CN113215089A

CN113215089A - Manufacturing method of edible chitosan 3D gel scaffold for cell culture meat

Info

Publication number: CN113215089A
Application number: CN202011080859.XA
Authority: CN
Inventors: 冯宪超; 周光宏; 陈琳; 陈燕; 李鳞子; 刘亚平; 陈晓红
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2020-10-11
Filing date: 2020-10-11
Publication date: 2021-08-06

Abstract

The invention relates to a method for manufacturing an edible chitosan 3D gel scaffold for cell culture meat, and the chitosan 3D gel scaffold manufactured by the method can be used for culturing skeletal muscle cells and providing support for manufacturing cell culture meat, belonging to the technical field of future food. Uniformly mixing chitosan with a certain concentration and sodium tripolyphosphate serving as a cross-linking agent according to a certain proportion, freezing at-1 to-80 ℃ to form hydrogel, and freeze-drying to obtain the chitosan 3D gel scaffold. The preparation method is simple and efficient, under the condition of not using toxic cross-linking agents, the chitosan 3D gel scaffold forms hydrogel through the electrostatic interaction between the chitosan and the sodium tripolyphosphate, other substances for promoting cell adhesion do not need to be added, and the obtained chitosan 3D gel scaffold has good cell adhesion and compatibility and can be used for skeletal muscle cell culture. The invention provides a novel, convenient and nontoxic manufacturing method of a 3D gel scaffold for research of cell culture meat.

Description

Manufacturing method of edible chitosan 3D gel scaffold for cell culture meat

Technical Field

The invention relates to the technical field of future food, in particular to a manufacturing method of an edible chitosan 3D gel scaffold for cell culture meat.

Background

As the world population and meat consumption increased, the Food and Agriculture Organization (FAO) projected that by 2050, the demand for meat would be as high as 70%, while traditional meat production capacity is approaching maximum levels and, ultimately, meat would become scarce and therefore more expensive, a luxury product and may exacerbate the already inequitous global food distribution. In the traditional meat production, only 5-25% of animals are processed into edible meat, so that the conversion rate of the traditional meat production is low, the traditional meat production usually needs a more intensive and more environmentally-friendly production mode, and the influence of the breeding industry on the environment can reach and exceed the level of the global stable recommended upper limit in 2050. In addition to this, a large part of global greenhouse gas emissions, land use, water and energy consumption derive from the production of traditional meat.

The cell culture meat technology is to produce meat and meat products by culturing animal muscle tissue in vitro by using tissue engineering technology. Therefore, the cell culture meat technology can effectively improve the production efficiency of meat, greatly reduce the land and energy consumption and the emission of greenhouse gases, and effectively relieve the problems of grains and environment caused by the breeding industry.

The traditional cell culture is generally carried out on a two-dimensional plane of a culture dish, and when the cells proliferate to a certain density, the growth is inhibited, so that the cells cannot proliferate and differentiate into muscle tissues in a large quantity. Extracellular matrix (ECM) is a structural network composed of various macromolecules in natural tissues, has functions of supporting cell anchoring and tissue assembly, and in novel 3D cell culture, a certain amount of ECM, such as collagen, RGD peptide, etc., is usually added to a 3D gel scaffold to ensure good cell adhesion. 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 3D gel scaffold with appropriate pore size, good cell adhesion and compatibility, which enables three-dimensional distribution of cells on the scaffold, and massive proliferation and differentiation to finally form an intact muscle tissue.

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 tripolyphosphate is a polyphosphate with chemical formula of Na₅P₃O₁₀It is commonly used as a moisture retaining agent, a quality improving agent, etc. in foods. Most scaffolds prepared using chitosan generally require the use of cross-linking agents such as glutaraldehyde, EDC/NHS, isocyanate, etc. to maintain the stability of the scaffold, and most of these cross-linking agents are toxic and not suitable for preparing edible 3D gel scaffolds.

To overcome these limitations and to produce edible 3D gel scaffolds suitable for cell culture meat, we invented a method of producing edible chitosan 3D gel scaffolds for cell culture meat: uniformly mixing chitosan and sodium tripolyphosphate solution according to a certain mass ratio, freezing at-1 to-80 ℃ to form hydrogel, and freeze-drying to obtain the 3D gel stent. The preparation method is simple and efficient, the cost is low, no toxic cross-linking agent is used, hydrogel is formed through the electrostatic interaction between the chitosan and the sodium tripolyphosphate, ECM does not need to be added, and the obtained chitosan 3D gel 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 3D gel scaffold for cell culture meat, aiming at the defects of high development cost, incapability of proliferating cells in a large amount and the like of the existing cell culture meat.

In order to achieve the above purpose, the present experiment discloses a method for manufacturing an edible chitosan 3D gel scaffold, which utilizes the electrostatic interaction between chitosan and sodium tripolyphosphate to form hydrogel, and the hydrogel is freeze-dried to obtain the 3D scaffold. Wherein the chitosan with the concentration of 0.5-3% is dissolved in 0.5-3% acetic acid solution; the concentration of the sodium tripolyphosphate is 0-80 mmol/L; the mass ratio of the chitosan solution to the sodium tripolyphosphate solution is 2: 1-10: 1.

The chitosan/sodium tripolyphosphate mixed solution is treated by a disperser at the rotating speed of 20000-30000 rpm and then treated by a cell crusher at the condition of 200-500 w for 1-20 min. And freezing the mixed solution at-1 to-80 ℃ for more than 2h to form hydrogel, and then freezing and drying to obtain the 3D gel stent.

The invention has the advantages that:

1. the invention selects chitosan and sodium tripolyphosphate as raw materials, both of which are safe and nontoxic, and can be widely applied to food additives, and the invention has the advantages of simple preparation and low cost, and has already realized commercialization. Therefore, the two raw materials are selected to manufacture the edible 3D gel scaffold, and the edible 3D gel scaffold has important value for the application in the fields of cell culture meat and tissue engineering;

2. ECM, such as collagen, RGD peptide, etc., is required to be added to a 3D scaffold generally used for cell culture. The 3D scaffold manufactured by the invention does not need to be added with ECM, still has good cell adhesion, and greatly reduces the cost of cell culture meat;

3. compared with other crosslinking modes, the method utilizes the electrostatic interaction between the chitosan and the sodium tripolyphosphate to form the hydrogel under the freezing condition, has no by-product and no toxic crosslinking agent, and has simple, green and nontoxic gelling condition.

Drawings

FIG. 1 is a photograph of a chitosan 3D gel scaffold;

FIG. 2 is a 50 SEM photograph of a chitosan 3D gel scaffold;

FIG. 3 is a 400 SEM photograph of a chitosan 3D gel scaffold;

FIG. 4 is a graph of gel strength of chitosan hydrogel, wherein A, B, C, D represents different concentrations of sodium tripolyphosphate;

FIG. 5 is a staining pattern of cell nuclei of C2C12 cells cultured on chitosan 3D gel scaffolds for 12D;

FIG. 6 is a 3D staining pattern of cell nuclei of C2C12 cells cultured by chitosan 3D gel scaffolds for 12D.

Detailed Description

The present invention will be described in further detail with reference to the following drawings and examples.

1. Manufacturing of chitosan 3D gel scaffold:

(1) dissolving chitosan in 0.5-3% acetic acid solution to obtain 0.5-3% chitosan solution; dissolving sodium tripolyphosphate in water to obtain a sodium tripolyphosphate solution with the concentration of 0-80 mmol/L;

(2) mixing chitosan and sodium tripolyphosphate solution according to the mass ratio of 2: 1-10: 1, treating the mixture by using a disperser at the rotating speed of 20000-30000 rpm, and then treating the mixture for 1-20 min by using a cell crusher under the condition of 200-500 w;

(3) and (3) freezing the mixed solution at-1 to-80 ℃ for more than 2h to form hydrogel, and then freezing and drying to obtain the 3D gel stent.

2. Skeletal muscle cell culture was performed on the prepared chitosan 3D gel 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 3D gel 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) after 12 days of culture, the scaffolds were stained for nuclei and observed using a confocal laser microscope, blue as nuclei.

Claims

1. A method of manufacturing an edible chitosan 3D gel scaffold for cell culture meat, comprising the steps of:

(1) dissolving chitosan in acetic acid solution at a certain concentration, and forming hydrogel through electrostatic interaction;

(2) uniformly mixing chitosan and a cross-linking agent sodium tripolyphosphate solution according to a certain mass ratio;

(3) freezing the mixed solution consisting of chitosan and sodium tripolyphosphate at-1 to-80 ℃ for more than 2h to form hydrogel;

(4) the hydrogel obtained after freezing was freeze-dried.

2. The method according to claim 1, wherein the chitosan is dissolved in 0.5-3% acetic acid solution at a concentration of 0.5-3%; the concentration of the sodium tripolyphosphate is 0-80 mmol/L; the mass ratio of the chitosan solution to the sodium tripolyphosphate solution is 2: 1-10: 1.

3. The method according to claim 1, wherein the chitosan/sodium tripolyphosphate mixed solution is treated with a disperser at 20000-30000 rpm and then treated with a cell disruptor at 200-500 w for 1-20 min.

4. The method according to claim 1, wherein the chitosan and the sodium tripolyphosphate are both food-grade materials and are widely applied to food additives; the manufacturing method of the edible 3D gel scaffold is simple, green and efficient, does not need to add any toxic cross-linking agent or extracellular matrix, has good cell adhesion and compatibility, and is suitable for the field of tissue engineering such as cell culture meat.