Edible biological scaffold, preparation method thereof and application of edible biological scaffold in preparation of cell culture meat
By preparing biological scaffolds using glutenin, chitosan, and agarose, the problem of the lack of edible scaffolds in cell-cultured meat production has been solved, enabling three-dimensional cell growth and large-scale expansion, and has broad application prospects.
Patent Information
- Application Number
- CN202512019810.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-30
- Publication Date
- 2026-03-13
AI Technical Summary
The lack of edible biological scaffold materials in current cell-cultured meat production limits its industrial application value.
Using glutenin, chitosan, and agarose as raw materials, a bioscaffold was prepared by dissolving, mixing, cooling and solidifying in acetic acid and then freeze-drying under vacuum to form a porous network structure.
It provides a safe, edible, low-cost, and simple-to-prepare biological scaffold that supports three-dimensional cell growth, promotes large-scale cell expansion, simulates the in vivo environment, and is suitable for cell-cultured meat production.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biological scaffold materials, and in particular to an edible biological scaffold, its preparation method, and its application in the preparation of cell-cultured meat. Background Technology
[0002] Cell-cultured meat is block-shaped meat prepared by culturing cells in vitro that closely resembles animal muscle tissue in appearance, nutrition, texture, and taste. It is also known as biocultured meat, cultured meat, or clean meat. Three-dimensional cell culture can simulate the growth environment of cells in vivo. Through three-dimensional culture, cells exhibit a three-dimensional growth state, which is conducive to constructing muscle tissue with realistic texture in vitro, achieving the purpose of in vitro shaping and construction of muscle tissue.
[0003] The production process of cell-cultured meat mainly includes four steps: cell bank construction, cell proliferation, cell differentiation, and cell harvesting. The industrialization of cell-cultured meat still faces several significant technical obstacles that limit its scale-up and commercial development: (1) research on cell culture and differentiation technologies; (2) development and optimization of non-animal-derived culture media; (3) development of efficient bioreactors; and (4) research and development of suitable scaffolds for in vitro 3D cell growth. Currently, research progress on cell scaffolds for cell-cultured meat mainly falls into two categories. One category involves developing novel scaffolds that promote cell adhesion and growth to achieve in vitro 3D culture and differentiation of seed cells. For example, GelMA is made from gelatin modified by methacrylation, retaining the RGD cell adhesion sequence and natural degradation properties of gelatin, while introducing photocrosslinkable double bond groups. Under the action of inedible photoinitiators, GelMA can rapidly crosslink to form a three-dimensional hydrogel network within 10-30 seconds. Another approach involves utilizing and developing microcarriers to achieve three-dimensional in vitro cell culture. Researchers have developed a technique for large-scale in vitro expansion of bovine myoblasts based on the traditional commercial microcarriers Cytodex1 and Synthemax II. Compared to planar culture, the use of microcarriers significantly increases the culture density of bovine myoblasts. Although the aforementioned scaffold materials and commercial microcarriers can achieve the proliferation of different types of seed cells, their components are non-food raw materials and therefore inedible, making them unsuitable for the production of cell-cultured meat. These factors greatly limit the industrial application value of these materials. Therefore, there is an urgent need to provide a new edible biological scaffold and its preparation method. Summary of the Invention
[0004] The purpose of this invention is to provide an edible biological scaffold, its preparation method, and its application in the preparation of cultured meat, in order to solve the problems existing in the prior art. This invention provides a new scaffold material for the production of cultured meat, which has the advantages of safe raw materials, edibility, wide availability, low cost, and simple preparation process, and has broad application prospects in the fields of food science and cell agriculture.
[0005] To achieve the above objectives, the present invention provides the following solution:
[0006] This invention provides a biological scaffold, the raw materials of which include glutenin, chitosan and agarose;
[0007] The mass ratio of the glutenin, chitosan and agarose is 3:(1-5):(1-5).
[0008] The present invention also provides a method for preparing the above-mentioned biological scaffold, comprising the following steps:
[0009] The glutenin and chitosan were dissolved separately in acetic acid solution and mixed well to obtain a mixed solution;
[0010] The agarose is heated and dissolved, then mixed with the mixed solution, cooled and solidified, and incubated to obtain the biological scaffold.
[0011] Furthermore, the concentration of the acetic acid solution is 0.1 mol / L.
[0012] The present invention also provides the application of the above-mentioned biological scaffold in the preparation of scaffold materials for culturing cells and / or preparing cell-cultured meat.
[0013] The present invention also provides a method for preparing a scaffold material, comprising the steps of pre-freezing and vacuum freeze-drying the above-mentioned biological scaffold.
[0014] Furthermore, the pre-freezing temperature is -80℃, and the time is 24 hours;
[0015] The vacuum freeze-drying temperature is -60℃ and the time is 72 h.
[0016] The present invention also provides a scaffold material obtained according to the above preparation method.
[0017] The present invention also provides an application of the above-mentioned scaffold material in the preparation of cultured meat.
[0018] The present invention also provides a method for preparing cell-cultured meat, comprising the steps of seeding cells onto the above-mentioned scaffold material and performing cell culture.
[0019] The present invention also provides cell-cultured meat obtained according to the above preparation method.
[0020] The present invention discloses the following technical effects:
[0021] This invention provides a biological scaffold prepared from glutenin, chitosan, and agarose. After vacuum freeze-drying, this scaffold forms a porous network structure. Experimental results show that this scaffold material exhibits outstanding hardness, toughness, chewiness, and swelling capacity, making it suitable as a scaffold for cell culture, enabling cells to proliferate rapidly and exhibit a three-dimensional growth morphology. This invention provides a novel scaffold material for the production of cultured meat, possessing advantages such as safe and widely available raw materials, low cost, and simple preparation process, showing broad application prospects in food science and cell agriculture. Attached Figure Description
[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.
[0023] Figure 1 This is a comparison chart of the stiffness of the biological scaffolds prepared in Examples 1-5 and Comparative Examples 1-5 in Experimental Example 1;
[0024] Figure 2 This is a comparison of the toughness of the biological scaffolds prepared in Examples 1-5 and Comparative Examples 1-5 in Experimental Example 1;
[0025] Figure 3 This is a comparison of the chewability of the biological scaffolds prepared in Examples 1-5 and Comparative Examples 1-5 in Experimental Example 1;
[0026] Figure 4 The graph shows the swelling rate test results of the biological scaffolds prepared in Examples 1-5 and Comparative Examples 1-5 in Experimental Example 2;
[0027] Figure 5 The graph shows the degradation rate detection results of the biological scaffolds prepared in Examples 1-5 and Comparative Examples 1-5 in Experimental Example 3;
[0028] Figure 6 This is a growth curve of chicken myoblasts inoculated onto the biological scaffolds prepared in Examples 1-5 and Comparative Examples 1-5 in Experimental Example 4;
[0029] Figure 7 The results of cell culture on the biological scaffold prepared in Example 1 of Experimental Example 5;
[0030] Figure 8The results of cell culture on the biological scaffold prepared in Example 2 of Experimental Example 5;
[0031] Figure 9 The results of cell culture on the biological scaffold prepared in Example 3 of Experiment 5;
[0032] Figure 10 The results of cell culture on the biological scaffold prepared in Example 4 of Experimental Example 5;
[0033] Figure 11 The results of cell culture on the biological scaffold prepared in Example 5 of Experiment 5;
[0034] Figure 12 The results of cell culture on the biological scaffold prepared in Comparative Example 1 in Experimental Example 5;
[0035] Figure 13 The results of cell culture on the biological scaffold prepared in Comparative Example 2 in Experimental Example 5;
[0036] Figure 14 The results of cell culture on the biological scaffold prepared in Comparative Example 3 in Experimental Example 5;
[0037] Figure 15 The results of cell culture on the biological scaffold prepared in Comparative Example 4 in Experimental Example 5;
[0038] Figure 16 The results of cell culture on the biological scaffold prepared in Comparative Example 5 are shown in Experimental Example 5. Detailed Implementation
[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0040] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0041] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0042] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.
[0043] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0044] The biological scaffold provided by this invention does not require the introduction of inedible cross-linking agents during its preparation process. All raw materials used are substances permitted to be added to food, possessing edible natural properties, and can be used in the production and processing of cell-cultured meat.
[0045] The mesh structure of the biological scaffold material provided by this invention can simulate the three-dimensional growth environment of animal cells to the greatest extent, has good stability, and can reproduce the in vivo environment of cells in vitro, which is conducive to the three-dimensional growth of cells in vitro, enabling chicken myoblasts to exhibit a three-dimensional growth state.
[0046] The biological scaffold material provided by this invention has good biocompatibility, supports cell adhesion, three-dimensional growth and proliferation, and thus exhibits a three-dimensional growth state, which can efficiently promote the large-scale and efficient expansion and culture of chicken myoblasts.
[0047] The biological scaffold material of the present invention can form a stable mesh structure and remains stable in a culture medium at 37°C for 168 hours without dissolving or collapsing. It has high stability and facilitates cell attachment and nutrient delivery.
[0048] In the raw materials for preparing the biological scaffold of this invention, glutenin, as the main storage protein in wheat, has unique functionality and nutritional value; chitosan and agarose can also be used as functional polysaccharides. In addition to serving as biological scaffold materials for three-dimensional cell growth, they also endow cell culture meat products with the effects of regulating intestinal flora and helping to alleviate inflammatory responses in vivo.
[0049] The raw materials for preparing the biological scaffold of this invention are all natural macromolecules, which are low in cost and can be promoted and applied on a large scale and at low cost in the field of cell agriculture. This will help reduce the production cost of cultured meat. Among them, glutenin can be extracted from wheat, chitosan can be extracted from the shells of marine arthropods such as shrimp and crabs, the shells and bones of mollusks, and the cell walls of higher plants, and has been commercially produced. Agarose can be obtained by further separating and purifying agar extracted from red algae.
[0050] The preparation process of the biological scaffold material of the present invention is simple, convenient, and easy to promote and apply.
[0051] The glutenin of this invention is derived from wheat, and the molecular weight of the high molecular weight glutenin subunits ranges from 80 to 130 kDa.
[0052] The chitosan of this invention has a degree of deacetylation of not less than 95% and a viscosity of 100-200 mpa.s.
[0053] The agarose of this invention is a high gel strength agarose with a gel strength greater than 1500 g / cm².
[0054] Example 1
[0055] 1. Preparation of raw materials
[0056] The chitosan has a glutenin content of 95% and a deacetylation degree of 95%, with a viscosity of 200 mPa·s. The mass ratio of chitosan to high gel strength agarose is 3:2:4.
[0057] 2. Preparation method
[0058] (1) Raw material processing: Prepare a glutenin powder solution with pH 3 and concentration of 3% using 0.1 M acetic acid, and prepare a chitosan powder solution with concentration of 2% using 0.1 M acetic acid;
[0059] (2) Raw material mixing: Add 3% glutenin solution and 2% chitosan solution to a beaker in sequence, and vortex mix at 26℃ for 40 min to obtain a mixed solution;
[0060] (3) Secondary reaction: Add 2% agarose aqueous solution (80℃) to the mixed solution, mix thoroughly, pour into a container, cool and solidify, and incubate at room temperature for 20 min;
[0061] (4) Freeze-drying: The biological scaffold from step (3) was pre-frozen at -80°C for 24 h and then freeze-dried under vacuum at -60°C for 72 h.
[0062] Example 2
[0063] 1. Preparation of raw materials
[0064] The chitosan has a glutenin content of 95% and a deacetylation degree of 95%, with a viscosity of 200 mPa·s. The mass ratio of chitosan to high gel strength agarose is 3:5:1.
[0065] 2. Preparation method
[0066] (1) Raw material processing: Glutenin powder was prepared into a solution with pH 3 and concentration of 3% using 0.1 M acetic acid; chitosan powder was prepared into a solution with concentration of 2% using 0.1 M acetic acid; and agarose powder was heated and dissolved into a 2% aqueous solution.
[0067] (2) Raw material mixing: Add 3% glutenin solution and 2% chitosan solution to a beaker in sequence, and vortex mix at 26℃ for 40 min to obtain a mixed solution;
[0068] (3) Secondary reaction: Add 2% agarose aqueous solution (80℃) to the mixed solution, mix thoroughly, pour into a container, cool and solidify, and incubate at room temperature for 20 min;
[0069] (4) Freeze-drying: The biological scaffold from step (3) was pre-frozen at -80°C for 24 h and then freeze-dried under vacuum at -60°C for 72 h.
[0070] Example 3
[0071] 1. Preparation of raw materials
[0072] The chitosan has a glutenin content of 95%, a degree of deacetylation of 95%, and a viscosity of 200 mPa·s. The mass ratio of chitosan to high gel strength agarose is 3:4:1.
[0073] 2. Preparation method
[0074] (1) Raw material processing: Glutenin powder was prepared into a solution with pH 3 and concentration of 3% using 0.1 M acetic acid; chitosan powder was prepared into a solution with concentration of 2% using 0.1 M acetic acid; and agarose powder was heated and dissolved into a 2% aqueous solution.
[0075] (2) Raw material mixing: Add 3% glutenin solution and 2% chitosan solution to a beaker in sequence, and vortex mix at 26℃ for 40 min to obtain a mixed solution;
[0076] (3) Secondary reaction: Add 2% agarose aqueous solution (80℃) to the mixed solution, mix thoroughly, pour into a container, cool and solidify, and incubate at room temperature for 20 min;
[0077] (4) Freeze-drying: The biological scaffold from step (3) was pre-frozen at -80°C for 24 h and then freeze-dried under vacuum at -60°C for 72 h.
[0078] Example 4
[0079] 1. Preparation of raw materials
[0080] The chitosan has a glutenin content of 95%, a degree of deacetylation of 95%, a viscosity of 200 mPa·s, and a mass ratio of chitosan to high gel strength agarose of 1:1:1.
[0081] 2. Preparation method
[0082] (1) Raw material processing: Glutenin powder was prepared into a solution with pH 3 and concentration of 3% using 0.1 M acetic acid; chitosan powder was prepared into a solution with concentration of 2% using 0.1 M acetic acid; and agarose powder was heated and dissolved into a 2% aqueous solution.
[0083] (2) Raw material mixing: Add 3% glutenin solution and 2% chitosan solution to a beaker in sequence, and vortex mix at 26℃ for 40 min to obtain a mixed solution;
[0084] (3) Secondary reaction: Add 2% agarose aqueous solution (80℃) to the mixed solution, mix thoroughly, pour into a container, cool and solidify, and incubate at room temperature for 20 min;
[0085] (4) Freeze-drying: The biological scaffold from step (3) was pre-frozen at -80°C for 24 h and then freeze-dried under vacuum at -60°C for 72 h.
[0086] Example 5
[0087] 1. Preparation of raw materials
[0088] The chitosan has a glutenin content of 95%, a degree of deacetylation of 95%, and a viscosity of 200 mPa·s. The mass ratio of chitosan to high gel strength agarose is 3:1:5.
[0089] 2. Preparation method
[0090] (1) Raw material processing: Prepare a glutenin powder solution with pH 3 and concentration of 3% using 0.1 M acetic acid, and dissolve the agarose powder by heating to a 2% aqueous solution;
[0091] (2) Raw material mixing: Add 3% glutenin solution and 2% chitosan solution to a beaker in sequence, vortex mix at 26℃ for 40 min to obtain a mixed solution;
[0092] (3) Secondary reaction: Add 15 mL of 2% agarose aqueous solution (80℃) to the mixed solution, mix thoroughly, pour into a container, cool and solidify, and incubate at room temperature for 20 min;
[0093] (4) Freeze-drying: The biological scaffold from step (3) was pre-frozen at -80°C for 24 h and then freeze-dried under vacuum at -60°C for 72 h.
[0094] Comparative Example 1
[0095] 1. Preparation of raw materials
[0096] The chitosan has a glutenin content of 85%, a degree of deacetylation of 85%, and a viscosity of 200 mPa·s. The mass ratio of chitosan to agarose is 3:4:0.
[0097] 2. Preparation method
[0098] (1) Raw material processing: Prepare a glutenin powder solution with pH 3 and concentration of 3% using 0.1 M acetic acid, and prepare a chitosan powder solution with concentration of 2% using 0.1 M acetic acid;
[0099] (2) Raw material mixing: 10 mL of 3% glutenin solution and 20 mL of 2% chitosan solution were added to a beaker in sequence and vortexed at 26℃ for 40 min to obtain a mixed solution;
[0100] (3) Freeze-drying: The mixed solution prepared in step (2) is placed into a container, treated at 121℃ and 0.1 MPa for 15 min, pre-frozen at -80℃ for 24 h, and then freeze-dried under vacuum at -60℃ for 72 h, and treated at 121℃ and 0.1 MPa for 15 min.
[0101] Comparative Example 2
[0102] 1. Preparation of raw materials
[0103] The chitosan has a glutenin content of 85%, a degree of deacetylation of 85%, and a viscosity of 200 mPa·s. The mass ratio of chitosan to agarose is 3:3:0.
[0104] 2. Preparation method
[0105] (1) Raw material processing: Prepare a glutenin powder solution with pH 3 and concentration of 3% using 0.1 M acetic acid, and prepare a chitosan powder solution with concentration of 2% using 0.1 M acetic acid;
[0106] (2) Raw material mixing: 10 mL of 3% glutenin solution and 15 mL of 2% chitosan solution were added to a beaker in sequence and vortexed at 26℃ for 40 min to obtain a mixed solution;
[0107] (3) Freeze-drying: The mixed solution prepared in step (2) is placed into a container, treated at 121℃ and 0.1 MPa for 15 min, pre-frozen at -80℃ for 24 h, and then freeze-dried under vacuum at -60℃ for 72 h, and treated at 121℃ and 0.1 MPa for 15 min.
[0108] Comparative Example 3
[0109] 1. Preparation of raw materials
[0110] The chitosan has a glutenin content of 85%, a degree of deacetylation of 85%, and a viscosity of 200 mPa·s. The mass ratio of chitosan to agarose is 3:2:0.
[0111] 2. Preparation method
[0112] (1) Raw material processing: Prepare a glutenin powder solution with pH 3 and concentration of 3% using 0.1 M acetic acid, and prepare a chitosan powder solution with concentration of 2% using 0.1 M acetic acid;
[0113] (2) Raw material mixing: 10 mL of 3% glutenin solution and 10 mL of 2% chitosan solution were added to a beaker in sequence and vortexed at 26℃ for 40 min to obtain a mixed solution;
[0114] (3) Freeze-drying: The mixed solution prepared in step (2) is placed into a container, treated at 121℃ and 0.1 MPa for 15 min, pre-frozen at -80℃ for 24 h, and then freeze-dried under vacuum at -60℃ for 72 h, and treated at 121℃ and 0.1 MPa for 15 min.
[0115] Comparative Example 4
[0116] 1. Preparation of raw materials
[0117] The chitosan has a glutenin content of 85%, a degree of deacetylation of 85%, and a viscosity of 200 mPa·s. The mass ratio of chitosan to agarose is 3:1:0.
[0118] 2. Preparation method
[0119] (1) Raw material processing: Prepare a glutenin powder solution with pH 3 and concentration of 3% using 0.1 M acetic acid, and prepare a chitosan powder solution with concentration of 2% using 0.1 M acetic acid;
[0120] (2) Raw material mixing: 10 mL of 3% glutenin solution and 5 mL of 2% chitosan solution were added to a beaker in sequence and vortexed at 26℃ for 40 min to obtain a mixed solution;
[0121] (3) Freeze-drying: The mixed solution prepared in step (2) is placed into a container, treated at 121℃ and 0.1 MPa for 15 min, pre-frozen at -80℃ for 24 h, and then freeze-dried under vacuum at -60℃ for 72 h, and treated at 121℃ and 0.1 MPa for 15 min.
[0122] Comparative Example 5
[0123] 1. Preparation of raw materials
[0124] The mass ratio of glutenin, chitosan and agarose is 3:0:0.
[0125] 2. Preparation method
[0126] (1) Raw material processing: Prepare a glutenin powder solution with pH 3 and concentration of 3% using 0.1 M acetic acid;
[0127] (2) Raw material mixing: Add 10 mL of 3% glutenin solution to a beaker, vortex, and obtain the vortexed solution;
[0128] (3) Freeze-drying: The vortexed solution prepared in step (2) was placed into a container, treated at 121℃ and 0.1 MPa for 15 min, pre-frozen at -80℃ for 24 h, and then freeze-dried under vacuum at -60℃ for 72 h, and treated at 121℃ and 0.1 MPa for 15 min.
[0129] Experimental Example 1: Determination of the textural properties of biological scaffold materials
[0130] The biological scaffold materials of the above embodiments and comparative examples were subjected to TPA (texture profile analysis) testing, as detailed below:
[0131] A compression experiment was conducted on a biological scaffold material with a diameter of 20 cm and a height of 20 cm. The statistical results are as follows: Figure 1 , Figure 2 and Figure 3 As shown above, the results indicate that, with the same sample height and diameter, the hardness of Examples 1-5 is significantly higher than that of Comparative Examples 1-5. Moreover, in terms of toughness and chewiness, Examples 2-5 are also significantly superior to Comparative Examples 2-5, and the final cell-cultured meat products produced will have a texture that is more in line with the texture of real meat.
[0132] Experiment Example 2: Detection of Swelling Rate of Biological Scaffold Materials
[0133] The biological scaffolds of each embodiment and comparative example were weighed after drying and placed in PBS solution, then incubated at 37°C until swelling equilibrium was reached. The samples were then removed, excess surface moisture was blotted with filter paper, and the mass after swelling was recorded using an electronic balance. The results are shown below. Figure 4 As shown in the figure. The results indicate that a decrease in chitosan content leads to a decrease in the swelling rate of the bioscaffold material, while an increase in agarose content can significantly improve the swelling rate of the bioscaffold material, which is more conducive to cell attachment, can better regulate the exchange of nutrients and metabolic waste, and promote cell proliferation.
[0134] Experiment Example 3: Stability Testing of Bioscaffold Materials in Culture Media
[0135] The bio-ink scaffolds of each embodiment and comparative example were weighed after drying and placed in PBS solution, then incubated in a 37°C incubator, with the PBS solution changed daily. At each set time point, they were removed, freeze-dried, and weighed again; the ratio of the freeze-dried scaffolds to the original weight was the percentage of remaining weight. Results are as follows: Figure 5 As shown. The results indicate that the biological scaffold materials in Examples 1-5 remained stable after culturing in the culture medium at 37°C for 168 h, with a remaining weight percentage between 85-90%, demonstrating good stability. In contrast, the biological materials in Comparative Examples 1-5 began to dissolve after culturing in the culture medium at 37°C for 12 h, with a remaining weight percentage between 20-30%, and completely dissolved in the culture medium after 96 h, exhibiting poor stability.
[0136] Experiment Example 4: Assay of Cell Proliferation Activity of Biological Scaffolds
[0137] The metabolic activity of chicken myoblasts after 1, 3, 5 and 7 days of culture on scaffolds of Examples 1-5 and Comparative Examples 1-5 was detected by the CCK-8 assay.
[0138] The cell-inoculated scaffolds were removed on the designated days, rinsed with PBS, and then placed in 500 μL of culture medium containing 10% CCK-8 reagent. The scaffolds were incubated at 37°C and 5% CO2 for 1 h. After incubation, 100 μL of sample was aliquoted into 96-well plates and analyzed using a microplate reader. The results are as follows: Figure 6 As shown, the proliferative activity of chicken myoblasts on the biological scaffolds prepared in Examples 1-5 is significantly stronger.
[0139] Experiment Example 5: Detection of the effect of biological scaffolds on promoting three-dimensional cell growth
[0140] The cell growth promotion effect of the bio-inks prepared in the above embodiments and comparative examples on the bio-scaffold materials was tested, as follows:
[0141] Chicken myoblasts were seeded into the bioscaffold materials of each example and comparative example, and cultured at a constant temperature of 37°C under 5% CO2 conditions. After 5 days of culture, the cytoskeleton and nucleus were stained with FITC-labeled phalloidin-DAPI, and the morphology and spatial distribution of the cells were observed under a laser confocal high-content analysis platform. The observation results of cell culture in the bioscaffold materials of Examples 1-5 are as follows: Figure 7 , Figure 8 , Figure 9 , Figure 10 , Figure 11 As shown, the observation results of cell culture using the biological scaffold materials in Comparative Examples 1-5 are as follows: Figure 12 , Figure 13 , Figure 14 , Figure 15 , Figure 16 As shown above, the results indicate that the cells on the bio-scaffolds of Examples 1-5 are growing well and numerous, with a large number of cells observed in different layers of the bio-ink, exhibiting a three-dimensional growth state; while the number of cells on the bio-ink scaffolds of Comparative Examples 1-5 is significantly reduced, and they are all monolayer adherent growth, without exhibiting a three-dimensional growth state.
[0142] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A biological scaffold, characterized in that, The raw materials for the biological scaffold include glutenin, chitosan, and agarose; The mass ratio of the glutenin, chitosan and agarose is 3:(1-5):(1-5).
2. A method for preparing a biological scaffold according to claim 1, characterized in that, Includes the following steps: The glutenin and chitosan were dissolved separately in acetic acid solution and mixed well to obtain a mixed solution; The agarose is heated and dissolved, then mixed with the mixed solution, cooled and solidified, and incubated to obtain the biological scaffold.
3. The preparation method according to claim 2, characterized in that, The concentration of the acetic acid solution is 0.1 mol / L.
4. The use of the biological scaffold of claim 1 in the preparation of scaffold materials for culturing cells and / or preparing cell-cultured meat.
5. A method for preparing a scaffold material, characterized in that, It includes the steps of pre-freezing and vacuum freeze-drying the biological scaffold described in claim 1.
6. The preparation method according to claim 5, characterized in that, The pre-freezing temperature is -80℃, and the time is 24 hours; The vacuum freeze-drying temperature is -60℃ and the time is 72 h.
7. A scaffold material obtained by the preparation method according to claim 5 or 6.
8. The use of the scaffold material according to claim 7 in the preparation of cultured meat.
9. A method for preparing cell-cultured meat, characterized in that, The method includes the step of seeding cells onto the scaffold material of claim 7 and culturing the cells.
10. Cell-cultured meat obtained by the preparation method according to claim 9.