CN113215090A - Manufacturing method of edible chitosan/sodium alginate/gelatin 3D scaffold for cell culture meat - Google Patents
Manufacturing method of edible chitosan/sodium alginate/gelatin 3D scaffold for cell culture meat Download PDFInfo
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Abstract
The invention relates to a method for manufacturing an edible chitosan/sodium alginate/gelatin 3D scaffold for cell culture meat, and the chitosan/sodium alginate/gelatin 3D scaffold manufactured by the method can be used for culturing skeletal muscle cells and provides a support for manufacturing cell culture meat, belonging to the technical field of future food. The method comprises the steps of uniformly mixing chitosan, sodium alginate, gelatin and collagen with certain concentration according to a certain proportion, reacting for more than 2 hours at room temperature, and freeze-drying to obtain the chitosan/sodium alginate/gelatin 3D scaffold. The preparation method is simple and efficient, the 3D scaffold is formed through electrostatic interaction among chitosan, gelatin and sodium alginate under the condition of not using toxic cross-linking agents, and the gelatin is used for partially replacing collagen to be used as a substance for promoting cell adhesion, so that the obtained chitosan/sodium alginate/gelatin 3D scaffold has good cell adhesion and compatibility, and can be used for skeletal muscle cell culture. The invention provides a manufacturing method of a novel and nontoxic 3D bracket for research of cell culture meat.
Description
Technical Field
The invention relates to the technical field of future food, in particular to a manufacturing method of an edible chitosan/sodium alginate/gelatin 3D bracket for cell culture meat.
Background
Meat is a precious source of animal protein, however, traditional meat production capacity is now approaching a maximum level, almost two thirds of agricultural land is used for livestock, and the remaining third is almost unsatisfied with human phytofood needs. Meat production is one of the major factors that contribute to the degradation of the global environment. Currently, livestock raised for meat production consumes 30% of the global ice-free land and 8% of the global fresh water, while producing 18% of the global total greenhouse gas (GHG) emissions. Animal husbandry is also one of the major factors responsible for the deforestation and the deterioration of wildlife habitats. Worldwide, 34% of the greenhouse gas emissions associated with animal husbandry are due to deforestation, of which 25% are methane emissions produced by intestinal fermentation of ruminants. The Food and Agriculture Organization (FAO) projected that by 2050 the demand for meat would be as high as 70%, and therefore, a more efficient way of meat production would be desirable to mitigate the environmental impact of traditional ways of meat production.
The negative impact of traditional meat production on the environment can be effectively alleviated by culturing animal muscle tissue in vitro by using a tissue engineering technique, which is called a cell culture meat technique. In addition to being environmentally friendly, cell culture meat has other benefits. The cell culture meat can reduce human and animal contact, thereby preventing animal disease transmission and epidemic zoonosis transmission. In addition, the cell culture meat can control nutrition, texture, taste and the like of meat products, thereby reducing nutrition-related diseases such as cardiovascular diseases and the like.
Extracellular matrix (ECM) is a complex network composed of macromolecules, provides a suitable place for survival and activity of cells, influences shape, metabolism, function, migration, proliferation and differentiation of cells through a signal transduction system, and can provide attachment sites for cells in 3D culture of the cells to promote cell proliferation. However, ECM with good biological activity usually needs to be obtained from animal tissue, and the extraction procedure is complicated and expensive. Therefore, there is a need for a substance that can replace ECM in whole or in part, and a 3D scaffold with appropriate pore size and good cell compatibility, so that cells can be three-dimensionally distributed on the scaffold and proliferate and differentiate in large quantities to finally form intact muscle tissue.
Gelatin is a hydrolysate of collagen, is a natural nutritional food thickener, contains 18 amino acids, has effect in promoting cell growth and proliferation, and has simple extraction process and low cost compared with collagen. The chitosan is a product of natural polysaccharide chitin with deacetylation, is a cationic polysaccharide, is widely applied to various fields of food additives, antibacterial agents, tissue engineering carrier materials and the like, and has excellent performances such as biocompatibility, antibacterial property, biodegradability and the like. Sodium alginate is a byproduct after extracting iodine and mannitol from brown algae such as kelp or gulfweed, is an anionic polysaccharide, and is commonly used as a thickener, a stabilizer and the like in food. In order to maintain the stability of the scaffold, most scaffolds prepared by using chitosan or sodium alginate usually need to use cross-linking agents, such as glutaraldehyde, EDC/NHS, calcium chloride, etc., and most of these cross-linking agents have certain toxicity and are not suitable for preparing edible 3D scaffolds.
To overcome these limitations and to produce edible 3D scaffolds suitable for cell culture meat, we invented a method of manufacturing edible chitosan/sodium alginate/gelatin 3D scaffolds for cell culture meat: uniformly mixing chitosan, gelatin and collagen solution according to a certain mass ratio, dropwise adding sodium alginate solution, stirring and reacting at room temperature for more than 2 hours, and freeze-drying to obtain the 3D scaffold. The preparation method is simple and efficient, no toxic cross-linking agent is used, the cost is reduced to a certain extent, the hydrogel is formed through the electrostatic interaction among the chitosan, the gelatin and the sodium alginate, and the obtained chitosan/sodium alginate/gelatin 3D scaffold has good cell adhesion and compatibility, can be used for skeletal muscle cell culture and is used for producing cell culture meat.
Disclosure of Invention
The invention aims to provide a method for manufacturing an edible chitosan/sodium alginate/gelatin 3D scaffold for cell culture meat, aiming at the defects of high research and development cost, incapability of massively proliferating cells and the like of the existing cell culture meat.
In order to achieve the purpose, the experiment discloses a manufacturing method of an edible chitosan/sodium alginate/gelatin 3D scaffold, which utilizes the electrostatic interaction among chitosan, gelatin and sodium alginate to form hydrogel, and the hydrogel is frozen and dried to obtain the 3D scaffold. Wherein the concentration of the chitosan is 0.5-3%, the concentration of the gelatin is 0.5-5%, the concentration of the collagen is 0.5-5%, and the chitosan, the gelatin and the collagen are dissolved in 0.5-3% acetic acid solution; the concentration of the sodium alginate is 0.5-3%.
The mass ratio of the gelatin solution to the collagen solution is 1: 5-5: 1; the mass ratio of the chitosan solution to the mixed solution of gelatin and collagen is 3: 1-1: 1; the mass ratio of the sodium alginate solution to the mixed solution of chitosan, gelatin and collagen is 1: 3-3: 1. And stirring the mixed solution at room temperature for reaction for more than 2 hours, and freeze-drying to obtain the 3D gel scaffold.
The invention has the advantages that:
1. the invention selects chitosan, sodium alginate, gelatin and collagen as raw materials, and the four raw materials are safe and nontoxic, are widely applied to food additives and are commercialized. Therefore, the edible 3D gel scaffold prepared from the raw materials has important value for the application in the fields of cell culture meat and tissue engineering;
2. the 3D scaffold prepared by the method partially replaces collagen with gelatin, has good cell adhesion and the function of promoting cell growth, and greatly reduces the cost of cell culture meat;
3. compared with other crosslinking modes, the invention can be prepared by utilizing the electrostatic interaction among chitosan, gelatin and sodium alginate under the condition of stirring at room temperature, has no by-product and does not need to add a crosslinking agent with toxicity, and the gelling condition is simple, green and nontoxic.
Drawings
FIG. 1 is a photograph of a chitosan/sodium alginate/gelatin 3D scaffold;
fig. 2 is a graph of the porosity of a chitosan/sodium alginate/gelatin 3D scaffold, wherein A, B, C, D represents gelatin and collagen in different ratios, respectively;
FIG. 3 is a staining pattern of viable/dead cells of a chitosan/sodium alginate/gelatin 3D scaffold cultured in myoblasts of C2C12 mice after 1, 3 and 7 days, wherein A, B represents gelatin and collagen in different proportions;
FIG. 4 is a staining pattern of cell nuclei of C2C12 cells cultured on chitosan/sodium alginate/gelatin 3D scaffolds for 1D;
FIG. 5 is a 3D staining map of cell nuclei of C2C12 cells cultured on chitosan/sodium alginate/gelatin 3D scaffolds for 1D;
FIG. 6 is a staining pattern of viable/dead cells of CHGSC pig muscle cells cultured by chitosan/sodium alginate/gelatin 3D scaffold after 1, 3 and 7 days, wherein A, B represents gelatin and collagen in different proportions;
FIG. 7 is a staining pattern of cell nuclei of CHGSC cells cultured on chitosan/sodium alginate/gelatin 3D scaffolds for 1D;
FIG. 8 is the 3D staining pattern of cell nucleus after chitosan/sodium alginate/gelatin 3D scaffold culture of CHGSC cells for 1D.
Detailed Description
The present invention will be described in further detail with reference to the following drawings and examples.
1. Manufacturing a chitosan/sodium alginate/gelatin 3D scaffold:
(1) dissolving chitosan, gelatin and collagen in 0.5-3% acetic acid solution to obtain 0.5-3%, 0.5-5% and 0.5-5% chitosan solution, gelatin solution and collagen solution respectively; dissolving sodium alginate in water to obtain a sodium alginate solution with the concentration of 0.5-3%;
(2) mixing gelatin and collagen solution in a mass ratio of 1: 5-5: 1, and mixing chitosan solution and the mixed solution of gelatin and collagen in a mass ratio of 3: 1-1: 1;
(3) mixing a sodium alginate solution and a mixed solution of chitosan, gelatin and collagen in a mass ratio of 1: 3-3: 1;
(4) and stirring the mixed solution at room temperature for more than 2 hours, and freeze-drying to obtain the 3D scaffold.
2. Skeletal muscle cell culture was performed on the fabricated chitosan/sodium alginate/gelatin 3D scaffolds:
(1) C2C12 mouse myoblast cell recovery centrifugation and heavy suspension in DMEM/F-12 medium containing 10% FBS and 1% penicillin/streptomycin solution at 37 ℃ in 5% CO2Culturing in an incubator until the cell confluence reaches about 80%, and obtaining cell suspension by trypsinization and centrifugation;
(2) washing the chitosan/sodium alginate/gelatin 3D scaffold with 1% penicillin/streptomycin-containing PBS solution for three times, adding the culture medium, soaking for 30min, removing the culture medium, inoculating cells, supplementing the culture medium after the cells adhere to the wall, and culturing at 37 ℃ and 5% CO2Culturing in an incubator;
(3) respectively staining the live cells/dead cells of the scaffold after culturing for 1, 3 and 7 days, and observing by using a fluorescence microscope, wherein green is live cells and red is dead cells;
(4) after 1d of incubation, the scaffolds were stained for nuclei and observed using a confocal laser microscope, blue as nuclei.
3. The porcine myocyte culture was performed on the manufactured chitosan/sodium alginate/gelatin 3D scaffolds:
(1) CHGSC porcine myocytes were resuscitated, centrifuged and resuspended in DMEM/F-12 medium containing 10% FBS and 1% penicillin/streptomycin solution at 37 ℃ in 5% CO2Culturing in an incubator until the cell confluence reaches about 80%, and obtaining cell suspension by trypsinization and centrifugation;
(2) washing the chitosan/sodium alginate/gelatin 3D scaffold with 1% penicillin/streptomycin-containing PBS solution for three times, adding the culture medium, soaking for 30min, removing the culture medium, inoculating cells, supplementing the culture medium after the cells adhere to the wall, and culturing at 37 ℃ and 5% CO2Culturing in an incubator;
(3) respectively staining the live cells/dead cells of the scaffold after culturing for 1, 3 and 7 days, and observing by using a fluorescence microscope, wherein green is live cells and red is dead cells;
(4) after 1d of incubation, the scaffolds were stained for nuclei and observed using a confocal laser microscope, blue as nuclei.
Claims (5)
1. A method of manufacturing an edible chitosan/sodium alginate/gelatin 3D scaffold for cell culture meat, comprising the steps of:
(1) dissolving chitosan, gelatin and collagen in an acetic acid solution at a certain concentration, dissolving sodium alginate in water at a certain concentration, and forming hydrogel through electrostatic interaction;
(2) uniformly mixing a gelatin solution and a collagen solution according to a certain mass ratio;
(3) uniformly mixing a chitosan solution and a mixed solution of gelatin and collagen according to a certain mass ratio;
(3) dropwise adding a sodium alginate solution into a mixed solution consisting of chitosan, gelatin and collagen under the condition of strong stirring;
(4) stirring the mixed solution at room temperature to react for a certain time;
(5) the mixed solution after the reaction was freeze-dried.
2. The method according to claim 1, wherein the chitosan concentration is 0.5-3%, the gelatin concentration is 0.5-5%, the collagen concentration is 0.5-5%, and the chitosan and the gelatin are dissolved in 0.5-3% acetic acid solution; the concentration of the sodium alginate is 0.5-3%.
3. The method according to claim 1, wherein the mass ratio of the gelatin solution to the collagen solution is 1: 5-5: 1; the mass ratio of the chitosan solution to the mixed solution of gelatin and collagen is 3: 1-1: 1; the mass ratio of the sodium alginate solution to the mixed solution of chitosan, gelatin and collagen is 1: 3-3: 1.
4. The method according to claim 1, wherein the mixed solution is stirred at room temperature for reaction for 2 hours or more.
5. The method of claim 1, wherein the chitosan, sodium alginate, gelatin and collagen are all food grade materials and have been commercially produced; the manufacturing method of the edible 3D bracket is simple, green and efficient, any toxic cross-linking agent is not required to be added, the gelatin is used for replacing collagen to serve as an extracellular matrix, the cost can be reduced, and the edible 3D bracket has good cell adhesion and compatibility and is suitable for the field of tissue engineering such as cell culture meat.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113215089A (en) * | 2020-10-11 | 2021-08-06 | 西北农林科技大学 | Manufacturing method of edible chitosan 3D gel scaffold for cell culture meat |
| CN113892643A (en) * | 2021-09-23 | 2022-01-07 | 西北农林科技大学 | Manufacturing method of edible sodium alginate/gelatin 3D gel scaffold adopting tea polyphenol coating and used for cell culture meat |
| CN114481363A (en) * | 2022-02-28 | 2022-05-13 | 上海食未生物科技有限公司 | Method for producing cell culture meat by using mushroom chitosan fiber scaffold |
| CN114645012A (en) * | 2022-04-18 | 2022-06-21 | 南京农业大学 | Production method of cell culture meat based on food-grade oriented scaffold material |
| CN114874978A (en) * | 2022-05-07 | 2022-08-09 | 南京周子未来食品科技有限公司 | Personalized customization of cell culture meat based on porous scaffold material and production method thereof |
| CN115418023A (en) * | 2022-09-15 | 2022-12-02 | 西北农林科技大学 | Preparation method of edible cell culture meat 3D support through ion crosslinking |
| CN115491347A (en) * | 2022-09-15 | 2022-12-20 | 江南大学 | Preparation method of blocky cell culture meat |
| WO2023225995A1 (en) * | 2022-05-27 | 2023-11-30 | 汕头得宝投资有限公司 | Use of sodium alginate-gelatin 3d scaffold in supporting differentiation of preadipocytes |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113215089A (en) * | 2020-10-11 | 2021-08-06 | 西北农林科技大学 | Manufacturing method of edible chitosan 3D gel scaffold for cell culture meat |
| CN113892643A (en) * | 2021-09-23 | 2022-01-07 | 西北农林科技大学 | Manufacturing method of edible sodium alginate/gelatin 3D gel scaffold adopting tea polyphenol coating and used for cell culture meat |
| CN114481363A (en) * | 2022-02-28 | 2022-05-13 | 上海食未生物科技有限公司 | Method for producing cell culture meat by using mushroom chitosan fiber scaffold |
| CN114645012A (en) * | 2022-04-18 | 2022-06-21 | 南京农业大学 | Production method of cell culture meat based on food-grade oriented scaffold material |
| CN114874978A (en) * | 2022-05-07 | 2022-08-09 | 南京周子未来食品科技有限公司 | Personalized customization of cell culture meat based on porous scaffold material and production method thereof |
| WO2023225995A1 (en) * | 2022-05-27 | 2023-11-30 | 汕头得宝投资有限公司 | Use of sodium alginate-gelatin 3d scaffold in supporting differentiation of preadipocytes |
| CN115418023A (en) * | 2022-09-15 | 2022-12-02 | 西北农林科技大学 | Preparation method of edible cell culture meat 3D support through ion crosslinking |
| CN115491347A (en) * | 2022-09-15 | 2022-12-20 | 江南大学 | Preparation method of blocky cell culture meat |
| CN115418023B (en) * | 2022-09-15 | 2023-08-29 | 西北农林科技大学 | Preparation method of edible cell culture meat 3D bracket by utilizing ionic crosslinking |
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