Modified agarose for cell culture meat as well as preparation method and application of modified agarose

Abstract

The invention discloses modified agarose for cell culture meat as well as a preparation method and application of the modified agarose, and relates to the technical field of biological food materials. The preparation method comprises the following steps: preparing agarose into an agarose-based hydrogel stent and an agarose-based thin film stent through physical cooling and drying methods, and then coating the agarose-based hydrogel stent and the agarose-based thin film stent with polydopamine coatings through a dip coating method. Therefore, the surfaces of the agarose-based hydrogel scaffold and the agarose-based film scaffold can adsorb serum protein and adhere cells. By utilizing the modified agarose-based scaffold, adhesion of skeletal muscle satellite cells can be realized, absorption of nutrient substances by the skeletal muscle satellite cells is greatly enhanced, and proliferation and differentiation activities of the skeletal muscle satellite cells are facilitated. In addition, the agarose containing the multinuclear myotube can be used as a cell culture meat module for preparing cell culture meat.

Description

Technical Field

[0001] The invention relates to the technical field of biological food materials, and in particular to a modified agarose for cell cultured meat and a preparation method and application thereof. Background Art

[0002] The growing contradiction between global meat supply and demand has raised concerns about issues associated with traditional animal husbandry, including environmental degradation, greenhouse gas emissions, animal welfare, public health, and food quality and safety. Therefore, there is an urgent need to develop alternatives to traditional meat products. Cell-cultured meat is produced in vitro without the need for long-term animal breeding and slaughter. Cell-cultured meat produced through scientific, efficient, and environmentally friendly cell biology, tissue engineering, and food technology is also highly nutritious and healthy, and has the potential to solve problems associated with traditional animal husbandry.

[0003] In the production of cell cultured meat, the cell scaffold can mimic the structure of cultured meat, which is conducive to the extensive adhesion, proliferation and differentiation of muscle cells on the scaffold surface and improves the texture of cultured meat. At present, the most commonly reported cell cultured meat scaffold materials include proteins (soy protein isolate, pea protein isolate, gluten, zein, gelatin and collagen), polysaccharides (plant cellulose, alginate, bacterial cellulose, chitosan, alginate and gellan gum) and synthetic polymers. Polysaccharides and proteins are coexisting biomacromolecules in food. However, polysaccharides generally show poor cell adhesion and are not conducive to cell proliferation and differentiation in the later stage of cell culture. For example, compared with gelatin, gluten and soy protein films, polysaccharide films made of bacterial cellulose, cellulose nanofibers and chitosan have poor cell adhesion properties. This is due to the lack of cell adhesion sites in polysaccharides, such as the RGD (arginine-glycine-aspartic acid) peptide structure in proteins. At present, this problem can be solved by preparing polysaccharide-protein complexes to make up for the lack of cell biological activity of polysaccharide materials. Nevertheless, the general preparation process of polysaccharide / protein complexes involves the use of toxic cross-linking agents and the optimization of many parameters (such as additives, temperature, pressure, and humidity), but it is worth noting that animal-derived protein components (gelatin and collagen) and toxic cross-linking agents should be avoided in cell scaffolds for cell-cultured meat. In addition, although plant-derived proteins can replace animal-derived proteins to prepare cell scaffolds, consumers may be allergic to plant-derived proteins, such as peanut protein and gluten. Compared with proteins from animal and plant sources, polysaccharides are abundant and inexpensive, and most natural polysaccharides have the advantages of low immunogenicity, low toxicity, and biodegradability. Therefore, the production of protein-free polysaccharide scaffolds is urgent to promote the high-value application of polysaccharide materials in cell-cultured meat.

[0004] Agarose (AG) is a neutral polysaccharide component of agar, which is widely found in food. It has good gelling and film-forming properties and has been selected as a representative model material for polysaccharide cell scaffolds. However, the low cell bioactivity of agarose scaffolds is its main disadvantage.

[0005] In the prior art, a patent application with publication number CN111701074A discloses a polydopamine hybrid agarose hydrogel and a preparation method and application. The hydrogel scaffold prepared by the invention has excellent biocompatibility and cell adhesion. The research is mainly carried out around the scaffold to promote tissue repair and regeneration in animal defective tissues. The scaffold induces local dedifferentiated cells, immune cells and other types of cells (vascular endothelial cells, fibroblasts, etc.) around the defective tissue of the animal to migrate into the inner and outer surfaces of the scaffold. Under the synergistic action of these cells, the cells are promoted to secrete growth factors and cytokines, and necrotic tissues and pathogens are removed, creating a good microenvironment for tissue repair and regeneration, and jointly promoting the repair and regeneration process of defective tissues, which has broad application prospects in the field of tissue engineering. However, the in vitro culture conditions and methods of specific cells are still unknown, and it is still unclear how the surface modification of biomaterials and the interaction between cells and materials regulate the growth and development of cells. At present, cell scaffolds play a vital role as a key platform for cell growth and differentiation in the production process of cell cultured meat. Data related to the growth and development of muscle cells on agarose / polydopamine (AG / PDA) scaffolds with bioactive surfaces are urgently needed to achieve precise regulation of cell growth and differentiation, thereby improving the yield and quality of cell culture and promoting its development in cell-cultured meat.

[0006] There is currently a lack of data on the adhesion, proliferation and differentiation of myocytes on agarose / polydopamine (AG / PDA) scaffolds with bioactive surfaces to promote in vitro protein-rich myofiber formation to support the future application of polysaccharides as cell scaffolds for cell cultured meat. Therefore, it is still a challenge to obtain agarose-based scaffold materials that facilitate the life activities of cells in vitro "adhesion-proliferation-differentiation" and achieve large-scale in vitro myofiber preparation. Summary of the invention

[0007] In response to the above-mentioned problem of how to prepare agarose-based scaffold materials with high cell biological activity and strong cell adhesion, the present invention proposes a modified agarose with strong adsorption capacity and long-term stability for cell culture meat and its preparation method and application, especially a modified agarose-based scaffold material for culturing skeletal muscle satellite cells as a cell scaffold.

[0008] The present invention first prepares an agarose-based hydrogel scaffold and an agarose-based film scaffold by combining a physical crosslinking method with a dehydration drying method, and then enhances the ability of the scaffold to adsorb proteins and growth factors by dipping and coating a polydopamine coating. The polydopamine (PDA) coating enables the agarose-based hydrogel scaffold and the agarose-based film scaffold to have the ability of viscosity cells, and can promote the efficient proliferation and differentiation of skeletal muscle satellite cells, and the two modified agarose-based scaffolds can maintain good mechanical properties and integrity after swelling with a large amount of water. At the same time, the PDA coating modification process is simple and efficient, does not involve the addition of toxic chemical crosslinking reagents and proteins, and realizes the modification of the agarose scaffold.

[0009] The present invention first provides a method for preparing modified agarose for cell meat culture, comprising the following steps:

[0010] (1) Preparing an agarose-based hydrogel scaffold: dissolving agarose to prepare a solution, pouring the solution into a mold, and allowing the solution to solidify to obtain an agarose-based hydrogel scaffold;

[0011] (2) Preparing an agarose-based film scaffold: dehydrating and drying the obtained agarose-based hydrogel scaffold to obtain an agarose-based film scaffold;

[0012] (3) Dipping and coating with polydopamine coating: The two scaffolds obtained in step (1) and step (2) are immersed in a dopamine (DA) solution to obtain an agarose-based hydrogel scaffold and an agarose-based film scaffold coated with polydopamine coating, respectively, which are the modified agarose.

[0013] Preferably, in step (1), agarose and water are dissolved to prepare a solution, and the concentration of the solution is 0.1wt%-5wt%;

[0014] The standing condition is: standing at room temperature for 1-2 hours.

[0015] The mold is not specifically limited. In the present invention, a mold cell culture plate six-well plate (circular, radius 1.74 mm) and a glass culture dish (circular, radius 4.5 mm) are used to obtain agarose-based hydrogel scaffolds and agarose-based film scaffolds with complete structures.

[0016] In step (2), the dehydration and drying conditions are: the dehydration and drying temperature is 25-37° C., and the dehydration and drying time is 48-72 hours.

[0017] Preferably, in step (3), the concentration of dopamine is 0.05-0.2 mg / ml.

[0018] Specifically, in step (3), when the polydopamine coating is applied by immersion, the two scaffolds obtained in step (1) and step (2) are first immersed in PBS for 0.5-1h, and then immersed in a dopamine solution for 1-2h. Thereafter, the scaffolds are repeatedly washed with PBS for 3-5 times, each time for 3 minutes, and the unbound and unstable polydopamine particles on the surface are removed by washing with PBS.

[0019] The invention also provides modified agarose prepared by the preparation method.

[0020] The present invention also provides the use of the modified agarose in preparing a culture scaffold for cell cultured meat, wherein the cells are skeletal muscle satellite cells, muscle stem cells, adipocytes, endothelial cells or fibroblasts.

[0021] The present invention also provides the use of the modified agarose as a cell culture scaffold in cell cultured meat, wherein the cells in the cell cultured meat are skeletal muscle satellite cells, muscle stem cells, adipocytes, endothelial cells or fibroblasts.

[0022] Beneficial results achieved by the present invention:

[0023] 1. The present invention combines a physical cross-linking method with a dehydration drying method to prepare an agarose-based hydrogel scaffold and an agarose-based film scaffold, and then immerses and coats a polydopamine coating, and finally successfully prepares a polydopamine-coated agarose-based hydrogel scaffold and an agarose-based film scaffold. The immersion-coated polydopamine coating can be adsorbed on the agarose-based hydrogel scaffold and the agarose-based film scaffold for a long time and stably. The mechanical properties of the two scaffolds are intact, so that the scaffold material will not degrade and collapse during the subsequent skeletal muscle satellite cell culture process, and a modified agarose scaffold material with strong cell adhesion properties is obtained while avoiding the use of toxic chemical cross-linking reagents and animal-derived proteins.

[0024] 2. The invention introduces a polydopamine coating into an agarose-based scaffold, which is beneficial to the genome amplification of skeletal muscle satellite cells and the expression of myogenic differentiation factors, and can efficiently promote the proliferation of skeletal muscle satellite cells.

[0025] 3. The invention introduces a polydopamine coating into the agarose-based scaffold, which is beneficial to the expression of myosin heavy chain protein of skeletal muscle satellite cells, improves the differentiation and fusion of skeletal muscle satellite cells during the differentiation induction stage, and contributes to the formation of multinucleated myotubes. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Preparation of agarose-based hydrogel scaffolds and agarose-based film scaffolds;

[0027] Figure 2 coating agarose-based hydrogel scaffolds with polydopamine;

[0028] Figure 3 This is a scanning electron micrograph of a polydopamine-coated agarose-based film scaffold;

[0029] Figure 4 is the determination of cell adhesion rate; wherein, A is the adhesion of cells on agarose hydrogel scaffold-polydopamine (0.2 mg / ml) (AG / PDA-0.2); B is the adhesion of cells on agarose hydrogel scaffold-polydopamine (0.1 mg / ml) (AG / PDA-0.1); C is the adhesion of cells on agarose hydrogel scaffold-polydopamine (0.05 mg / ml) (AG / PDA-0.05); D is the adhesion of cells on agarose hydrogel scaffold (AG); E is the adhesion of cells on traditional tissue cell culture plates (TCP); F is the statistical analysis of cell adhesion rate;

[0030] Figure 5 For SEM analysis of cell adhesion status; Figure 5 A is scanning electron microscopy analysis of polydopamine-coated agarose film scaffolds without adherent cells; Figure 5 B is scanning electron microscopy analysis of polydopamine-coated agarose film scaffolds containing adherent cells;

[0031] Figure 6 To culture cells on a polydopamine-coated agarose-based hydrogel scaffold; wherein A is a cell proliferation test of porcine skeletal muscle satellite cells on a polydopamine-coated agarose gel scaffold; B is a cell differentiation test of porcine skeletal muscle satellite cells on a polydopamine-coated agarose gel scaffold;

[0032] Figure 7 To culture cells on a polydopamine-coated agarose-based film scaffold; wherein A is a cell viability test of porcine skeletal muscle satellite cells on a polydopamine-coated agarose film scaffold; B is a cell differentiation test of porcine skeletal muscle satellite cells on a polydopamine-coated agarose film scaffold;

[0033] Figure 8 It is a mechanical test of polydopamine coated agarose-based film scaffold; wherein A is the stress-strain curve of polydopamine coated agarose-based film scaffold containing myotubes formed by fusion of porcine skeletal muscle satellite cells and myotubes not formed by fusion of porcine skeletal muscle satellite cells; B is the stress-strain curve of agarose film scaffolds with different concentrations;

[0034] Fig. 9It is a food-like processing of cell cultured meat; wherein A is unstained monolayer cell cultured meat (polydopamine-coated agarose-based film containing myotubes formed by fusion of pig skeletal muscle satellite cells); B is stained monolayer cell cultured meat (polydopamine-coated agarose-based film containing myotubes formed by fusion of pig skeletal muscle satellite cells); C is the appearance of stained monolayer cell cultured meat and real pig lean meat;

[0035] Fig.10 The present invention is a schematic diagram of the preparation method of the modified agarose and its application in cell culture meat. DETAILED DESCRIPTION

[0036] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0037] The schematic diagram of the preparation method of the modified agarose of the present invention and its application in cell culture meat is as follows Fig.10 shown.

[0038] Example 1: Preparation of agarose-based hydrogel scaffolds and agarose-based film scaffolds

[0039] Add 2g of agarose powder (AG) and 100ml of deionized water to a beaker, and stir while heating until the agarose dissolves to obtain an agarose solution with a concentration of 2wt%. Pour the agarose solution into the mold, let it stand at room temperature for 1h, then demold the prepared hydrogel and rinse it with deionized water to obtain an agarose-based hydrogel scaffold. Then place the agarose-based hydrogel scaffold in a constant temperature drying oven at 37°C for 48h to obtain a dry agarose-based film scaffold. Then immerse the agarose-based film scaffold in sterile deionized water for 2h to finally obtain an agarose-based film scaffold. The experimental results are as follows. Figure 1 shown.

[0040] Example 2: Polydopamine-coated agarose-based hydrogel scaffold

[0041] The obtained agarose-based hydrogel scaffold was immersed in 75% ethanol for 2 hours, and then repeatedly immersed in sterile deionized water for 5 minutes. The operation of washing the ethanol with sterile water was performed three times to remove all residual ethanol. Then the sterilized agarose-based hydrogel scaffold was immersed in TRIS buffer containing different concentrations of dopamine (0.05, 0.1, 0.2 mg / ml) at room temperature for 2 hours. Then it was washed with 0.01M PBS three times, each time for 5 minutes. The polydopamine (PDA)-coated agarose-based hydrogel scaffold was directly used for cell inoculation and culture. The obtained agarose-based hydrogel scaffolds with different concentrations of polydopamine coating were as follows: Figure 2 shown.

[0042] Example 3: Polydopamine coated agarose-based film scaffold

[0043] The obtained agarose-based film scaffold was immersed in 75% ethanol for 2 hours, and then repeatedly immersed in sterile deionized water for 5 minutes. The operation of washing the ethanol with sterile water was performed three times to remove all residual ethanol. Then the sterilized agarose-based film scaffold was immersed in TRIS buffer containing different concentrations of dopamine (0.05, 0.1, 0.2 mg / ml) at room temperature for 2 hours. Then it was washed with 0.01M PBS three times, each time for 5 minutes. The polydopamine-coated agarose-based film scaffold was directly used for cell inoculation and culture. The scanning electron microscopy images of the agarose-based film scaffold and the polydopamine-coated agarose-based film scaffold are shown in the following figure. Figure 3 As shown, dopamine was self-oxidized to polydopamine and adhered to the surface of the agarose-based film scaffold to form white particles, indicating that the polydopamine coating was successfully modified on the surface of the agarose-based film scaffold.

[0044] Example 4: Determination of cell adhesion rate

[0045] The porcine skeletal muscle satellite cells were inoculated on the polydopamine-coated agarose-based hydrogel scaffold for 24 h, and the non-adherent cells were washed with 0.01 M PBS, and the cell adhesion rate was calculated to determine the effect of the polydopamine-coated agarose-based hydrogel scaffold on the adhesion of porcine skeletal muscle satellite cells. The first cell adhesion rate (%) was calculated according to the following formula. The experimental results are shown in Figure 4 As shown, the cell adhesion on the agarose hydrogel scaffold is very low, while the cell adhesion rate of the polydopamine-coated agarose-based hydrogel scaffold is not significantly different from that of the traditional tissue cell culture plate, and the cell adhesion rate is nearly 100%, indicating that the polydopamine-coated agarose hydrogel can enhance the ability of the scaffold to adsorb proteins.

[0046]

[0047] Example 5: Adhesion state of cells on agarose-based hydrogel scaffolds and agarose-based film scaffolds

[0048] Porcine skeletal muscle satellite cells were inoculated on agarose-based hydrogel scaffolds and polydopamine-coated agarose-based hydrogel scaffolds for 24 h, and the non-adherent cells were washed with 0.01 M PBS. The cell adhesion state was directly observed under an optical microscope. Figure 5 As shown in A, cells adhered to the polydopamine-coated agarose-based hydrogel scaffold are stretched and have flat features.

[0049] The polydopamine-coated agarose-based film scaffold was pre-frozen at -80°C for 12 hours, then freeze-dried in a vacuum freeze dryer for 48 hours, and the freeze-dried scaffold was bonded to the conductive glue and sprayed with gold before analysis by scanning electron microscopy at 3 kV accelerating voltage. Figure 5 As shown in B, pig skeletal muscle satellite cells were observed to adhere to the polydopamine-coated agarose-based film scaffold, and the cells were stretched and had a flattened characteristic adhesion state.

[0050] Example 6: Culturing cells on polydopamine-coated agarose-based hydrogel scaffolds

[0051] Porcine skeletal muscle satellite cells were cultured at 10,000 cells / cm 2 The cells were seeded at a density of 1.50 μg / mL on a polydopamine-coated agarose-based hydrogel scaffold and cultured in a growth medium (90% DMEM (containing 1% penicillin and 1% streptomycin), 10% FBS) for 3 days. The growth medium (90% DMEM (containing 1% penicillin and 1% streptomycin), 10% FBS) was replaced every two days. During this period, a significant increase in cell activity and a large number of cells that proliferated significantly were observed ( Figure 6 ); then the cells were cultured in a culture incubator with differentiation medium (98% DMEM (containing 1% penicillin and 1% streptomycin), 2% horse serum) for 5 days, and the differentiation medium (98% DMEM (containing 1% penicillin and 1% streptomycin), 2% horse serum) was changed every other day. During this period, a large number of cells were observed to differentiate and fuse to form multinucleated myotubes ( Figure 6 ).

[0052] Example 7: Culturing cells on polydopamine-coated agarose-based film scaffolds

[0053] Porcine skeletal muscle satellite cells were cultured at 10,000 cells / cm 2 The cells were seeded at a density of 1.50 μg / mL on a polydopamine-coated agarose-based film support and cultured in growth medium (90% DMEM (containing 1% penicillin and 1% streptomycin), 10% FBS) for 5 days. The growth medium (90% DMEM (containing 1% penicillin and 1% streptomycin), 10% FBS) was replaced every two days. During this period, a significant increase in cell activity was observed, indicating that the cells had undergone a large number of proliferation ( Figure 7); then the cells were cultured in a culture incubator with differentiation medium (98% DMEM (containing 1% penicillin and 1% streptomycin), 2% horse serum) for 5 days, and the differentiation medium (98% DMEM (containing 1% penicillin and 1% streptomycin), 2% horse serum) was changed every other day. During this period, a large number of cells were observed to differentiate and fuse to form multinucleated myotubes ( Figure 7 ).

[0054] Example 8: Mechanical testing of polydopamine-coated agarose-based film scaffolds

[0055] The polydopamine-coated agarose-based film scaffold containing the myotubes formed by the fusion of porcine skeletal muscle satellite cells in Example 7 was cut into a dumbbell shape with a thickness of 0.1 mm using a standard mold. Before testing, carefully check whether the film scaffold has cracks or deformation. The tensile test was carried out on a universal material testing machine (Zwick / Roell Z020, Zwick). The mechanical sensor was calibrated to 50N and the tensile speed was 1mm / min. Each group of scaffolds was tested 3 times, and the average value was taken to eliminate the error. The study found that the polydopamine-coated agarose-based film scaffold of porcine skeletal muscle satellite cells prepared by the present invention has a certain mechanical strength, and the scaffold with the required mechanical properties can be prepared by adjusting the concentration of agarose ( Figure 8 ).

[0056] Example 9: Processing of cell cultured meat into food

[0057] The polydopamine-coated agarose-based film scaffold containing myotubes formed by fusion of pig skeletal muscle satellite cells in Example 7 was harvested, which was a single layer of cell cultured meat. After repeated washing with PBS and water and dyeing with food additives, multiple layers of cell cultured meat slices were stacked to form a cell cultured meat product. The experimental results are shown in FIG. Fig. 9 shown.

[0058] In the embodiment of the present invention, the porcine skeletal muscle satellite cells can be replaced by muscle stem cells, adipocytes, endothelial cells, fibroblasts, etc.; the culture medium of the growth medium can be replaced by F12+DMEM.

[0059] The above are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should be regarded as the protection scope of the present invention.

Claims

1. A method for preparing modified agarose for cell cultured meat, characterized in that: The following steps are involved: (1) Preparing an agarose-based hydrogel scaffold: dissolving agarose to prepare a solution, pouring the solution into a mold, and allowing the solution to solidify to obtain an agarose-based hydrogel scaffold; (2) Preparing an agarose-based film scaffold: dehydrating and drying the obtained agarose-based hydrogel scaffold to obtain an agarose-based film scaffold; (3) Dipping and coating with polydopamine coating: The two scaffolds obtained in step (1) and step (2) are immersed in a dopamine solution to obtain an agarose-based hydrogel scaffold and an agarose-based film scaffold coated with polydopamine coating, respectively, which are the modified agarose.

2. The method for preparing modified agarose for cell cultured meat according to claim 1, characterized in that: In step (1), agarose is dissolved to prepare a solution, wherein the concentration of the solution is 0.1 wt%-5 wt%; The standing condition is: standing at room temperature for 1-2 hours.

3. The method for preparing modified agarose for cell cultured meat according to claim 1, characterized in that: In step (2), the dehydration and drying conditions are: the dehydration and drying temperature is 25-37° C., and the dehydration and drying time is 48-72 hours.

4. The method for preparing modified agarose for cell cultured meat according to claim 1, characterized in that: In step (3), the concentration of dopamine is 0.05-0.2 mg / ml.

5. The method for preparing modified agarose for cell cultured meat according to claim 1, characterized in that: In step (3), when the polydopamine coating is applied by immersion, the two scaffolds obtained in step (1) and step (2) are first immersed in PBS for 0.5-1 h, and then immersed in a dopamine solution for 1-2 h.

6. The modified agarose prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the modified agarose according to claim 6 in preparing a cell culture scaffold for cell cultured meat.

8. The use according to claim 7, characterized in that: The cells are skeletal muscle satellite cells, muscle stem cells, adipocytes, endothelial cells or fibroblasts.

9. Use of the modified agarose according to claim 6 as a cell culture scaffold in cell cultured meat.

10. The use according to claim 9, characterized in that: The cells in the cell-cultured meat are skeletal muscle satellite cells, muscle stem cells, adipocytes, endothelial cells or fibroblasts.

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