F68, a commonly used shear protectant in suspension cell proliferation culture); astragalus polysaccharide (Maclean, A860847); inulin (MCE, HY-N7075); vitamin C (MCE, HY-B0166G); selenium-enriched yeast (Yuanye Biotechnology, S29789); insulin (MCE, HY-P1156).

[0092] Preparation of an anti-cell flocculation agent solution with a concentration of 25 mg / mL: Weigh 2.5 g of sodium dextran sulfate and dissolve it in 90 mL of distilled water. After complete dissolution, add distilled water to make up the volume to 100 mL. Finally, filter the solution through a 0.22 μm filter membrane to remove bacteria, and you will get an anti-cell flocculation agent solution with a concentration of 25 mg / mL.

[0093] Preparation of F68 solution with a concentration of 100 mg / mL: Weigh 5 g of F68 and dissolve it in 45 mL of distilled water. After it is completely dissolved, add distilled water to make up the volume to 50 mL. Finally, filter the solution through a 0.22 μm filter membrane to remove bacteria, and you will get an F68 solution with a concentration of 100 mg / mL.

[0094] Preparation of Astragalus polysaccharide solution with a concentration of 10 mg / mL: Weigh 1 g of Astragalus polysaccharide and add it to 90 mL of distilled water. After it is completely dissolved, add distilled water to make up the volume to 100 mL. Finally, filter the solution through a 0.22 μm filter membrane to remove bacteria, and you will get an Astragalus polysaccharide solution with a concentration of 10 mg / mL.

[0095] Preparation of an inulin solution with a concentration of 250 mg / mL: Weigh 25 g of inulin and add it to 90 mL of distilled water. After it is completely dissolved, add distilled water to make up the volume to 100 mL. Finally, filter the solution through a 0.22 μm filter membrane to remove bacteria, and you will get an inulin solution with a concentration of 250 mg / mL.

[0096] Preparation of a vitamin C solution with a concentration of 17.612 mg / mL: Weigh 1.7612 g of vitamin C and add it to 90 mL of distilled water. After it is completely dissolved, add distilled water to make up the volume to 100 mL. Finally, filter the solution through a 0.22 μm filter membrane to remove bacteria, and you will get a vitamin C solution with a concentration of 17.612 mg / mL.

[0097] Preparation of a 0.05 mg / mL selenium-enriched yeast solution: Weigh 5 mg of selenium-enriched yeast and add it to 90 mL of distilled water. After it is completely dissolved, add distilled water to make up the volume to 100 mL. Finally, filter the solution through a 0.22 μm filter membrane to remove bacteria, and you will get a 0.05 mg / mL selenium-enriched yeast solution.

[0098] Preparation of insulin solution with a concentration of 0.0116 mg / mL: Weigh 1.16 mg of insulin and add it to 90 mL of distilled water. After it is completely dissolved, add distilled water to make up the volume to 100 mL. Finally, filter the solution through a 0.22 μm filter membrane to remove bacteria, and you will get an insulin solution with a concentration of 0.0116 mg / mL.

[0099] Example 1: Preparation of culture medium for inducing the adhesion of suspended cells.

[0100] 1. Preparation of ordinary culture medium:

[0101] The ordinary culture medium (designated as medium 1) consists of the following components: DMEM medium, fetal bovine serum 10% v / v, anti-cell clumping agent 25ug / mL, and poloxamer 188 1mg / mL;

[0102] The preparation method of 500mL No. 1 culture medium includes: mixing 50mL FBS, 0.5mL of anti-cell clumping agent solution with a concentration of 25mg / mL and 5mL of F68 solution with a concentration of 100mg / mL, and then using DMEM to make up to 500mL.

[0103] 2. Preparation of culture medium for inducing suspension cell adhesion:

[0104] The culture medium for inducing suspension cell adhesion (designated as medium 2) consisted of the following components: DMEM medium, fetal bovine serum 10% v / v, astragalus polysaccharide 100ug / mL, inulin 2.5mg / mL, vitamin C 176.12ug / mL, selenium-enriched yeast 0.5ug / mL, and insulin 0.0116ug / mL;

[0105] The preparation method of 500mL of culture medium No. 2 includes: mixing 50mL of FBS, 5mL of Astragalus polysaccharide solution with a concentration of 10mg / mL, 5mL of inulin solution with a concentration of 250mg / mL, 5mL of vitamin C solution with a concentration of 17.612mg / mL, 5mL of selenium-enriched yeast solution with a concentration of 0.05mg / mL, and 0.5mL of insulin solution with a concentration of 0.0116mg / mL, and then adjusting the volume to 500mL using DMEM.

[0106] 3. Observe the growth status of chicken embryo fibroblast cell lines in different culture media:

[0107] CEF cell lines were divided into 2x10 5 / well of the culture medium was seeded into a 6-well plate, 2 mL of medium No. 1 was added, and the plate was incubated at 39°C with 5% CO2. After 2 hours of incubation, the growth status of the chicken embryo fibroblast cell line was observed. Figure 1 (A).

[0108] CEF cell lines were divided into 2x10 5 / well of the culture medium was seeded into a 6-well plate, 2 mL of medium No. 2 was added, and the plate was incubated at 39°C with 5% CO2. After 2 hours of incubation, the growth status of the chicken embryo fibroblast cell line was observed. Figure 1 B);

[0109] Depend on Figure 1 It can be seen that the CEF cell line using medium No. 1 always remained in a suspended cell state, while the CEF cell line using medium No. 2 remained in an adherent cell state, proving that medium No. 2 provided by the present invention can induce suspended cells (chicken embryo fibroblast cell line) to adhere again under plate conditions.

[0110] Example 2: Preparation and density screening of gelatin-modified soybean textured protein scaffold.

[0111] The culture medium No. 1 used in this embodiment is the same as the culture medium No. 1 in Example 1; the culture medium No. 2 used in this embodiment is the same as the culture medium No. 2 in Example 1.

[0112] 1. Preparation of gelatin-modified soybean textured protein scaffold:

[0113] Soy protein textured fibers were cross-linked with a 5% (w / w) gelatin aqueous solution for 12 hours to obtain the cross-linked product.

[0114] The cross-linked products were sterilized by cobalt-60 gamma irradiation to obtain the initial gelatin-modified soybean textured protein.

[0115] The initial gelatin-modified soy textured protein was soaked in sterile water and swelled after fully absorbing water to obtain swollen gelatin-modified soy textured protein.

[0116] The expanded gelatin-modified soybean textured protein was cut into small pieces of 1cm x 1cm x 1cm. Then, the expanded gelatin-modified plant textured protein was soaked in culture medium No. 2 to obtain the gelatin-modified soybean textured protein scaffold. The soaking time was 30min at 39℃.

[0117] 2. Density screening:

[0118] CEF cell lines were seeded at densities of 1E6 cells / mL, 3E6 cells / mL, and 5E6 cells / mL onto gelatin-modified soybean textured fibrous scaffolds, respectively. Medium No. 2 was added (the amount of medium No. 2 added was just enough to cover the gelatin-modified soybean textured fibrous scaffolds). The scaffolds were then incubated at 39°C in a 5% CO2 incubator. After one day of incubation, block-shaped cultured meat (such as…) was obtained. Figure 2 (As shown), and the number of cells that did not adhere to the gelatin-modified soybean textured fibrous scaffold was collected. The cell adhesion rate was calculated as a percentage of the total seeded cells. The results are as follows: Figure 3 As shown, the formula for calculating cell adhesion rate is as follows:

[0119] Cell adhesion rate = (1 - number of cells not adhered to the gelatin-modified soybean textured scaffold / total number of inoculated cells) × 100%, where the total number of inoculated cells refers to the number of CEF cell lines initially inoculated. For example, when the density is 1E6 cells / mL, the total number of inoculated cells is 1E6 cells; when the density is 3E6 cells / mL, the total number of inoculated cells is 3E6 cells; and when the density is 5E6 cells / mL, the total number of inoculated cells is 5E6 cells.

[0120] Depend on Figure 2-3 It is known that a density of 3E6 cells / mL is optimal, with a cell adhesion rate as high as 96.8%. This indicates that the present invention can induce suspension cells (CEF cell line) to adhere to the scaffold material (gelatin-modified soybean textured fibrous scaffold), achieving efficient integration of suspension cells and scaffold material.

[0121] Example 3: Preparation of block-shaped cell cultured meat.

[0122] 1. This embodiment uses a 2L bioreactor from Yingde Biotechnology, which has two modes: intermittent stirring and constant-speed stirring. The stirring function is achieved by a magnetically coupled stirrer, and it is equipped with electrodes for temperature, pH, dissolved oxygen, and liquid level, enabling automatic monitoring of key parameters during the cultivation process. The culture medium No. 1 used in this embodiment is the same as that in Example 1; the culture medium No. 2 used in this embodiment is the same as that in Example 1.

[0123] Figure 4 This is a flowchart illustrating the preparation of block-shaped cultured meat in Example 3 of the present invention, as shown below. Figure 4 As shown, the method for preparing block-shaped cultured meat includes the following steps:

[0124] 1200 mL of medium No. 1 was added to a sterilized 2L bioreactor in advance. Then, CEF cell lines were inoculated into medium No. 1 at an inoculation rate of 2E5 / mL in a clean bench and cultured for 6 days at 120 rpm, 39℃, pH=7.2, and 60% dissolved oxygen. During the culture period, medium No. 1 was replaced every 2 days, and cell density was measured daily. When the cell density reached its peak, the proliferated cells were obtained.

[0125] After proliferation culture, the cells were centrifuged at 1000 rpm for 5 min, the supernatant was carefully discarded, the cell pellet was collected, and then the cell pellet was resuspended in 50 mL of culture medium No. 2 to obtain a cell suspension with a density of 3E6 cells / mL.

[0126] A cell suspension with a density of 3E6 cells / mL was transferred to a 2L bioreactor and seeded onto a 1cm x 1cm x 1cm gelatin-modified soybean textured scaffold (the gelatin-modified soybean textured scaffold in Example 2, also known as an edible scaffold). Culture medium No. 2 was added to cover the gelatin-modified soybean textured scaffold. The cells were cultured at varying speeds for 24 hours under alternating stirring conditions (70 rpm, 5 min; 0 rpm, 1 h, 24 cycles), at 39°C, pH 7.2, and 60% dissolved oxygen, allowing the cells to adhere to the surface of the gelatin-modified plant textured scaffold. Subsequently, the cells were cultured at a constant speed (95 rpm) for 8 days under constant speed stirring conditions, at 39°C, pH 7.2, and 60% dissolved oxygen, forming block-shaped cultured meat.

[0127] 2. Characterization of block-shaped cultured meat

[0128] (1) HE staining

[0129] HE staining (Hematoxylin-Eosin Staining) is a commonly used histological staining method for observing the morphology and structure of tissues and cells.

[0130] The block-shaped cell cultured meat from Example 3 above was subjected to conventional steps (tissue fixation-dehydration-clearing-wax impregnation-embedding-mounting-dewaxing and hydration-hematoxylin-eosin staining-dehydration and clearing-mounting) and then subjected to histological structural analysis. Figure 5 Image showing HE staining results of cultured meat from block cells.

[0131] Depend on Figure 5 As can be seen, the block-shaped cell culture meat in Example 3 exhibited obvious cell adhesion and distribution: the cell nuclei were dark blue, the cell scaffold was light blue, and the cytoplasm and extracellular matrix were pink. This demonstrates that the method of the present invention alters the adhesion molecules on the cell surface, enabling suspended cells (CEF cell line) to adhere to the scaffold material (gelatin-modified plant fibroin scaffold) and to secrete extracellular matrix normally.

[0132] (2) DAPI staining

[0133] DAPI staining is a commonly used fluorescent staining technique for observing the morphology of cell nuclei and chromosomes.

[0134] After freezing the block-shaped cultured meat from Example 3 above, surface and internal tissue sections of the gelatin-modified plant fibroin scaffold were taken. The surface and internal tissue sections were stained with 1 μg / mL DAPI for 5 min each, and washed three times with PBS for five minutes each time. The adhesion and migration of suspended cells (CEF cell line) on the scaffold material (gelatin-modified plant fibroin scaffold) were observed under a fluorescence microscope. Figure 6 As shown.

[0135] Depend on Figure 6 It is known that CEF cell lines can adhere to, proliferate, and migrate into the interior of a scaffold material (gelatin-modified plant fibroin scaffold). This demonstrates that the method of this invention alters the adhesion molecules on the cell surface, enabling suspended cells (CEF cell lines) to adhere normally to the scaffold material (gelatin-modified plant fibroin scaffold) and to perform normal cell activities: proliferation and migration.

[0136] (3) Dil staining

[0137] Dil is a lipophilic fluorescent dye that can bind to the cell membrane and emit fluorescence, used to observe the morphology and structure of the cell membrane.

[0138] The sections of the block-shaped cell cultured meat from Example 3 were stained with 1 μg / mL Dil solution for 20 min; washed three times with PBS for five minutes each time to remove unbound Dil; and the morphology of the suspension cells (CEF cell line) on the scaffold material (gelatin-modified plant fibroin scaffold) was observed using a fluorescence microscope. Figure 7 As shown.

[0139] Depend on Figure 7 It can be seen that the CEF cell line maintains good cell morphology on the gelatin-modified plant textured protein scaffold. This demonstrates that the block-shaped cell cultured meat prepared using the method of this invention can maintain good morphological structure.

[0140] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing block-shaped cultured meat, characterized in that, Includes the following steps: Suspension cells were inoculated into culture medium No. 1 for proliferation culture. When the cell density reached its peak, the proliferated cells were obtained. The suspension cells were chicken embryo fibroblast cell lines. After proliferation culture, the cells were centrifuged, the cell pellet was collected, and the cell pellet was resuspended in medium No. 2 to obtain a cell suspension. Cell suspension was inoculated onto a gelatin-modified plant textured protein scaffold, and after adding medium No. 2, it was cultured at variable speed and then at constant speed to obtain block-shaped cultured meat; the gelatin-modified plant textured protein scaffold was prepared by a method including the following steps: cross-linking plant textured protein with gelatin; The formulation of culture medium No. 1 consists of the following components: basal culture medium, fetal bovine serum 8-12% v / v, anti-cell clumping agent 23-25 ​​ug / mL, and poloxamer 188 0.8-1 mg / mL; The formulation of culture medium No. 2 consists of the following components: basal culture medium, fetal bovine serum 8-12% v / v, astragalus polysaccharide 95-100 ug / mL, inulin 2-2.5 mg / mL, vitamin C 166.12-176.12 ug / mL, selenium-enriched yeast 0.2-0.5 ug / mL, and insulin 0.0106-0.0116 ug / mL.

2. The method for preparing block-shaped cultured meat according to claim 1, characterized in that, The cell suspension density was 1E6-5E6 cells / mL.

3. The method for preparing block-shaped cultured meat according to claim 1, characterized in that, In the centrifugation process, the rotation speed is 800-1000 rpm and the time is 5-8 min; And / or, the basal culture medium includes any one of DMEM medium, DMEM / F12 medium, and MEM medium.

4. The method for preparing block-shaped cultured meat according to claim 1, characterized in that, The gelatin-modified plant fibroin scaffold is obtained by a preparation method including the following steps: Plant textured protein was cross-linked with gelatin and sterilized to obtain the initial gelatin-modified plant textured protein. The initial gelatin-modified plant textured protein was soaked in sterile water and swelled after fully absorbing water to obtain swelled gelatin-modified plant textured protein. The expanded gelatin-modified plant textured protein was soaked in culture medium No. 2 to obtain a gelatin-modified plant textured protein scaffold. Culture medium No. 2 is the culture medium No. 2 described in claim 1.

5. The method for preparing block-shaped cultured meat according to claim 4, characterized in that, During the soaking treatment, the temperature is 38-39℃ and the time is 20-30 minutes; And / or, the plant textured protein is any one of soybean textured protein, peanut textured protein, and wheat textured protein.

6. The method for preparing block-shaped cultured meat according to claim 1, characterized in that, The inoculation amount of the suspended cells is 2E5-3E5 cells / mL.

7. The method for preparing block-shaped cultured meat according to claim 1, characterized in that, The process of inoculating suspension cells into culture medium No. 1 for proliferation culture, and obtaining proliferated cells when the cell density reaches its peak, specifically includes: Suspension cells were inoculated into medium No. 1 and cultured for 4-6 days at 110-120 rpm, 38-39℃, pH 7.2-7.4, and 50%-60% dissolved oxygen. Medium No. 1 was replaced every 1-2 days during the culture period. When the cell density reached its peak, the cultured cells were obtained.

8. The method for preparing block-shaped cultured meat according to claim 1, characterized in that, The cell suspension adheres to the surface of a gelatin-modified plant textured protein scaffold and is then cultured at a constant rate to obtain block-shaped cultured meat, specifically comprising: After the cells adhered to the surface of a gelatin-modified plant textured protein scaffold, they were cultured at a constant rate for 7-10 days at 90-110 rpm, 38-39℃, pH=7.2-7.4, and 50%-60% dissolved oxygen. During the constant rate culture, the culture medium No. 2 was replaced every 1-2 days to obtain block-shaped cultured meat.

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