Directional porous bionic gelatin scaffold, preparation method and application thereof

Abstract

The invention relates to the technical field of cell culture, in particular to an oriented porous bionic gelatin scaffold for controlling cell orientation arrangement, a preparation method and application thereof, and the oriented porous bionic gelatin scaffold can be used for cell culture meat production. The preparation method comprises the following steps: mixing and solidifying gelatin and glutamine transaminase, directionally freezing, freeze-drying, sterilizing, inoculating and culturing cells and the like. Parameters and culture medium components in each step are also explained. Compared with the prior art, a regular directional porous structure is formed through a hydrogel scaffold and directional freezing, directional arrangement and growth of fish muscle satellite cells are guided, the cell activity is high, and the proliferation and differentiation effects are good; the gelatin material is adopted, so that the cost is low, the biocompatibility is good, and the gelatin material is superior to collagen and wheat glutelin; the preparation process is simple, does not need complex equipment, is suitable for large-scale production, and promotes the development of cell culture meat.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell culture technology, in particular to a preparation method of a directional porous biomimetic gelatin scaffold and application thereof, especially to a directional porous biomimetic gelatin scaffold for controlling cell orientation arrangement, a preparation method and application thereof. BACKGROUND

[0002] Cell culture meat is a promising disruptive technology that can solve the sustainability and environmental problems of current traditional livestock farming. Unlike other alternative proteins, cell culture meat is produced by animal stem cells in vitro and is a product closest to natural meat. The key step of cell culture meat synthesis is the culture of cells on a suitable three-dimensional scaffold, which should provide a microenvironment similar to the in vivo cell growth environment to promote cell adhesion, growth and differentiation. In order to simulate the in vivo growth environment of cells, it is necessary to understand the structure of natural muscle tissue. Natural muscle tissue is composed of parallelly arranged multinucleated muscle fibers of relatively equal size, which are surrounded by extracellular matrix. This structural feature determines the key factor of three-dimensional biomimetic in vitro model production: promoting cell directional growth and differentiation to form uniaxially arranged myotubes.

[0003] In muscle tissue engineering, muscle cells can be pre-aligned by a scaffold matrix with a specific structure to promote directional arrangement and growth of cells, and then promote cell-cell fusion and formation of aligned myotubes. Directional freeze-drying is an effective method for preparing anisotropic porous scaffolds. Unlike the isotropic structure produced by traditional freeze-drying, directional freeze-drying can produce a three-dimensionally arranged porous structure by controlling the direction of ice crystal growth. For skeletal muscle systems, directional freeze-drying has become an alternative to other commonly used techniques (such as 3D printing and electrospinning) due to its simple process, no need for complex equipment, and the ability to produce properly oriented macropores in a suitable range. With the development of cell culture meat industry, directional freeze-drying technology has also been gradually applied to cell culture meat. Unlike other applications, the main problem of applying directional freeze-drying technology to cell culture meat is the selection of materials. The material should have biocompatibility, a large surface area, non-toxic and edible, and allow the maximum diffusion of culture medium.

[0004] Currently, researchers have only used collagen hydrogel and wheat gluten to prepare scaffolds with aligned porous structures by directional freeze-drying method. Among them, collagen contains a large amount of RGD peptides, which helps cell adhesion. However, collagen hydrogel is expensive and not suitable for large-scale production. Although wheat gluten is relatively low in cost, it has poor biocompatibility due to the lack of RGD peptides. Therefore, it is necessary to find a material that is both inexpensive and has good biocompatibility to prepare a biomimetic scaffold with directional porous structure by directional freeze-drying method. SUMMARY

[0005] To solve the above technical problems, the present application provides a kind of directional porous biomimetic gelatin scaffold, preparation method and application thereof, and the biomimetic scaffold with directional porous structure is low in cost, higher in biocompatibility and simple in preparation required equipment.After sterilization, the scaffold is inoculated with cells and after culture, cells can grow directionally, which can be used for the production of cell culture meat or for the manufacture of biological scaffold or for the manufacture of biological blood vessels.

[0006] A kind of directional porous biomimetic gelatin scaffold, including hydrogel scaffold with directional porous structure, and proliferative cell attached and grown on the hydrogel scaffold;

[0007] The proliferative cell is fish muscle satellite cell.

[0008] Further, the hydrogel scaffold is the mixed prepolymer of gelatin solution and glutamine transaminase solution arranged in mold, which is obtained by vacuum freeze drying.

[0009] Further, the fish muscle satellite cell is large yellow croaker fish muscle satellite cell.

[0010] The application of a kind of directional porous biomimetic gelatin scaffold is used for cell culture meat or for the manufacture of biological scaffold or for the manufacture of biological blood vessels.

[0011] A kind of preparation method of directional porous biomimetic gelatin scaffold, comprising the following steps,

[0012] Step 1) prepolymer preparation, mix gelatin solution with mass fraction of 3-9% and glutamine transaminase solution with final concentration of 0.5-1.8 U / mL, put into mold and solidify at temperature of 24-37 ℃ for 20-60 min to form hydrogel prepolymer;

[0013] Step 2) directional freezing, place the solidified hydrogel prepolymer and its mold on the heat conduction plate above liquid nitrogen to perform directional freezing to form frozen hydrogel;

[0014] Step 3) freeze drying, place the frozen hydrogel in vacuum freeze dryer to perform freeze drying for 36-48 h to form hydrogel scaffold with directional porous structure;

[0015] Step 4) sterilization treatment, immerse the scaffold in 75% ethanol for 24-48 h, wash with PBS and then sterilize by ultraviolet light and dry;

[0016] Step 5) cell inoculation, inoculate fish muscle satellite cells at 5-20 × 10 6 / cm³, and incubate and culture.

[0017] Further, in step 2), the heat-conducting plate is one of a copper plate, an iron plate, an aluminum plate, an alloy metal with a heat conductivity ≥200 W / (m·K), or an alloy metal with a heat conductivity ≥200 W / (m·K) and a non-metal; the bottom of the mold directly contacts the heat-conducting plate, and the liquid nitrogen level does not exceed the surface of the heat-conducting plate.

[0018] Further, in step 3), the hydrogel support has a porosity ≥80%, a pore size of 50-200 μm, and a pore channel arranged along a freezing direction.

[0019] Further, the arrangement is perpendicular to the heat-conducting plate.

[0020] Further, the incubation is performed in a 25-29℃ incubator, and the incubation time is 1-1.5 h.

[0021] Further, the culture includes proliferation culture and differentiation culture performed in sequence.

[0022] The proliferation culture is performed for 3-5 d, and the differentiation culture is performed for 6-8 d; the culture temperature is 25-127℃; and the volume concentration of CO2 is 4-16%.

[0023] More specifically, the heat-conducting plate can also be made of a material that is easy to obtain and has good heat conductivity.

[0024] Preferably, the proliferation culture medium used in the proliferation culture is DMEM / F12 culture medium containing 10% fetal bovine serum and 1×antibiotics (100 U / mL penicillin G sodium salt and 0.1 mg / mL streptomycin sulfate), and the differentiation culture medium used in the differentiation culture is DMEM / F12 culture medium containing 8% horse serum, 10 ng / mL IGF-1, 50 nM necrosulfonamide, 200 μM ascorbic acid, and 1×antibiotics (100 U / mL penicillin G sodium salt and 0.1 mg / mL streptomycin sulfate).

[0025] Compared with the prior art, the present application has the following advantages:

[0026] 1) The structure of the support is precisely controllable, and cell directional arrangement is achieved

[0027] The present application uses liquid nitrogen-heat-conducting plate directional freezing technology to make the water in the gel form ice crystals perpendicular to the metal plate, and form a regular directional porous structure after freeze-drying. Fish muscle satellite cells can be arranged along the pores of the support, and the cell stretching degree and arrangement consistency in the directional support are significantly higher than those in the non-directional support, thus solving the problem of disordered cell growth in the traditional support.

[0028] 2) The material and process have significant advantages and are suitable for large-scale application

[0029] Low cost and high biocompatibility: gelatin is chosen as the base material, which reduces the cost by more than 60% compared to collagen, and contains natural cell adhesion sites, which is better than wheat gluten which lacks biological activity; glutamine transaminase crosslinking further improves the stability of the scaffold, avoiding the toxic residue of chemical crosslinking agents.

[0030] Simple process and easy operation: no need for complex equipment such as 3D printing and electrospinning, just through directional freezing and freeze-drying, the design of wrapping the mold with thermal insulation material (except the bottom) further reduces energy consumption, which is suitable for industrialized production.

[0031] 3) Excellent cell culture effect, improving the quality of cultured meat

[0032] Strong cell activity and proliferation ability: live and dead cell staining shows that the proportion of live cells in the directional scaffold is significantly higher than that in the traditional scaffold, and the cell density is increased by 30%-50% after 5 days of proliferation culture, and continuous myotube structures are formed after 6-8 days of differentiation culture.

[0033] Adapt to the characteristics of fish muscle satellite cells: for the culture of fish muscle satellite cells such as large yellow croaker, the pore structure of the scaffold and the formula of the culture medium work together to improve the directional differentiation efficiency of the cells by more than 40%, providing a fibrous structure similar to natural muscle for cultured fish meat.

[0034] 4) Promote breakthroughs in cell culture meat technology

[0035] For the first time, directional freeze-drying method is applied to fish muscle satellite cell scaffold, solving the problem of high cost of collagen and poor compatibility of wheat gluten in the prior art, providing a special biomimetic scaffold for fish cultured meat.

[0036] The size of the scaffold can be flexibly adjusted by the mold, and the shape and size of the cultured meat are directly determined by the scaffold, meeting the needs of standardized production. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The electron micrograph of the gelatin scaffold prepared in Example 1 after directional freeze-drying;

[0038] Figure 2 The live and dead cell staining chart of the cells after 5 days of culture in Example 1, green represents live cells, and red represents dead cells;

[0039] Figure 3 The electron micrograph of the gelatin scaffold prepared in Comparative Example 1 after traditional freeze-drying.

[0040] Figure 4 The live and dead cell staining chart of the cells after 5 days of culture in Comparative Example 1, green represents live cells, and red represents dead cells.

[0041] Figure 5 Figure 9 is a comparison chart of the arrangement of cells after 5 days of culture in Example 1 and Comparative Example 1; A: immunofluorescence staining of cells after 5 days of culture in Example 1, red represents actin, and blue represents the nucleus; B: immunofluorescence staining of cells after 5 days of culture in Comparative Example 1, red represents actin, and blue represents the nucleus; C: nuclear aspect ratio of cells after 5 days of culture in Example 1 and Comparative Example 1; D: nuclear orientation analysis of cells after 5 days of culture in Example 1 and Comparative Example 1.

[0042] Figure 6 Figure 10 is an electron micrograph of the gelatin scaffold prepared in Example 2 after directional freeze-drying.

[0043] Figure 7 Figure 11 is a live and dead cell staining chart of cells after 5 days of culture in Example 2, green represents live cells, and red represents dead cells.

[0044] Figure 8 Figure 12 is an electron micrograph of the gelatin scaffold prepared in Comparative Example 2 after conventional freeze-drying.

[0045] Figure 9 Figure 13 is a live and dead cell staining chart of cells after 5 days of culture in Comparative Example 2, green represents live cells, and red represents dead cells. DETAILED DESCRIPTION

[0046] The application will be further described below in conjunction with specific examples. The following examples are only used to illustrate the application and are not intended to limit the scope of application. Modifications or replacements of the methods, steps or conditions of the application, without departing from the spirit and essence of the application, all belong to the scope of the application.

[0047] The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials available from commercial channels unless otherwise specified.

[0048] DMEM / F12 medium (PM150312, Procell); fetal bovine serum (WISENT); antibiotics (Solarbio); horse serum (HyClone); IGF-1 (Solarbio); necrosulfonamide (Macklin); ascorbic acid (Aladdin); fish gelatin (Yin Ye Bio); glutamine transaminase (Dibo); PBS buffer (NEST); paraformaldehyde (Yin Ye Bio); immunostaining permeation liquid (Beyotime); rhodamine-labeled phalloidin (Yixing Bio); live / dead cell double staining kit (BestBio)

[0049] Example 1

[0050] Firstly, a gelatin mother liquor with a mass fraction of 10% and a glutamine transaminase mother liquor with a concentration of 10 U / mL were prepared, and the gelatin mother liquor, the glutamine transaminase mother liquor and deionized water were mixed and poured into a mold, the final concentrations of gelatin and glutamine transaminase were 5% and 1 U / mL respectively, and the final volume of the pre-polymer was 0.5 mL. The mold containing the pre-polymer solution was placed in a 30℃ incubator for 40 min to solidify the gelatin.

[0051] The iron plate was placed in a foam box and liquid nitrogen was poured into it to pre-cool the iron plate. After pre-cooling was completed, the mold containing the gelatin was placed on the iron plate for directional freezing for 5 min. The bottom of the mold was in contact with the metal plate, and the rest of the surface was covered with a layer of thermal insulation material. After freezing was completed, the mold containing the gelatin was placed in a vacuum freeze dryer for freeze drying for 40 h to obtain a gelatin scaffold with a directional porous structure.

[0052] The gelatin scaffold was cut into a scaffold with a size of 8 mm x 4 mm x 1 mm (LWH), and the scaffold was immersed in 75% ethanol for 48 h for sterilization. After sterilization was completed, the scaffold was washed 3 times in phosphate buffered saline (PBS) and dried under a UV lamp. After drying, the large yellow croaker muscle satellite cells were inoculated onto the dried scaffold at an inoculation amount of 5 x 106 cells / cm3, and the scaffold with cells was incubated in a 27℃ incubator for 1 h. After incubation, culture medium was added for culture, and proliferation culture was performed for 5 days and differentiation culture was performed for 6 days. The sample at the 5th day of proliferation culture was taken for live and dead cell staining and cytoskeleton staining.

[0053] The SEM images of the cross section and the longitudinal section of the gelatin scaffold obtained in this example are shown in Figure 1 , the live and dead cell staining at the 5th day of proliferation culture is shown in Figure 2 , the cytoskeleton staining at the 5th day of proliferation culture is shown in Figure 5 A, the nucleus aspect ratio is shown in Figure 5 C, the nucleus aspect ratio indicates the degree of stretching of the cells; and the nucleus orientation distribution is shown in Figure 5 D. The SEM images show that the scaffold prepared by directional freeze drying has a directional arrangement of pore structure inside. The live and dead cell staining and the cytoskeleton staining show that the cell growth has a certain directionality. The cell aspect ratio and the nucleus orientation distribution show that the cells are elongated and arranged along the pore structure when the cells grow in the directional freeze-dried scaffold.

[0054] Example 2

[0055] Firstly, a gelatin mother liquor with a mass fraction of 10% and a glutamine transaminase mother liquor with a concentration of 10 U / mL were prepared, and the gelatin mother liquor, the glutamine transaminase mother liquor and deionized water were mixed and poured into a mold, and the final concentrations of gelatin and glutamine transaminase were 3% and 1 U / mL respectively, and the final volume of the pre-polymer was 0.5 mL. The mold containing the pre-polymer solution was placed in a 30℃ incubator for 40 min to solidify the gelatin.

[0056] The iron plate was placed in a foam box, and liquid nitrogen was poured into the iron plate to pre-cool the iron plate. After pre-cooling was completed, the mold containing the gelatin was placed on the iron plate for directional freezing for 5 min. The bottom of the mold was in contact with the metal plate, and the remaining surface was covered with a layer of thermal insulation material. After freezing was completed, the mold containing the gelatin was placed in a vacuum freeze dryer for freeze drying for 40 h to obtain a gelatin scaffold with a directional porous structure.

[0057] The gelatin scaffold was cut into a scaffold with a size of 8 mm x 4 mm x 1 mm (LWH), and the scaffold was immersed in 75% ethanol for 48 h for sterilization. After sterilization was completed, the scaffold was washed in phosphate buffer (PBS) for 3 times, and was placed under a UV lamp to dry. After drying, the large yellow croaker muscle satellite cells were inoculated onto the dried scaffold at an inoculation amount of 5 x 106 cells / cm3, and the scaffold with the cells was incubated in a 27℃ incubator, and the incubation time was preferably 1 h. After incubation was completed, culture medium was added for culture, and proliferation culture was performed for 5 days, and differentiation culture was performed for 6 days. The sample at the 5th day of proliferation culture was taken for live and dead cell staining.

[0058] The SEM images of the cross section and the longitudinal section of the gelatin scaffold obtained in the example are shown in Figure 6 , the live and dead cell staining at the 5th day of proliferation culture is shown in Figure 7 , the cytoskeleton staining at the 5th day of proliferation culture is shown in

[0059] Comparative Example 1

[0060] The comparative example described herein is similar to Example 1, and the difference is that the mold containing the gelatin is not placed on the iron plate pre-cooled by liquid nitrogen for directional freezing for 5 min, but is directly placed in liquid nitrogen for freezing for 5 min.

[0061] The SEM images of the cross section and the longitudinal section of the gelatin scaffold obtained in the comparative example are shown in Figure 3 , the live and dead cell staining at the 5th day of proliferation culture is shown in Figure 4 , the cytoskeleton staining at the 5th day of proliferation culture is shown in Figure 5 B, and the nucleus aspect ratio is shown in Figure 5 C, the nucleus aspect ratio indicates the degree of stretching of the cells; and the nucleus orientation distribution is shown in Figure 5D. SEM images can see that the scaffold prepared by traditional freeze-drying method has no regular arrangement of pore structure inside. Live and dead cell staining and cytoskeleton staining can see that cell growth has no directionality. Cell aspect ratio and nucleus orientation distribution can see that cells have no obvious direction when growing in the traditional freeze-drying scaffold.

[0062] Comparative Example 2

[0063] The present comparative example is similar to Example 2, except that the mold containing gelatin is not placed on the iron plate pre-cooled by liquid nitrogen for directional freezing for 5 min, but is directly placed in a refrigerator at about 80°C for freezing for 24 h.

[0064] SEM images of the cross section and longitudinal section of the gelatin scaffold obtained in the present comparative example are shown in Figure 8 ; and live and dead cell staining of the 5th day of proliferation culture is shown in Figure 9 . SEM images can see that the scaffold prepared by traditional freeze-drying method has no regular arrangement of pore structure inside. Live and dead cell staining can see that cell growth has no directionality.

[0065] The present application has the following advantages over the prior art:

[0066] 1) The scaffold structure is precisely controllable, realizing directional arrangement of cells

[0067] The directional porous structure is significant: through the liquid nitrogen-heat conduction plate directional freezing technology, the water in the gel forms ice crystals perpendicular to the metal plate, and after freeze-drying, a regular directional porous structure is formed Figure 1 、 6 Electron microscope images), and the pore arrangement consistency is much better than that of the disordered structure prepared by traditional freeze-drying method Figure 3 、 8 .

[0068] Guiding cell orientation growth: fish muscle satellite cells can be arranged directionally along the scaffold pores Figure 5 A), and the cell stretching degree and arrangement consistency in the directional scaffold are significantly higher than those in the non-directional scaffold Figure 5 C, D), solving the problem of disordered cell growth in the traditional scaffold Figure 5 B).

[0069] 2) The material and process have significant advantages and are suitable for large-scale application

[0070] Low cost and high biocompatibility: gelatin is selected as the base material, which reduces the cost by more than 60% compared with collagen, and contains natural cell adhesion sites, which is better than wheat gluten which lacks biological activity; glutamine transaminase crosslinking further improves the stability of the scaffold, avoiding the toxicity of chemical crosslinking agents.

[0071] Simple process: No need for 3D printing, electrospinning and other complex equipment, only through directional freezing (5-10 minutes) and freeze-drying (36-48 hours) can be prepared, suitable for industrialized production.

[0072] 3) Excellent cell culture effect, support culture meat quality improvement

[0073] Strong cell activity and proliferation ability: live and dead cell staining shows (Figure 2), the proportion of living cells in the directional scaffold (green) is significantly higher than that in the traditional scaffold (Figure 3), the cell density is increased by 30%-50% after 5-day proliferation culture, and continuous myotube structure is formed after 6-8-day differentiation culture. Figure 2 、 7 Figure 4 、 9 Strong cell activity and proliferation ability: live and dead cell staining shows (Figure 2), the proportion of living cells in the directional scaffold (green) is significantly higher than that in the traditional scaffold (Figure 3), the cell density is increased by 30%-50% after 5-day proliferation culture, and continuous myotube structure is formed after 6-8-day differentiation culture.

[0074] Adapt to the characteristics of fish muscle satellite cells: for the culture of fish muscle satellite cells such as large yellow croaker, the pore structure (pore size adapted to cell growth needs) and culture medium formula (containing IGF-1, ascorbic acid and other differentiation factors) of the scaffold synergistically improve the cell directional differentiation efficiency by more than 40%, providing a fibrous structure similar to natural muscle for fish meat culture.

[0075] 4) Promote breakthrough in cell culture meat technology

[0076] Fill the technical gap: for the first time, directional freeze-drying method is applied to fish muscle satellite cell scaffold, solving the problem of high cost of collagen and poor compatibility of wheat gluten in existing technology, providing a special biomimetic scaffold for fish culture meat.

[0077] Strong controllability of products: the size of the scaffold can be flexibly adjusted through the mold (such as 8mm x 4mm x 1mm in the examples), the shape and size of the culture meat are directly determined by the scaffold, meeting the needs of standardized production.​

Claims

1. A directional porous biomimetic gelatin scaffold, characterized by: It includes a hydrogel scaffold with a directional porous structure, and proliferating cells attached and grown on the hydrogel scaffold; The proliferating cells are fish muscle satellite cells.

2. The oriented porous biomimetic gelatin scaffold according to claim 1, characterized in that: The hydrogel scaffold is a hydrogel scaffold obtained by vacuum freeze-drying a prepolymer of a gelatin solution and a transglutaminase solution set in a mold.

3. The oriented porous biomimetic gelatin scaffold according to claim 1, characterized in that: The fish muscle satellite cells are large yellow croaker muscle satellite cells.

4. An application of a directional porous biomimetic gelatin scaffold, characterized by: Used for cell culture meat or for the manufacture of biological scaffolds or for the manufacture of biological blood vessels.

5. A method for preparing a directional porous biomimetic gelatin scaffold, characterized by: The following steps are included: Step 1) Prepolymer preparation: a gelatin solution having a mass fraction of 3 to 9% is mixed with a transglutaminase solution having a final concentration of 0.5 to 1.8 U / mL, and the mixture is placed in a mold and cured at a temperature of 24 to 37° C. for 20 to 60 minutes to form a hydrogel prepolymer; Step 2) Directional freezing: placing the solidified hydrogel prepolymer and its mold on a heat conducting plate above liquid nitrogen for directionally freezing to form a frozen hydrogel; Step 3) freeze drying: placing the frozen hydrogel in a vacuum freeze dryer for freeze drying for 36 to 48 hours to form a hydrogel scaffold with an oriented porous structure; Step 4) Sterilization: Immerse the stent in 75% ethanol for 24-48 hours, wash with PBS, and then sterilize with UV light and air dry. Step 5) Cell seeding: 5-20×10 6 Fish muscle satellite cells were inoculated at 100 μg / cm³ and cultured after incubation.

6. The method for preparing a directional porous biomimetic gelatin scaffold according to claim 5, characterized in that: In step 2), the heat conducting plate is one of a copper plate, an iron plate, an aluminum plate, an alloy metal with a thermal conductivity of ≥200 W / (m·K), or an alloy metal non-metal with a thermal conductivity of ≥200 W / (m·K); the bottom directly contacts the heat conducting plate, and the remaining surface is wrapped with insulation material, and the liquid nitrogen level does not exceed the surface of the heat conducting plate.

7. The method for preparing a directional porous biomimetic gelatin scaffold according to claim 5, characterized in that: In step 3), the hydrogel scaffold has a porosity of ≥80%, a pore size of 50 to 200 μm, and pores are directionally arranged along the freezing direction.

8. The method for preparing a directional porous biomimetic gelatin scaffold for controlling cell orientation according to claim 7, characterized in that: The orientation is perpendicular to the heat conducting plate.

9. The method for preparing a directional porous biomimetic gelatin scaffold according to claim 5, characterized in that: In step 5), the incubation is to incubate the scaffold with cells in an incubator at 25-29° C. for 1-1.5 hours.

10. The method for preparing a directional porous biomimetic gelatin scaffold according to claim 5, characterized in that: The culture includes proliferation culture and differentiation culture performed sequentially; The proliferation culture time is 3 to 5 days, and the differentiation culture time is 6 to 8 days; the culture temperature is 25 to 127° C.; and the volume concentration of CO 2 in the culture is 4 to 16%.

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