A method for preparing a gel scaffold for cell cultured meat

Abstract

A method for preparing a composite gel scaffold based on gelatin-chitosan-corn starch belongs to the field of cell culture meat technology. The scaffold is prepared by cross-linking gelatin, chitosan, and corn starch with genipin followed by freeze-drying. The preparation method includes steps such as dissolving, mixing, heating and stirring, cross-linking reaction, gel formation, swelling in ultrapure water, and freeze-drying. The raw materials used in this scaffold are safe and non-toxic, and the resulting scaffold possesses high mechanical properties, a suitable porous structure, and good biocompatibility, supporting the adhesion, proliferation, and differentiation of animal cells. The scaffold prepared by this invention has a simple process, controllable cost, and good prospects for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of cell culture meat technology, specifically relating to a hydrogel scaffold for cell culture meat and its preparation method. Background Technology

[0002] Cultured meat, based on the mechanisms of animal muscle growth and repair, is obtained by culturing stem cells in vitro. It possesses components and nutrients similar to real muscle tissue while meeting sustainability requirements, making it a highly efficient and green future food production technology. The structure of cultured meat places specific demands on the performance of scaffold materials. The three-dimensional production of cultured meat requires three-dimensional structural materials to provide physical support, mimicking the extracellular matrix, reproducing the natural three-dimensional microenvironment of cells, and promoting more natural cell behavior and tissue formation. Existing structured technologies, such as 3D-printed scaffolds and electrospinning, face challenges due to high costs and difficulty in mass production. Hydrogel scaffolds, on the other hand, possess a physical structure similar to the extracellular matrix, good biocompatibility, and their fabrication process is universally applicable, low-cost, and suitable for large-scale production. Therefore, hydrogels are an excellent scaffold material for tissue engineering.

[0003] As a sustainable meat production method, cultured meat requires scaffold materials to provide a biomimetic microenvironment for cells. Gelatin has good biocompatibility but poor thermal stability, while chitosan promotes cell adhesion but has poor formability. To overcome the limitations of single materials, the development of composite scaffolds has become an important research direction. Corn starch, as a widely available, low-cost, and fully edible natural polysaccharide, has excellent thickening, gelling, and film-forming properties. Currently, there are no reports on edible tissue engineering hydrogel scaffolds filled with corn starch. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for preparing a gelatin and chitosan-filled corn starch gel scaffold. The gelatin, chitosan, and corn starch in the scaffold provided by this invention are all safe and non-toxic components, ensuring food safety as part of a food product. The gel preparation process is simple, with adjustable parameters, enabling industrial-scale production. The scaffold prepared using this technology exhibits good mechanical properties, supporting the adhesion, proliferation, and differentiation of muscle cells within the scaffold, and shows clear application prospects in the field of cultured meat.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] S1 chitosan powder was dissolved in acetic acid solution to prepare chitosan solution. Gelatin powder was added to chitosan solution and heated and stirred to obtain mixed solution.

[0007] S2. Add corn starch to the mixture and heat while stirring;

[0008] S3 Add genipin to step S2 and stir to obtain a compound solution;

[0009] The S4 compound solution was injected into a mold to form a gel, and then the gel was placed in ultrapure water to swell and freeze-dried to obtain a porous scaffold.

[0010] Preferably, in step S1, the mass fraction of the acetic acid solution is 1% (w / v), the mass fraction of the chitosan solution is 0-4% (w / v), and the mass fraction of the gelatin solution is 0-8% (w / v).

[0011] Preferably, the stirring temperature in step S1 is 50°C and the stirring time is 30 min.

[0012] Preferably, the concentration of corn starch in step S2 is 1-5%.

[0013] Preferably, the stirring temperature in step S2 is 90℃ and the time is 30min.

[0014] Preferably, the amount of genipin added in step S3 is 1.0-5.0 mmol / L.

[0015] Preferably, the low-temperature curing temperature in step S4 is 4°C.

[0016] Preferably, the freeze-drying time in step S4 is 24-48 hours.

[0017] This invention provides a method for preparing cell-cultured meat, specifically including the following steps:

[0018] (1) The cell culture meat scaffold described above was soaked in PBS and high concentration of double antibodies and sterilized by ultraviolet irradiation to obtain a sterile cell culture meat scaffold.

[0019] (2) Add DMEM medium to the sterile cell culture scaffold to swell, and aspirate the medium after swelling equilibrium.

[0020] (3) Take cells and seed them on a cell culture scaffold, and perform cell adhesion, proliferation and differentiation treatment in an environment of 37℃ and 5% CO2.

[0021] In one embodiment of the present invention, in step (1), sterilization refers to incubating the cell culture scaffold under ultraviolet light overnight to kill microorganisms that affect cell growth.

[0022] In one embodiment of the present invention, in step (2), the volume ratio of DMEM culture medium to scaffold is 3-6:1 until the color of the hydrogel scaffold is close to the color of the culture medium.

[0023] In one embodiment of the present invention, in step (3), the seeding cell density is 1×10⁻⁶. 5 -1×106 per mL.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] This invention involves mixing gelatin and chitosan solutions of different concentrations, adding corn starch for filling, and covalently modifying the mixture with genipin at 90°C to form a compound gel. After the gel swells, it is freeze-dried to prepare a cell culture scaffold.

[0026] This invention utilizes food-derived natural polysaccharides and proteins as raw materials to successfully prepare a composite gel material with high mechanical strength, controllable porous structure, and excellent biocompatibility. By adjusting the concentration of each component, the mechanical properties, pore structure, and biocompatibility of the scaffold can be effectively controlled, thus providing an important basis for its application as a cell scaffold material. Attached Figure Description

[0027] Figure 1 The diagram shows the rheological properties of the composite gel.

[0028] Figure 2 This is a scanning electron microscope image of the composite gel.

[0029] Figure 3 CCK 8 diagram of the prepared cell culture scaffold.

[0030] Figure 4 The live and dead staining images of the prepared cell culture scaffold.

[0031] Figure 5 This is a flowchart illustrating the preparation of a cell culture scaffold for the present invention. Detailed Implementation

[0032] The following examples further illustrate specific implementations of the present invention, but the implementation and protection of the present invention are not limited thereto. Unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the field.

[0033] Example 1

[0034] S1: Dissolve chitosan in a 1% acetic acid solution and stir overnight to ensure complete dissolution, thus preparing a 2% chitosan solution.

[0035] S2: Add gelatin powder to the chitosan solution so that the gelatin concentration accounts for 2% of the total system concentration, and stir at 50°C to dissolve and obtain a homogeneous solution.

[0036] S3: Stir the solution at 80–90℃ for 20 min, add genipin and continue stirring for 20 min to obtain the compound solution.

[0037] S4: The solution was injected into the mold and refrigerated at -4°C overnight. The resulting gel swelled in ultrapure water and then dried in a freeze dryer for 48 hours to obtain a porous scaffold.

[0038] Example 2

[0039] The preparation method is the same as in Example 1, except that the gelatin concentration accounts for 4% of the total system concentration.

[0040] Example 3

[0041] The preparation method is the same as in Example 1, except that the gelatin concentration is 6% of the total system concentration.

[0042] Example 4

[0043] The preparation method is the same as in Example 1, except that the gelatin concentration accounts for 8% of the total system concentration.

[0044] Example 5

[0045] S1: Dissolve chitosan in a 1% acetic acid solution and stir overnight to ensure complete dissolution, thus preparing a 2% chitosan solution.

[0046] S2: Add gelatin powder to chitosan solution so that the gelatin concentration accounts for 6% of the total system concentration, stir at 50°C for 30 minutes to dissolve and obtain a homogeneous solution.

[0047] S3: Disperse corn starch in the above solution, with the starch concentration accounting for 1% of the total system concentration.

[0048] S4: Stir the solution at 80–90℃ for 20 min, add genipin and continue stirring for 20 min to obtain the compound solution.

[0049] S5: The solution was injected into the mold and refrigerated at -4°C overnight. The resulting gel swelled in ultrapure water and then dried in a freeze dryer for 48 hours to obtain a porous scaffold.

[0050] Example 6

[0051] The preparation method is the same as in Example 5, except that the starch concentration is 2% of the total system concentration.

[0052] Example 7

[0053] The preparation method is the same as in Example 5, except that the starch concentration accounts for 3% of the total system concentration.

[0054] Test methods

[0055] The rheological properties of the composite hydrogel were determined using a rotational rheometer, and the linear viscoelastic region (LVR) was identified in frequency scanning mode. Figure 1As shown, when the gelatin concentration increases from 2% to 8%, both the storage modulus (G') and loss modulus (G'') increase in a concentration-dependent manner. With increasing gelatin concentration, the deformation resistance of the network structure is significantly enhanced. G' and G'' are highest when 2% starch is added. Therefore, increasing the gelatin concentration and introducing an appropriate amount of corn starch can effectively enhance the viscoelasticity of the gel, providing experimental evidence for the performance regulation of tissue engineering scaffolds.

[0056] Figure 2 The images are scanning electron microscope (SEM) images. Microscopic observation revealed different structures in the gel samples with varying amounts of gelatin and starch. As the gelatin concentration increased, the number of pores gradually increased, and the connectivity between pores initially strengthened before stabilizing, resulting in a denser gel network. The continuity and integrity of the network structure were significantly enhanced at 6% and 8% gelatin addition. The network continuity and integrity were also significantly enhanced at 2% starch addition.

[0057] Cell compatibility of the cell-meat scaffold was assessed using CCK-8 assay and live / dead cell staining. The sterilized cell-meat scaffold was first pre-soaked in DMEM / F12 complete medium for 12 hours, then... 5 C2C12 cells were seeded at a density of [number] cells / mL. Cell viability was assessed using the CCK-8 assay after 2 days of culture. Figure 3 As shown, the cell survival rate was significantly improved with the addition of 6% gelatin. This may be because the mechanical properties and pore structure of the scaffold were more suitable for cell growth at this level.

[0058] Live and dead cell staining: After aseptic treatment of the cell culture scaffold, stain with 1×10⁻⁶ cells. 5 C2C12 cells were inoculated at a density of cells / mL, and after culturing for 2 days, live / dead staining was performed. Figure 4 As shown, green fluorescence indicates live cells, while red fluorescence indicates dead cells. The presence of abundant green fluorescence in the staining results indicates good cell growth, and the difference in fluorescence intensity also suggests that cells can grow into the scaffold. Specifically, with a 2% gelatin addition level, a small number of dead cells were observed to aggregate at the scaffold edge, which may be related to the lower mechanical strength and insufficient structural support of this group of gels. Cell growth was better with a 6% gelatin addition level, indicating that the prepared scaffold with 6% gelatin addition has good biocompatibility and cell adhesion.

Claims

1. A gel scaffold for cultured meat, characterized in that, The scaffold is made by cross-linking gelatin, chitosan, and corn starch with genipin, followed by swelling and freeze-drying.

2. A method for preparing the gel scaffold of claim 1, characterized in that, Includes the following steps: (1) Chitosan powder is dissolved in acetic acid solution to prepare chitosan solution. Gelatin powder is added to chitosan solution and heated and stirred to obtain mixed solution. (2) Add corn starch to the mixture and heat and stir. (3) Add genipin to step (2), stir, and obtain a compound solution; (4) The compound solution is injected into the mold to form a gel, and then the gel is placed in ultrapure water to swell and freeze-dried to obtain a porous scaffold.

3. The preparation method according to claim 2, characterized in that, The acetic acid solution in step (1) has a mass fraction of 1-2%, the chitosan has a mass fraction of 0-4%, and the gelatin has a mass fraction of 0-8%.

4. The preparation method according to claim 2, characterized in that, In step (1), the stirring temperature is 40-70℃ and the stirring time is 20-60min. In step (2), the stirring temperature is 80-90℃ and the stirring time is 20-60min.

5. The preparation method according to claim 2, characterized in that, In step (2), the amount of corn starch added is 1-5% of the total mass of the system.

6. The preparation method according to claim 2, characterized in that, In step (3), the concentration of genipin added is 1.0-5.0 mmol / L.

7. A method for preparing cell-cultured meat, characterized in that, Includes the following steps: (1) The gel scaffold described in claim 1 is sterilized to obtain a sterile scaffold; (2) The sterile scaffold was swollen and equilibrated in DMEM medium; (3) The cells were loaded at a rate of 1×10 5 -1×10 6 Cells were seeded at a density of 1 cell / mL on the swollen scaffold and cultured at 37°C and 5% CO2 to allow them to adhere, proliferate and differentiate on the scaffold.

8. The preparation method according to claim 7, characterized in that, The sterilization process described in step (1) includes immersing the stent in a PBS solution containing double antibodies and then irradiating it with ultraviolet light overnight.

9. The preparation method according to claim 7, characterized in that, The volume ratio of DMEM culture medium to scaffold in step (2) is 3:1-6:

1.

10. The cell-cultured meat according to claim 7, characterized in that, The cells are one or more of the following animal cells: muscle cells, adipocytes, fibroblasts, mesenchymal stem cells, and pluripotent stem cells.

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