CN114317394A - Microcarrier for three-dimensional cell culture and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biological materials and future foods, and discloses an edible soluble microcarrier for three-dimensional cell culture, and a preparation method and application thereof. The method comprises the following steps: directly mixing the edible plant-derived ionic crosslinked gel-type polysaccharide and the edible non-animal-derived protein to prepare a mixed solution, then preparing the mixed solution into liquid drops, and carrying out crosslinking reaction to obtain the microcarrier. The invention also discloses a method for carrying out surface modification on the microcarrier to obtain the modified microcarrier. The microcarrier provided by the invention is edible, soluble, good in biocompatibility and cell adhesion, can support cell adhesion, growth and proliferation, can be used for rapidly producing cells on a large scale, reduces the cost of cell culture meat products, is especially used in the field of cell culture meat, and has a wide application prospect.

Description

A kind of microcarrier for cell three-dimensional culture and its preparation method and application

technical field

The invention relates to a microcarrier for three-dimensional cell culture, a preparation method and application thereof, in particular to an edible and dissolvable microcarrier for cell three-dimensional culture, a preparation method and application thereof, and in particular The application of the micro-agglomerate tissue formed by spontaneous agglomeration belongs to the field of biological materials and future food technology.

Background technique

Traditional animal husbandry produces a large amount of greenhouse gases and pollutes soil and water resources. In addition, the production and consumption of animal meat products may lead to the transmission of pathogens and endanger public health. Compared with traditional cultured meat, cell cultured meat is a new biotechnology based on the principle of tissue engineering, which simulates the process of cell growth and differentiation in a clean environment in vitro, and cultivates animal muscle tissue for the production of meat products. At the same time, it can greatly reduce the consumption of land, water resources and greenhouse gas emissions. In addition, cell-cultured meat can provide a nutritional structure and eating experience similar to animal meat, so it can effectively alleviate the environmental and food safety problems caused by traditional aquaculture without requiring consumers to change their inherent eating habits.

One of the difficulties in the cell culture meat industry is to reduce costs and achieve large-scale production. The number of cells contained in 10-100 kg of animal meat is 10 ¹² to 10 ¹³ , while the traditional two-dimensional cell culture method is difficult to produce enough cells for meat product production in a short time, and the time, space and labor The high cost has become one of the important factors limiting the price of cell cultured meat products.

In addition, in order to promote cell growth and simulate the taste of animal meat, the production process of cell-cultured meat often requires the introduction of bioscaffolds. However, in order to obtain cell-cultured meat products with high cell content and taste close to real animal meat, a high cell seeding density is usually required, and there are problems such as uneven seeding and easily hindered nutrient transport inside the scaffold, resulting in the use of scaffolds. Volume is limited.

Due to its large specific surface area, microcarriers can provide much higher surface area than two-dimensional culture systems for cell adhesion and growth under the same volume conditions. For example, during the culture process, by adding microcarriers to the culture system, the cells can spontaneously migrate from the existing microcarriers in the culture system to the newly added microcarriers, and the rapid proliferation of cells can be achieved with simple operations. It has great potential in large-scale cell culture.

At present, the common commercial microcarriers can be divided into cross-linked dextran microcarriers, cellulose microcarriers, protein microcarriers, and microcarriers based on synthetic polymers according to the raw materials. Among them, cross-linked dextran microcarriers are represented by Cytodex series, which are surface-modified with denatured collagen or diethylaminoethyl (DEAE). The whole is positively charged, which is conducive to cell adhesion. vector. Cellulose microcarriers, represented by Cytopore, are positively charged by DEAE modification and have a porous structure, which can protect cells from shear stress in addition to providing a surface area for cell adhesion. Most protein microcarriers use gelatin or denatured collagen as the matrix. For example, the Tantti 3D cell culture scaffold uses natural collagen as the raw material and has a three-dimensional macroporous structure, which can be used for in vitro cell amplification. In addition to natural macromolecules, artificial synthetic materials, such as microcarriers prepared from glass and polystyrene, have high repeatability and specificity. However, their applications are limited due to the lack of sites for cell recognition and adhesion on their surfaces. . However, the existing commercial microcarriers are mostly used in the field of medicine and cannot meet the relevant regulations and requirements of the food industry.

Microcarriers that can be used in the food field can be roughly divided into three categories: 1) Inedible and insoluble microcarriers, which can only be used for cell expansion in the early stage of production, and the amplified cells need to be digested and combined with the microcarriers. Complete separation, but easy to cause cell loss in the process of digestion, low harvest efficiency, and microcarrier fragments may remain in the cell pellet and enter the end product; 2) Inedible and soluble microcarriers, because they are inedible, cannot be When added to the end product, the microcarriers still need to be degraded or dissolved to harvest cells. The processing steps are cumbersome, and the degradation or dissolution of the microcarriers needs to be precisely controlled and harmful substances are easily generated, thereby causing damage to cells; 3) Edible microcarriers , can be directly added to the end product; and because the microcarrier directly enters the end product, it is easy to affect the taste, taste, color, etc. of the meat product. Therefore, the microcarrier used for cell culture needs to support the rapid growth and proliferation of cells. In addition to the properties of food, it also needs to have a beneficial contribution to food flavor and so on. However, the existing microcarriers cannot simultaneously have the advantages of being edible, soluble and high in cell adhesion, and cannot meet the relevant requirements of the food industry. Therefore, for example, in the field of cell cultured meat, there is an urgent need to develop microcarriers that can be used in cell culture and have the characteristics of being edible, soluble, and having high adhesion to cells.

SUMMARY OF THE INVENTION

In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide an edible and dissolvable microcarrier for three-dimensional cell culture and its preparation method and application, which can be used for the rapid and mass production of cells to solve the problem of Various problems in the prior art, such as poor cell adhesion of the microcarriers, or the need to separate the microcarriers because the microcarriers are not edible, lead to cumbersome processing steps or incomplete separation, etc. production and cost reduction.

In order to achieve the above object and other related objects, the present invention provides a microcarrier for three-dimensional cell culture, the microcarrier is a microcarrier comprising edible plant-derived ionomer gel-type polysaccharide and edible plant-derived microcarriers. Proteins are directly mixed to make a mixed solution to generate droplets, which are then obtained after cross-linking and/or immobilization reactions.

Wherein, the edible plant-derived ion-crosslinked gelling polysaccharide is selected from one or more of sodium alginate, gellan gum, carrageenan, pectin, and polymannuronic acid. The pectin may be low ester pectin.

Wherein, the edible non-animal-derived protein is selected from one or more of vegetable protein and microbial protein.

The vegetable protein is selected from the genus Soybean, Pea, Zea, Solanum, Oryza, Arachis, Avena, Rosaceae, Cowpea, Chickpea, Mint, Chenopodium, Vetch Genus, Lentil, Cannabis, Jugland, Corylus, Sesame, Cashew; including but not limited to soy protein isolate, glutenin, pea protein, zein, potato protein, rice protein, peanut protein, oat protein, almond protein, mung bean protein, chickpea protein, chia seed protein, quinoa protein, broad bean protein, lentil protein, edamame protein, hemp protein, walnut protein, hazelnut protein, sesame protein, chia seed protein, One or more of cashew protein, etc.

The microbial protein includes, but is not limited to, one or more of yeast protein, mushroom protein, fungal protein, algal protein, and the like.

Wherein, the mass ratio of the edible plant-derived ion-crosslinked gelling polysaccharide to the edible plant-derived protein is 1-50:1-10.

Another aspect of the present invention provides a method for preparing a microcarrier for three-dimensional cell culture, the method comprising the following steps:

(1) preparing a mixed solution containing ion cross-linked gelatinous polysaccharide and protein;

(2) The droplet generation method is used to generate protein-polysaccharide composite droplets;

(3) collecting the droplets into a cross-linking agent and/or a fixative, and performing a cross-linking reaction and/or a fixation reaction to obtain the microcarrier for three-dimensional cell culture.

The method of the present invention further comprises the step of modifying the surface of the microcarrier with a surface modifier selected from the group consisting of carbohydrates, proteins, polypeptides, polysaccharides, synthetic polymer compounds, and extracellular matrix. One or more of the points.

Another aspect of the present invention is to provide a microcarrier for three-dimensional cell culture prepared by the above method.

Another aspect of the present invention is to provide an application of the above-mentioned microcarrier for three-dimensional cell culture in cultured cells, cultured cell-microcarrier aggregates, and cultured meat.

Another aspect of the present invention is to provide a method for culturing cells using the above-mentioned microcarriers for three-dimensional cell culture, the method comprising:

(1) the microcarrier inoculated cells are cultured for a certain period of time;

(2) harvesting cells;

Or, the cell-seeded microcarriers spontaneously agglomerate to form cell-microcarrier clumps, which are harvested to obtain cell-microcarrier clumps;

Or, the cell-seeded microcarriers spontaneously agglomerate to form cell-microcarrier clumps, and the cell-microcarrier clumps are bonded and extruded to obtain cell cultured meat products.

Another aspect of the present invention is to provide a product obtained by inoculating cells on the above-mentioned microcarriers for three-dimensional cell culture.

Compared with the prior art, the beneficial effects of the present invention include:

1. The microcarriers constructed in the present invention for three-dimensional cell culture are edible and soluble microcarriers. The invention uses polysaccharides and proteins that can be ionically cross-linked into gels to prepare microcarriers, and has the advantages of mild conditions and reversible reaction. The microcarriers prepared by the method of the invention can be completely dissolved by EDTA or enzyme-EDTA and cells can be harvested. Avoid incomplete cell digestion, difficult to separate cells, or easy to cause cell damage during digestion, resulting in microcarrier fragments that are difficult to separate from cells and other problems, which can improve the efficiency of cell harvesting.

2. In the preparation method of the microcarrier provided by the present invention, the polysaccharide and the protein are directly mixed to make a mixed solution, and then droplets are generated for cross-linking/fixation reaction to obtain the microcarrier, and the microcarrier with controllable performance can be prepared by a one-step method. carrier, reducing process steps and production costs.

3. The preparation method of the microcarrier provided by the present invention can adapt to a variety of large-scale production processes, continuously produce microcarriers with uniform size, good biocompatibility and cell adhesion, and can support cell adhesion, growth and proliferation. , which can be used for rapid and large-scale production of cells, reducing the cost of cell cultured meat products.

4. The present invention utilizes the interaction between cells to spontaneously agglomerate the microcarriers inoculated with cells to form cell-microcarrier clumps, and can directly obtain cell cultured meat products through bonding and extrusion molding, avoiding the use of biological scaffolds for cell seeding density. Problems such as high demand, uneven inoculation, and hindered internal nutrient transport simplify the operation process, which is conducive to cost reduction and large-scale production.

5. The raw materials used in the present invention are all commercial food raw materials, which are suitable for the production of large-scale production of cell cultured meat.

Description of drawings

Fig. 1 is a flow chart of the preparation method of edible and dissolvable microcarriers for cell culture and micro-agglomerate tissue formed by spontaneous agglomeration.

FIG. 2 is an optical microscope picture of the microcarrier obtained in Example 1. FIG.

FIG. 3 is an optical microscope picture of the microcarrier obtained in Example 3. FIG.

4 is a stained picture of living cells that adhere and grow on the surface of the microcarriers (prepared in Example 1) in Example 7, and the sampling times are the 1st, 3rd, 5th, and 7th days after inoculation (in each panel, Scale bar is 200 μm).

Figure 5 is a stained picture of living cells adhering and growing on the surface of the microcarriers (prepared in Example 2) in Example 8. The sampling times were on the 1st, 3rd, 5th, and 7th days after inoculation, respectively, and the observation began on the 5th day. Cells grow qualitatively and orderly on the surface of the microcarriers (each panel, the scale bar is 200 μm).

Figure 6 is a stained picture of living cells growing on the surface of the microcarriers (prepared in Example 4) in Example 9, and the sampling times are the 1st, 3rd, 5th, and 7th days after inoculation (in each panel, the scale is 200 μm).

Figure 7 is a stained picture of live cells growing on the surface of microcarriers (prepared in Example 5) in Example 10, and the sampling time is the 1st, 3rd, 5th, and 7th days after inoculation (each small picture, the scale is 200 μm ).

Figure 8 is a stained picture of living cells growing on the surface of microcarriers (prepared in Example 5) in Example 11. The sampling time is the 5th day after inoculation. It was observed that multiple microcarriers spontaneously aggregated to form cell-microcarriers clumps.

FIG. 9 is an optical microscope picture of the microcarrier obtained in Comparative Example 1. FIG.

Figure 10 is the stained picture of the living cells of the polysaccharide-protein composite microcarriers prepared in Comparative Example 2 that adhere and grow on the surface. The sampling time was on the 1st, 3rd, 5th, and 7th days after inoculation, and only a small amount of cell adhesion was observed. On the surface of the microcarriers, and the phenomenon of intercellular self-aggregation was observed, indicating that the selection of polysaccharide species has an important influence on the adhesion of cells on the surface of the microcarriers (in each panel, the scale is 200 μm).

Figure 11 is a picture of live cell staining of the polysaccharide-protein (fish collagen peptide) composite microcarriers and polysaccharide-protein (gelatin) composite microcarriers prepared in Comparative Example 3 that adhere and grow on the surface, and the sampling time is the first, 3, 5, and 7 days; for the polysaccharide-protein (fish collagen peptide) composite microcarriers, it was observed that only a small amount of cells adhered to the surface of the microcarriers on days 1-5 after inoculation, and most of the cells were cell-to-cell. Adhesion and agglomeration, a small amount of cell proliferation was observed on the 7th day; for the polysaccharide-protein (gelatin) composite microcarrier, it was observed that only a small amount of cells adhered to the surface of the microcarrier from the 1st to the 5th day after seeding. Rapid cell proliferation was observed on day 7 (bar is 200 μm in each panel).

12 is an optical microscope picture of the microcarriers obtained under different cross-linking and immobilization conditions in Comparative Example 4 after being stirred on a magnetic stirrer for 24 hours. For the microcarriers obtained by the cross-linking and fixing method in Example 1, the morphology was complete after stirring for 24 hours (A); The microcarriers prepared from the aqueous solution of 100% as the cross-linking fixative, a large number of fragments appeared after stirring for 24h (B); for the microcarriers obtained by heating and precipitation in a water bath at 80°C, they were almost completely dissolved (C) after stirring for 24h (each). In the small figure, the scale bar in the figure is 200 μm).

Figure 13 is a picture of live cell staining of the polysaccharide-protein composite microcarriers prepared in Comparative Example 5 adhering and growing on the surface. The sampling time is the 1st, 4th, and 7th days after inoculation, corresponding to pictures A to C in turn (each small picture , the scale bar is 200 μm).

Detailed ways

The embodiments of the present invention are described below through specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

In the present invention, the mass volume percent concentration (g/mL) refers to the mass of a certain substance in a certain volume of solution; for example, the mass volume percent concentration (g/mL) of a certain substance is 1%, which means that the substance is in 100 mL of solution. The mass in is 1 g. Other analogies.

The invention provides a microcarrier for three-dimensional cell culture and a preparation method thereof. The method comprises: directly mixing edible plant-derived ion-crosslinked gelling polysaccharide and edible non-animal-derived protein , make a mixed solution, generate droplets, and prepare microcarriers through suitable cross-linking and immobilization reaction conditions. The microcarriers are used for cell culture, can support the rapid proliferation of cells, and spontaneously aggregate to form cell-microcarrier clumps through the interaction between cells. According to the actual product form requirements, the microcarriers can be dissolved to harvest the cells for subsequent processing, or the cell cultured meat products can be obtained directly by gluing and extruding the clumps. The microcarrier prepared by the method of the present invention is used for cell culture meat, the operation is fast and simple, and large-scale production is easy to be realized, and it has great potential in the field of cell culture meat. Since animal-derived proteins can be better recognized by animal cells, in the prior art, in order to provide suitable growth and reproduction carriers for animal cells, animal-derived proteins are usually used to prepare microcarriers. Since polysaccharides are usually negatively charged, and animal-derived proteins are usually positively charged, in order to avoid the charge interaction between polysaccharides and animal-derived proteins, which may cause unfavorable phenomena such as aggregates or agglomeration, the existing methods usually prepare polysaccharides first. Microcarriers, and then use animal-derived proteins or polypeptides as modifiers to modify the surface of the microcarriers to prepare microcarriers containing both polysaccharides and proteins. The existing method uses animal-derived protein as raw material to prepare microcarriers. On the one hand, the prepared microcarriers are inedible and will increase the complexity of subsequent applications; promotion. The invention avoids animal-derived proteins in the prior art, adopts non-animal-derived proteins and polysaccharide molecules that can be ionically cross-linked to form gels, both have negative charges, and positive and negative electric agglutination reactions do not occur, and the use of polysaccharides and proteins The direct mixing method to prepare microcarriers is simple to operate, can greatly improve production efficiency and reduce production costs. The prepared microcarriers are edible and soluble, and can be better used for cell adhesion and growth and reproduction for subsequent applications.

The method for preparing a microcarrier for three-dimensional cell culture provided by the present invention includes: using a polysaccharide molecule that can be ionically crosslinked to form a gel as a base material, compounding with a protein according to a certain ratio, forming the mixture into droplets, and entering the mixture into a liquid containing crosslinking gel. After cross-linking and immobilization in a solution (collection bath) of a linking agent and/or a fixing agent, with or without surface modification, a protein-polysaccharide composite microcarrier is formed, that is, the microcarrier for three-dimensional cell culture. The obtained microcarriers are inoculated with cells, cultured for a certain period of time, and cell-microcarrier clumps are formed spontaneously, and the cells or clumps are harvested for subsequent processing.

Specifically, the method for preparing a microcarrier for three-dimensional cell culture according to the present invention includes the following steps:

(1) preparing a mixed solution containing ion cross-linked gelatinous polysaccharide and protein;

(2) The droplet generation method is used to generate protein-polysaccharide composite droplets;

(3) collecting the droplets into a solution containing a cross-linking agent and/or a fixative, and performing a cross-linking reaction and/or a fixation reaction to obtain the microcarrier for three-dimensional cell culture.

In step (1), the ion cross-linked gelatinous polysaccharide is an edible plant-derived ion-crosslinked gelatinous polysaccharide, including but not limited to sodium alginate, gellan gum, carrageenan, pectin, polysaccharide. One or more of mannuronic acid, etc. In some preferred embodiments, the ionically cross-linked gelling polysaccharide is sodium alginate. In some preferred embodiments, the ionomer gelatin polysaccharide is gellan gum. In some preferred embodiments, the ionomer gel-type polysaccharide is carrageenan. In some preferred embodiments, the ionomer gel polysaccharide is low-ester pectin. In some preferred embodiments, the ionomer gel polysaccharide is a mixture of sodium alginate and gellan gum. In some preferred embodiments, the ionomer gel-type polysaccharide is a mixture of sodium alginate, gellan gum and carrageenan. In some preferred embodiments, the ionomer gel-type polysaccharide is a mixture of sodium alginate, gellan gum, carrageenan and low-ester pectin. In some preferred embodiments, the ionomer gelatin polysaccharide is a mixture of gellan gum and carrageenan. In some preferred embodiments, the ionomer gel-type polysaccharide is a mixture of gellan gum and low-ester pectin. In some preferred embodiments, the ionomer gel-type polysaccharide is a mixture of carrageenan and low-ester pectin. In some preferred embodiments, the ionomer gel polysaccharide is a mixture of gellan gum, carrageenan and low-ester pectin.

In step (1), the protein is non-animal derived protein including but not limited to soybean protein isolate, glutenin, pea protein, zein, potato protein, rice protein, peanut protein, oat protein, almond protein, mung bean Protein, chickpea protein, chia seed protein, quinoa protein, broad bean protein, lentil protein, edamame protein, hemp seed protein, walnut protein, hazelnut protein, sesame protein, chia seed protein, cashew protein, yeast protein, One or more of mushroom protein, fungal protein, algae protein, etc. In some preferred embodiments, the protein is soy protein isolate. In some preferred embodiments, the protein is a yeast protein. In some preferred embodiments, the protein is glutenin. In some preferred embodiments, the protein comprises both soy protein isolate and yeast protein.

In step (1), the mass ratio of the ionically cross-linked gelatinous polysaccharide and the protein is 1-50:1-10. In some preferred embodiments, the mass ratio of the ionomer gelatin polysaccharide and protein is 20:1. In some preferred embodiments, the mass ratio of the ionomer gel polysaccharide and the protein is 1:2.5. In some preferred embodiments, the mass ratio of the ionomer gelatin polysaccharide and the protein is 1:10.

In step (1), as an option, the ion-crosslinked gelling polysaccharide can be dissolved in a solution first, and the solution can be any edible solution capable of dissolving the ion-crosslinked gelling polysaccharide; For example, the solution is water. In some preferred embodiments, the ionomer gel-type polysaccharide is dissolved in water to form an aqueous polysaccharide solution. In some preferred embodiments, the mass volume percent concentration (g/mL) of the ion-crosslinked gel polysaccharide aqueous solution is 0.1-5%. In some preferred embodiments, the mass volume percent concentration (g/mL) of the aqueous solution of the ionomer gel-type polysaccharide is 0.5%. In some preferred embodiments, the mass volume percent concentration (g/mL) of the aqueous solution of the ionomer gel-type polysaccharide is 1%. In some preferred embodiments, the mass volume percent concentration (g/mL) of the ion-crosslinked gel polysaccharide aqueous solution is 2%.

In step (1), as an option, the protein can be dissolved in a solution first, and the solution can be any edible solution that can dissolve the protein; for example, the solution is an alkaline solution, water; The alkaline solution includes an aqueous solution of edible soda ash (ie, sodium carbonate) or baking soda (ie, sodium hydroxide). In some preferred embodiments, the protein is dissolved in an alkaline solution. In some preferred embodiments, the protein is dissolved in a soda ash solution. In some preferred embodiments, the protein is dissolved in a baking soda solution. In some preferred embodiments, the mass volume percent concentration (g/mL) of the protein in the alkaline solution is 0.5-20%, which can be 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 , 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20%. In some preferred embodiments, the mass volume percent concentration (g/mL) of the protein in the alkaline solution is 5%. In some preferred embodiments, the mass volume percent concentration (g/mL) of the protein in the alkaline solution is 10%.

In step (2), the method for generating droplets is not limited, as long as droplets of suitable diameter can be generated. The methods for generating droplets include, but are not limited to, spray-freezing method, spray-drying method, electrostatic spray method, coaxial airflow atomization method, emulsification method, microfluidic method, and microcapsule granulation method. In some preferred embodiments, the method of generating droplets is a spray-freezing method. In some preferred embodiments, the method of generating droplets is electrostatic spraying. In some preferred embodiments, the method for generating droplets is microencapsulation granulation. In some preferred embodiments, the method for generating droplets is a coaxial airflow atomization method. In some preferred embodiments, the method for generating droplets is an emulsification method.

The droplets are micro-liquids, and the particle size of the droplets can be any suitable particle size. In some embodiments, the particle size of the droplets is 50-2000 μm, which can be 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 μm. In some preferred embodiments, the particle size of the droplets is 200-500 μm, that is, the same system contains droplets with a particle size of 200-500 μm. In some preferred embodiments, the particle size of the droplets is 200-800 μm, that is, the same system contains droplets with a particle size of 200-800 μm. . In some preferred embodiments, the droplets have a particle size of 200 μm. In some preferred embodiments, the droplets have a particle size of 300 μm. In some preferred embodiments, the droplets have a particle size of 400 μm. In some preferred embodiments, the droplets have a particle size of 500 μm. In some preferred embodiments, the droplets have a particle size of 600 μm. In some preferred embodiments, the droplets have a particle size of 700 μm. In some preferred embodiments, the droplets have a particle size of 800 μm.

In step (3), the cross-linking agent includes but is not limited to ions, and the ions can make the ion cross-linked gelatinous polysaccharide described above to carry out ion cross-linking reaction, including but not limited to calcium ions, magnesium ions, etc. One or more of ions, potassium ions, etc. In some embodiments, ionic-containing crosslinking agents are used. In some preferred embodiments, only calcium ion-containing crosslinking agents are used. In some preferred embodiments, calcium chloride-containing crosslinking agents are used. In some preferred embodiments, an aqueous calcium chloride-containing crosslinking agent is used.

In step (3), the fixative includes, but is not limited to, proteins, which can make the above-mentioned proteins precipitate and fix, and the proteins include but are not limited to transglutaminase, glucose oxidase, polyphenols One or more of oxidase, catalase, horseradish peroxidase, lactoperoxidase, phenolic compounds, and genipin. In some preferred embodiments, the fixative containing the fixative is a solution comprising proteins. In some preferred embodiments, the fixative-containing fixative is a solution comprising transglutaminase. In some preferred embodiments, the fixative-containing fixative is an aqueous solution comprising transglutaminase.

In some preferred embodiments, in step (3), a cross-linking agent and a fixing agent, or a solution containing a cross-linking agent and a fixing agent are used simultaneously. The cross-linking agent and the fixing agent can be used separately, that is, the cross-linking agent is used first and then the fixing agent is used, or the fixing agent is used first and then the cross-linking agent is used, and the number of cross-linking and fixing is not limited. One ion cross-linking, and then another protein fixation; the cross-linking agent and the fixative can exist in the same solution, and the cross-linking and fixation reactions are carried out at the same time. That is to say, the ions and proteins can exist in the same solution or in different solutions; when they exist in the same solution, the droplets perform ion cross-linking and protein cross-linking reactions simultaneously in the solution; when When ions and proteins are present in different solutions, the droplets can be ionically crosslinked in an ion-containing crosslinking solution first, and then the protein immobilized in a protein-containing fixative, or the droplets can be first immobilized in a protein-containing fixative. Protein immobilization in solution, followed by ionic crosslinking in ion-containing crosslinking solution. In some preferred embodiments, the cross-linking fixative is a solution comprising calcium ions and transglutaminase. In some preferred embodiments, the cross-linking fixative is an aqueous solution comprising calcium chloride and transglutaminase.

Wherein, in the cross-linking solution, the mass volume percentage concentration (g/mL) of the ions is 0.2-15%, which can be 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0%. Preferably, the mass volume percent concentration (g/mL) of the ions is 0.5-8%. Further preferably, the mass volume percentage concentration (g/mL) of the ions is 0.8-5%, which can be 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0%. In some preferred embodiments, the mass-volume percent concentration (g/mL) of the ions is 1%. In some preferred embodiments, the mass volume percent concentration (g/mL) of the ions is 1.5%. In some preferred embodiments, the mass-volume percent concentration (g/mL) of the ions is 2%.

Wherein, in the fixative solution, the mass volume percent concentration (g/mL) of the enzyme protein is 0.01-5%, and the mass volume percent concentration (g/mL) may be 0.01-0.5%, 0.5- 1%, 0.01-1%, 1-2%, 2-3%, 3-4%, 4-5%, 0.2-5%; 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0%. Preferably, the mass volume percent concentration (g/mL) of the protein is 0.5-3%. Further preferably, the mass volume percent concentration (g/mL) of the protein is 0.5-1.5%, which can be 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5%. In some preferred embodiments, the mass-volume percent concentration (g/mL) of the protein is 0.5%. In some preferred embodiments, the mass-volume percent concentration (g/mL) of the protein is 1%. In some preferred embodiments, the mass-volume percent concentration (g/mL) of the protein is 1.5%. In some preferred embodiments, the mass-volume percent concentration (g/mL) of the protein is 2%. In some preferred embodiments, the mass-volume percent concentration (g/mL) of the protein is 2.5%.

In step (3), the temperature of the cross-linking or fixing reaction is 2 to 55°C, which can be 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 , 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 55°C. In some preferred embodiments, the temperature of the crosslinking or fixing reaction is 22-55°C. In some preferred embodiments, the temperature of the crosslinking or immobilization reaction is 4°C. In some preferred embodiments, the temperature of the cross-linking or fixing reaction is room temperature 22-28°C. In some preferred embodiments, the temperature of the crosslinking or immobilization reaction is 30°C. In some preferred embodiments, the temperature of the crosslinking or immobilization reaction is 35°C. In some preferred embodiments, the temperature of the crosslinking or immobilization reaction is 40°C. In some preferred embodiments, the temperature of the crosslinking or immobilization reaction is 45°C. In some preferred embodiments, the temperature of the crosslinking or immobilization reaction is 50°C. In some preferred embodiments, the temperature of the cross-linking or fixing reaction is 40-55°C. In some preferred embodiments, the temperature of the crosslinking or immobilization reaction is 55°C.

In step (3), the time of the cross-linking or immobilization reaction is 5-500 min. In some preferred embodiments, the time of the cross-linking or immobilization reaction is 10-400 min. In some preferred embodiments, the time of the cross-linking or immobilization reaction is 20-300 min. In some preferred embodiments, the time of the cross-linking or immobilization reaction is 20-200 min. In some preferred embodiments, the time of the cross-linking or immobilization reaction is 5-120 min, which can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 , 75, 80, 85, 90, 95, 100, 105, 110, 115, 120min. In some preferred embodiments, the time of the cross-linking or immobilization reaction is 30 min. In some preferred embodiments, the time of the cross-linking or immobilization reaction is 60 min. In some preferred embodiments, the time of the cross-linking or immobilization reaction is 90 min. In some preferred embodiments, the time of the cross-linking or immobilization reaction is 120 min.

In the preparation method of the microcarrier of the present invention, after the step (3), it further includes the step (4) of modifying the surface of the microcarrier: adding the microcarrier obtained in the step (3) into the surface modifier solution , for surface modification.

In step (4), the surface modifier is any material capable of surface modification of the microcarrier, which can change various properties of the microcarrier, including but not limited to changing the adsorption performance of cells on the microcarrier, Growth performance, proliferation performance, cell arranging performance, etc.; such changes include increases or decreases, or any changes in performance considered to provide the overall performance of the microcarrier. The surface modification materials include, but are not limited to, one or more of carbohydrates, proteins, polypeptides, polysaccharides, synthetic polymer compounds, and extracellular matrix components.

In the surface modifier, the protein is selected from collagen, laminin, fibronectin, vitronectin, elastin, chondronectin, and gelatin.

In the surface modifier, the polypeptide is selected from polylysine, polyornithine, RGD (arginine-glycine-aspartic acid), YIGSR (tyrosine-isoleucine- Glycine-Serine-Arginine), IKVAV (Isoleucine-Lysine-Valine-Alanine-Dehydroretinol).

In the surface modifier, the polysaccharide is selected from chitosan, quaternized chitosan, lactose, N-acetylglucosamine, and alginate.

In the surface modifier, the synthetic polymer compound is selected from polyurethane, polyacrylate, and polyetherimide (PEI).

In some preferred embodiments, the surface modifier is one or more of polylysine, chitosan, chitosan oligosaccharide, and zein, wherein chitosan and chitosan oligosaccharide are animal and Non-animal sources. In some preferred embodiments, the chitosan and chitosan oligosaccharides are of non-animal origin. In some preferred embodiments, the surface modification material is polylysine. In some preferred embodiments, the surface modification material is non-animal derived chitosan. In some preferred embodiments, the surface modification material is non-animal derived chitosan oligosaccharide. In some preferred embodiments, the surface modification material is zein.

The solvent for dissolving the surface modifier is one or more of water, acetic acid, ethanol and the like. In some preferred embodiments, the solvent in which the surface modifier is dissolved is water. In some preferred embodiments, the solvent in which the surface modifier is dissolved is acetic acid. In some preferred embodiments, the solvent in which the surface modifier is dissolved is an aqueous solution of acetic acid.

The mass volume percent concentration (g/mL) of the surface modifier is 0.01-20%, which can be 0.01, 0.02, 0.06, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0% . In some preferred embodiments, the mass volume percent concentration (g/mL) of the surface modifier is 0.1-5%. In some preferred embodiments, the mass volume percent concentration (g/mL) of the surface modifier is 0.1%. In some preferred embodiments, the mass volume percent concentration (g/mL) of the surface modifier is 0.5%. In some preferred embodiments, the mass volume percent concentration (g/mL) of the surface modifier is 1%. In some preferred embodiments, the mass volume percent concentration (g/mL) of the surface modifier is 1.5%. In some preferred embodiments, the mass volume percent concentration (g/mL) of the surface modifier is 2%. In some preferred embodiments, the mass volume percent concentration (g/mL) of the surface modifier is 2.5%. In some preferred embodiments, the mass volume percent concentration (g/mL) of the surface modifier is 3%. In some preferred embodiments, the mass volume percent concentration (g/mL) of the surface modifier is 4%. In some preferred embodiments, the mass volume percent concentration (g/mL) of the surface modifier is 5%. In some preferred embodiments, the surface modification material is polylysine, the solvent system is water, and the mass volume percent concentration (g/mL) of polylysine is 0.1-1%. In some preferred embodiments, the surface modification material is chitosan oligosaccharide, the solvent system is water, and the mass volume percent concentration (g/mL) of chitosan oligosaccharide is 0.1-10%. In some preferred embodiments, when the material is chitosan, the solvent system is an aqueous solution of acetic acid, the acetic acid mass volume percentage concentration (g/mL) is 1-90%, and the chitosan mass volume percentage concentration (g/mL) /mL) is 0.1 to 5%. In some preferred embodiments, when the material is zein, the solvent system is 70-80% alcohol, and the mass volume percent concentration (g/mL) of zein is 5-30%.

The temperature of the surface modification is 10-80°C, which can be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80°C. In some preferred embodiments, the temperature of the surface modification is 10°C. In some preferred embodiments, the temperature of the surface modification is room temperature 22-28°C. In some preferred embodiments, the temperature of the surface modification is 20°C. In some preferred embodiments, the temperature of the surface modification is 25°C. In some preferred embodiments, the temperature of the surface modification is 30°C. In some preferred embodiments, the temperature of the surface modification is 35°C. In some preferred embodiments, the temperature of the surface modification is 40°C. In some preferred embodiments, the temperature of the surface modification is 45°C. In some preferred embodiments, the temperature of the surface modification is 50°C. In some preferred embodiments, the temperature of the surface modification is 55°C. In some preferred embodiments, the temperature of the surface modification is 60°C. In some preferred embodiments, the temperature of the surface modification is 65°C. In some preferred embodiments, the temperature of the surface modification is 70°C.

The surface modification time is 5-30 min. In some preferred embodiments, the surface modification time is 10-260 min. In some preferred embodiments, the surface modification time is 20-200 min. In some preferred embodiments, the surface modification time is 20-120 min, which can be 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 , 95, 100, 105, 110, 115, 120min. In some preferred embodiments, the surface modification time is 30 min. In some preferred embodiments, the surface modification time is 40 min. In some preferred embodiments, the surface modification time is 50 min. In some preferred embodiments, the surface modification time is 60 min. In some preferred embodiments, the time for the surface modification is 90 min. In some preferred embodiments, the surface modification time is 120 min.

The preparation of the microcarrier of the present invention also includes the step of natural drying or drying. The drying can be performed in any suitable manner, for example, the drying temperature can be 25-100°C.

In a specific embodiment, the method for preparing the microcarriers of the present invention and the method for culturing cells using the prepared microcarriers (see Figure 1 for the preparation flow chart) include the following steps:

(1) Dissolve the ion cross-linked gelatinous polysaccharide in water at a certain mass volume percentage concentration (g/mL);

(2) Dissolve the protein at a certain mass volume percentage concentration (g/mL) under alkaline conditions, or directly use the protein solid;

(3) Evenly mix the solution with the polysaccharide solution, or directly disperse the protein protein solid in the polysaccharide solution, so that the mass fraction of protein and polysaccharide reaches a certain ratio;

(4) using a droplet generation method to generate protein-protein-polysaccharide composite system microdroplets, and collect them in a collection bath containing the ions that can be cross-linked with polysaccharides to form gels;

(5) crosslinking the obtained droplets in a collecting bath for a certain period of time;

(6) Dissolve the protein cross-linking enzyme in water at a certain mass volume percentage concentration (g/mL), and under a certain temperature condition, soak the microcarriers in the protein immobilization solution for a certain period of time;

(7) Dissolve the surface modification material in a suitable solvent system with a certain mass volume percentage concentration (g/mL) to prepare a surface modification solution;

(8) Soak the fully cross-linked microcarriers in the surface modification solution for a certain period of time, and carry out surface modification under certain temperature conditions to obtain the protein protein-polysaccharide composite microcarriers.

The present invention also provides a method for cross-linking and immobilizing the microcarrier, and the method is as described in the step (3) of preparing the microcarrier.

The present invention also provides a microcarrier for three-dimensional cell culture prepared by the above-mentioned method, wherein the microcarrier is a protein-polysaccharide composite microcarrier, which is a macroporous gel type. The pore size of the microcarrier is 0.03-60 μm, preferably 0.1-50 μm, more preferably 0.2-30 μm, and more preferably 0.5-20 μm. The particle size of the microcarrier is 50-2000 μm; preferably 200-800 μm, more preferably 200-500 μm. Macroporous microcarriers have good permeability, which is conducive to the diffusion of nutrients and cell metabolites, and can provide a good growth environment for cells.

The invention also provides the application of the microcarrier for three-dimensional cell culture in culturing cells, culturing cell-microcarrier clumps, and culturing meat.

The present invention also provides a method for culturing cells using the microcarrier for three-dimensional cell culture, the method comprising: inoculating cells with the microcarrier, and culturing for a certain period of time to collect the cells.

The present invention also provides a method for culturing cell-microcarrier agglomerates and/or cell culture meat using the microcarrier for three-dimensional cell culture, the method comprising:

(1) the microcarrier inoculated cells are cultured for a certain period of time;

(2) the microcarriers inoculated with cells spontaneously agglomerate to form cell-microcarrier clumps, and harvest to obtain cell-microcarrier clumps;

Or, the cell-microcarrier clumps are bonded and extruded to obtain the cell cultured meat product.

In the present invention, in the method for culturing cells, culturing cell-microcarrier aggregates or cell culture meat using the microcarriers for three-dimensional cell culture, the cells include but are not limited to blood matrix proteins, hepatocytes, muscle cells, Osteoblasts, fibroblasts, adipocytes, odontoblasts, adult neuronal progenitor cells, neural stem cells, pluripotent stem cells from the subventricular forebrain region, ependymal neural stem cells, hematopoietic stem cells, hepatic hematopoietic stem cells, bone marrow Stem cells, adipose fibroblasts, adipose stem cells, mesenchymal stem cells, embryonic stem cells, bone marrow stromal cells, islet cell-producing stem cells, pancreatic-derived pluripotent islet-producing stem cells, muscle side population cells, bone marrow-derived recovered cells, blood derived mesenchymal precursor cells, bone marrow derived side population cells, muscle precursor cells, circulating skeletal stem cells, neural progenitor cells, multipotent adult progenitor cells, mesoderm progenitor cells, spinal cord progenitor cells and spore-like cells, various A population of cells formed by mixing and any combination thereof.

In some preferred embodiments, the microcarriers are used for culturing meat, and the inoculated cells are one or more of osteoblasts, fibroblasts, and adipocytes. In some preferred embodiments, the microcarriers are used for culturing meat, and the inoculated cells are porcine muscle satellite cells. In some preferred embodiments, the microcarriers are used for culturing cells for regenerative medicine, and the inoculated cells are mesenchymal stem cells.

The performance and structure of microcarriers have important effects on cell culture, especially the matrix material, surface shape, surface modification structure, charge, and hydrophilicity of microcarriers have important effects on cell attachment, growth and proliferation. In the prior art, animal-derived protein is used as one of the matrix materials for constructing microcarriers, because animal-derived matrix materials can better identify and adhere to animal cells, which is conducive to further cell growth and reproduction. However, plant-derived protein as a matrix material usually cannot provide a more suitable recognition site for animal cells, which is not conducive to the adhesion of animal cells. The present invention innovatively uses plant-derived proteins and polysaccharides as matrix materials prepared as microcarriers, and uses suitable cross-linking agents and/or fixatives to cross-link and/or cross-link proteins and polysaccharides by adjusting parameters during the preparation method. Fixing, in a more preferred manner, the surface of the microcarrier is modified with a surfactant, the prepared microcarrier can be well recognized and adsorbed by animal cells, and the formed three-dimensional structure provides favorable conditions for cell growth and clump formation The space environment, which in turn provides the basis for culturing cells, cell-microcarrier aggregates and cell culture meat.

The present invention adopts natural polysaccharide materials (such as sodium alginate, which is a natural edible material and has good biocompatibility) that can be cross-linked with ions to form hydrogels, and cross-linked with calcium ions to form gels network, rapid reaction, mild conditions, and reversible process; it can be dissolved by using ethylenediaminetetraacetic acid (EDTA). Natural protein-based materials, such as soy protein, yeast protein, etc., can be added to meat products to provide the taste and flavor of meat products. Mixing ionically cross-linked polysaccharide and protein, using the fast and reversible gelation process of polysaccharide and the flavor of protein, the microcarrier prepared by specific method and controlling appropriate parameter conditions has the ability to be suitable for large-scale production of cell cultured meat. related properties, such as high biological activity, can promote cell adhesion, growth and proliferation, through the interaction between cells, microcarriers can spontaneously agglomerate to form cell-microcarrier clumps, and the clumps can be glued and squeezed. Compression molding can directly obtain end products such as cell culture meat. The invention can simplify the inoculation process, does not need complex operations such as separation, and has unlimited culture volume, which is beneficial to the production of large-scale and commercial products.

As used herein, the "ionocrosslinked gelling polysaccharide" refers to a polysaccharide capable of forming a stable network structure within a molecule or between molecules in the presence of ions.

As used herein, the "protein immobilization" refers to the ability to denature and separate out proteins in the presence of a protein immobilizer, or to catalyze the formation of stable chemical bonds (such as disulfide bonds, etc.) within protein molecules and between protein molecules and protein molecules. network structure.

As used herein, the modification refers to chemical reactions and physical effects on the surface of the microcarriers by using surface modifiers, thereby changing the surface states of the microcarriers, such as the surface atomic layer structure and functional groups, surface hydrophobicity, electrical properties, chemical adsorption and reaction properties.

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments, Rather than limiting the scope of the invention; in the specification and claims of the present invention, the singular forms "a", "an" and "the" include the plural forms unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is to be understood that, unless otherwise indicated herein, both endpoints of each numerical range and any number between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment and materials used in the embodiments, according to the mastery of the prior art by those skilled in the art and the description of the present invention, the methods, equipment and materials described in the embodiments of the present invention can also be used Any methods, devices and materials similar or equivalent to those of the prior art can be used to implement the present invention.

Unless otherwise specified, the experimental methods, detection methods and preparation methods disclosed in the present invention all adopt the conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field. conventional technology.

Example 1: Preparation of Soy Protein Isolate-Sodium Alginate/Polylysine Surface Modified Microcarrier

Weigh 1 g of sodium alginate into 100 ml of deionized water, stir at room temperature until completely dissolved, and prepare a sodium alginate solution with a concentration of 1%. Weigh 0.1g of edible soda ash into 100ml of deionized water, stir to dissolve, then weigh 5g of soy protein isolate solid, add it to the alkali solution, stir at room temperature until completely dissolved, and prepare a soy protein isolate solution with a concentration of 5%. Measure 20ml of sodium alginate solution with a concentration of 1%, and measure 80ml of a soybean protein isolate solution with a concentration of 5% by volume by volume (g/mL), mix the two solutions evenly, and prepare the sodium alginate concentration as The composite solution of 0.2% and soybean protein isolate concentration of 4%, the mass ratio of soybean protein isolate and sodium alginate is 20:1. Micro-droplets are prepared by spray-freezing method to obtain micro-droplets with a particle size of 200-500 μm. Weigh 5g of calcium chloride solid and add it into 500ml of deionized water to prepare a 1% calcium chloride solution as an ionic cross-linking solution. microcarriers. Weigh 2.5 g of transglutaminase into 500 ml of water, dissolve at room temperature, and prepare a protein fixative solution. The calcium ion-crosslinked microcarriers were collected into the protein fixative solution, heated to 40°C in a water bath for 2 h, and the ionically crosslinked and protein-fixed microcarriers were obtained. Weigh 0.1 g of polylysine into 100 ml of deionized water, stir at room temperature until it is completely dissolved, and prepare a polylysine solution with a concentration of 0.1% as a surface modification material. Immerse the immobilized microcarriers in a 0.1% polylysine solution and stir at room temperature for 30 min to obtain polysaccharide-protein composite microcarriers with polylysine surface-modified ion cross-linking and protein immobilization, with a particle size of 200~ 500 μm, and its optical microscope picture is shown in Figure 2.

Example 2: Preparation of yeast protein-low ester pectin microcarriers

2 g of low-ester pectin was weighed into 100 ml of deionized water, heated in a water bath at 60° C. and stirred until completely dissolved to prepare a low-ester pectin solution with a concentration of 2%. Weigh 5 g of yeast protein solids into 100 ml of 2% low-ester pectin solution, stir to make the solid particles evenly dispersed, and the mass ratio of yeast protein to low-ester pectin is 2.5:1. Weigh 25 g of solid calcium chloride into 500 ml of deionized water to prepare a 5% calcium chloride solution for use as a collection bath. Microdroplets were prepared by electrostatic spraying, and the resulting microdroplets were cross-linked and fixed in a collecting bath at room temperature for 30 minutes to obtain ionically cross-linked polysaccharide-protein composite microcarriers with a particle size of 200-800 μm.

Example 3: Preparation of Glutenin-Gellan Gum Microcarriers/Chitosan Surface Modified Microcarriers

Weigh 1 g of gellan gum into 100 ml of deionized water, stir at room temperature until completely dissolved, and prepare a gellan gum solution with a concentration of 1%. Weigh 0.1g of baking soda into 100ml of deionized water, stir to dissolve, then weigh 10g of glutenin solid, add it to the alkali solution, stir at room temperature until completely dissolved, and prepare a glutenin solution with a concentration of 10%. Measure 50ml of gellan gum solution with a concentration of 1% and 50ml of glutenin solution with a concentration of 10%. Mix the two solutions evenly to prepare a compound with a gellan gum concentration of 0.2% and a glutenin concentration of 1%. solution, the mass ratio of glutenin to gellan gum is 10:1. Weigh 5 g of calcium chloride solid into 500 ml of deionized water to prepare a 1% calcium chloride solution, which is used as a collection bath. Micro-droplets were prepared by microcapsule granulation, and cross-linked and fixed in a collecting bath at room temperature for 60 min to obtain ionically cross-linked microcarriers with a particle size of 200-500 μm. Measure 2ml of acetic acid into 100ml of deionized water, weigh 0.5g of chitosan and add it to 100ml of 2% acetic acid solution, stir at room temperature until completely dissolved, and prepare a chitosan solution with a concentration of 0.5% as a surface modification material . Soak the cross-linked microcarriers in the collection bath in 0.5% chitosan solution, and stir at 60° C. for 2 h to obtain ionically crosslinked polysaccharide-protein composite microcarriers modified on the surface of chitosan, with a particle size of 200~ 500 μm, and its optical microscope picture is shown in Figure 3.

Example 4: Preparation of Zein Surface-Modified Sodium Alginate Porous Microcarriers

Weigh 0.5 g of sodium alginate into 100 ml of deionized water, stir at room temperature until completely dissolved, and prepare a sodium alginate solution with a concentration of 0.5%. Weigh 5g of zein solids and add them into 100ml of 0.5% sodium alginate solution, stir to make the solid particles evenly dispersed, and the mass ratio of zein to sodium alginate is 10:1. 5 g of calcium chloride solid was weighed into 500 ml of deionized water to prepare a 1% calcium chloride solution, which was used as a collection bath. Microdroplets were prepared by coaxial airflow atomization, and the resulting microdroplets were cross-linked and fixed in a collecting bath at room temperature for 30 minutes to obtain ionically cross-linked microcarriers with a particle size of 200-800 μm. The collected ionically cross-linked microcarriers were washed with deionized water, soaked and washed with 75% alcohol to remove zein to obtain porous microcarriers. Weigh 5 g of zein solid, add 100 ml of 75% alcohol, dissolve at room temperature, and prepare a 5% zein solution. The porous microcarriers were soaked in 5% zein solution, filtered to remove excess liquid, and dried at 80°C to obtain ion-crosslinked porous polysaccharide-protein composite microcarriers modified on the surface of zein, with a particle size of 200 ~800μm.

Example 5: Preparation of Soy Protein Isolate-Yeast Protein-Sodium Alginate Microcarrier

Weigh 1 g of sodium alginate into 100 ml of deionized water, stir at room temperature until completely dissolved, and prepare a sodium alginate solution with a concentration of 1%. Weigh 0.1g of edible soda ash into 100ml of deionized water, stir to dissolve, then weigh 5g of soy protein isolate solid, add it to the alkali solution, stir at room temperature until completely dissolved, and prepare a soy protein isolate solution with a concentration of 5%. Measure 20ml of sodium alginate solution with a concentration of 1%, measure 20ml of soybean protein isolate solution with a concentration of 5%, mix the two solutions, add 60ml of deionized water, mix well, and prepare a sodium alginate concentration of 0.2% , Soybean protein isolate concentration of 1% compound solution. 1 g of yeast protein solid was added to 100 ml of the above composite solution, and the solid particles were uniformly dispersed by stirring, wherein the mass ratio of the total mass of protein to sodium alginate was 10:1. To the mixed solution of polysaccharide and protein, edible oil with a volume of 10 times the mixed solution is added to prepare an emulsion system, and micro-droplets are prepared by an emulsification method to obtain micro-droplets with a particle size of 200-500 μm. Weigh 5g of calcium chloride solid and add it into 500ml of deionized water to prepare a 1% calcium chloride solution. Add 100 ml of the above-mentioned emulsification system to 500 ml of 1% calcium chloride solution, and stir at room temperature for 30 minutes to cross-link the droplets to obtain a polysaccharide-protein composite microcarrier.

Example 6: Dissolution of Soluble Microcarriers

Weigh 3.8g of EDTA solid and add it to 100ml of deionized water. After stirring with slight heat to dissolve, add 900ml of deionized water to prepare an EDTA solution with a concentration of 10mM. The microcarriers obtained in Example 1 were added to a 10 mM EDTA solution, shaken up to make the microcarriers fully contact with the solution, and allowed to stand at room temperature for 5 min to completely dissolve the microcarriers.

Example 7: Culture of porcine muscle satellite cells on microcarriers

Pig muscle satellite cells were seeded on the protein-polysaccharide composite microcarrier prepared in Example 1 at a density of 10 ⁴ /mg, in a complete medium (90% DMEM, 10% FBS (that is, every 100 ml of complete medium containing 90ml DMEM medium+10ml FBS)) for 7 days.

On the 1st, 3rd, 5th, and 7th days after inoculation, the living cells were stained and observed by light microscope and fluorescein diacetate, and the results are shown in FIG. 4 . It can be seen from Figure 4 that most of the cells adhered to the surface of the microcarriers and spread on the first day after inoculation, and then the number of cells was observed to increase significantly with the extension of the culture time on the 3rd, 5th, and 7th days. 1 The prepared microcarriers can provide a surface for adherent cells to adhere to and can support the rapid growth and proliferation of cells.

Example 8: Orientation of cells on proteoglycan composite microcarriers

C2C12 cells were seeded on the proteoglycan composite microcarrier obtained in Example 2 at a density of 10 ⁴ /mg, and cultured in a complete medium (90% DMEM, 10% FBS (that is, every 100 ml of complete medium contained 90 ml of DMEM). culture medium + 10 ml FBS)) for 7 days.

The cells were stained with fluorescein diacetate on the 1st, 3rd, 5th, and 7th days after inoculation, and the living cells were observed by a light microscope. The results are shown in FIG. 5 . It can be seen from Figure 5 that on the first day after seeding, most of the cells adhered to the surface of the microcarriers and spread, and then a significant increase in the number of cells was observed on the 3rd, 5th, and 7th days with the extension of the culture time, and on the 5th day. Dianhou cells are arranged in an orderly and directional arrangement along the surface pattern of the microcarrier.

Example 9: Adhesive growth of cells on proteoglycan composite microcarriers with porous structure

C2C12 cells were seeded on the polysaccharide-protein composite microcarriers with porous structure obtained in Example 4 at a density of 10 ⁴ /mg, in a complete medium (90% DMEM, 10% FBS (that is, per 100 ml of complete medium). Cultured in 90 ml DMEM medium + 10 ml FBS)) for 7 days.

The cells were stained with fluorescein diacetate on the 1st, 3rd, 5th, and 7th days after inoculation, and the living cells were observed by a light microscope. The results are shown in FIG. 6 . It can be seen from Figure 6 that cells can adhere to the surface of the microcarriers and spread on the first day after inoculation, and then the number of cells was observed to increase with the extension of the culture time on the third, fifth and seventh days, indicating that the cells contained a variety of proteins. The microcarriers of the components are capable of supporting cell growth and proliferation.

Example 10: Effects of various protein components on the adhesion and growth of cells on microcarriers

C2C12 cells were seeded on the proteoglycan composite microcarrier obtained in Example 5 at a density of 10 ⁴ /mg, and were cultured in a complete medium (90% DMEM, 10% FBS (that is, every 100 ml of complete medium contained 90 ml of DMEM). culture medium + 10 ml FBS)) for 7 days.

The cells were stained with fluorescein diacetate on the 1st, 3rd, 5th, and 7th days after inoculation, and the living cells were observed by a light microscope. The results are shown in FIG. 7 . It can be seen from Figure 7 that cells can adhere to the surface of the microcarriers and spread on the first day after inoculation, and then the number of cells was observed to increase with the extension of the culture time on the third, fifth and seventh days, indicating that the cells contained a variety of proteins. The microcarriers of the components are capable of supporting cell growth and proliferation.

Example 11: Spontaneous aggregation of protein-polysaccharide composite microcarriers to form cell-microcarrier clumps

Porcine skeletal muscle cells were seeded on the proteoglycan composite microcarrier obtained in Example 5 at a density of 10 ⁴ /mg, in a complete medium (90% DMEM, 10% FBS (that is, 90 ml per 100 ml of complete medium). DMEM medium+10ml FBS)) for 7 days.

The cells were stained with fluorescein diacetate on the 5th day after seeding, and the living cells were observed by a light microscope, and the results are shown in FIG. 8 . It can be seen from Figure 8 that on the 5th day, multiple microcarriers spontaneously agglomerated through cell-to-cell interactions to form cell-microcarrier clumps; the staining of live cells found that the surface of the clumps and between the microcarriers were wrapped by a large number of living cells. . The number of cell-microcarrier clumps was observed to increase and the volume of the clumps formed continued to increase with the extension of the culture time.

Comparative Example 1 Protein-only preparation of microcarriers

On the basis of Example 1, instead of using sodium alginate, only protein was used to prepare microcarriers, and the collection bath of the cross-linking fixative solution was different. Specifically, the existing mixed solution commonly used for protein cross-linking and fixation was used, which contained mass percent The following components: 4% acetic acid, 5% calcium chloride, 8% sodium chloride, 50% ethanol in water.

The optical microscope picture of the prepared protein microcarrier is shown in FIG. 9 . It can be seen from FIG. 9 that the particle size of the prepared protein microcarrier without polysaccharide is only 1-2 mm, and the specific surface area that can provide cell adhesion and proliferation is much lower than that of the polysaccharide-protein microcarrier in Example 1 of the present invention, which is applied to large-scale The production efficiency is too low, indicating that polysaccharide addition is necessary for regulating the particle size of microcarriers and preparing microcarriers with suitable particle size distribution.

Comparative Example 2 Effects of different polysaccharide species on microcarriers

On the basis of Example 2, only low-ester pectin was replaced with sodium alginate, and other conditions were the same as those of Example 2, to prepare a polysaccharide-protein composite microcarrier.

C2C12 cells were seeded on the microcarriers prepared by this method at a density of 10 ⁴ /mg, in full medium (90% DMEM, 10% FBS (ie, 90 ml DMEM medium + 10 ml FBS per 100 ml full medium)) cultured for 7 days.

The cells were stained with fluorescein diacetate on the 1st, 3rd, 5th, and 7th days after inoculation, and the living cells were observed by a light microscope. The results are shown in FIG. 10 . It can be seen from Figure 10 that only a very small amount of cells adhered to the surface of the microcarriers on the 1st to 7th days after inoculation, and the phenomenon of intercellular self-aggregation was observed, indicating that the selection of polysaccharide species has an important effect on the adhesion of cells to the surface of the microcarriers. Significant influence.

Comparative Example 3 Effects of Different Kinds of Animal-derived Proteins on Microcarriers

(1) Polysaccharide-protein (fish collagen peptide) composite microcarrier

On the basis of Example 2, only the yeast protein was replaced with animal-derived fish collagen peptides, and other conditions were the same as those of Example 2, to prepare a polysaccharide-protein composite microcarrier.

C2C12 cells were seeded on the microcarriers prepared by this method at a density of 10 ⁴ /mg, in full medium (90% DMEM, 10% FBS (ie, 90 ml DMEM medium + 10 ml FBS per 100 ml full medium)) cultured for 7 days.

Cells were stained with fluorescein diacetate on days 1, 3, 5, and 7 after inoculation, and living cells were observed by light microscopy. The results are shown in the left column of Figure 11 . It can be seen from Fig. 11 that only a small amount of cells adhered to the surface of the microcarriers on the 1st to 5th day after seeding, and most of the cells adhered and agglomerated with each other, and a small amount of cell proliferation was observed on the 7th day.

(2) Polysaccharide-protein (gelatin) composite microcarrier

On the basis of Example 2, the yeast protein was replaced with gelatin, and other conditions were the same as those of Example 2, to prepare a pectin-gelatin composite microcarrier.

C2C12 cells were seeded by the same method, that is, C2C12 cells were seeded on the microcarriers prepared by this method at a density of 10 ⁴ /mg, in a complete medium (90% DMEM, 10% FBS (that is, every 100 ml of complete medium contained 90ml DMEM medium+10ml FBS)) for 7 days.

Cells were stained with fluorescein diacetate on days 1, 3, 5, and 7 after inoculation, and live cells were observed by light microscopy. The results are shown in the right column of Figure 11 . It can be seen from FIG. 11 that only a small amount of cells adhered to the surface of the microcarriers on the 1st to 5th day after seeding, and rapid cell proliferation was observed on the 7th day. This indicates that yeast protein can more effectively promote cell adhesion and proliferation in a short time than animal-derived collagen.

Comparative Example 4 Effects of different cross-linking solutions, components of fixatives, and cross-linking and fixation conditions on microcarriers

(1) Example 1

The microcarriers obtained after cross-linking and fixing of ions and proteins obtained in Example 1 (that is, before adding polylysine) were dispersed in deionized water, and stirred on a magnetic stirrer for 24h. As shown in Figure A in Figure 12.

(2) Different cross-linking fixatives

With reference to the method of Example 1, the calcium chloride solution is replaced by an aqueous solution (cross-linked fixative) containing 4% acetic acid, 5% calcium chloride, 8% sodium chloride, 50% ethanol by mass percentage, respectively, and a spray is about to be used. - The microdroplets prepared by the freezing method are collected into the above-mentioned cross-linking fixative solution, and cross-linked and fixed for 30 min at room temperature.

Then, the immobilized microcarriers were dispersed in deionized water, and stirred on a magnetic stirrer for 24 hours. It was observed that the microcarriers were broken and a large number of fragments were formed, as shown in Figure B in Figure 12 .

(3) Different fixed conditions

Calcium chloride cross-linked microcarriers were prepared with reference to the method in Example 1, and then the microcarriers were heated in a water bath at 80° C. for 2 hours by thermal fixation to precipitate and fix proteins.

Then, the immobilized microcarriers were dispersed in deionized water, stirred on a magnetic stirrer for 24 hours, and it was observed that the microcarriers were almost completely dissolved, as shown in Figure C in Figure 12 . It shows that the microcarrier prepared by the cross-linking immobilization reagent and method of Example 1 of the present invention has more superior mechanical properties than other methods, and can maintain the shape under stirring conditions.

Comparative Example 5 Effects of different modifiers on microcarriers

Referring to Example 1, the surface modification material polylysine was replaced with mushroom-derived chitosan, and the others were the same as in Example 1, to obtain an edible and soluble microcarrier without animal-derived ingredients.

Referring to the method of Example 6, a dissolution experiment was performed on the soluble microcarriers, and it was found that the microcarriers prepared in this example were completely dissolved.

Referring to the method of Example 7, C2C12 cells were seeded on the proteoglycan composite microcarrier obtained in Example 5 at a density of 10 ⁴ /mg, in a complete medium (90% DMEM, 10% FBS (that is, per 100 ml). The complete medium contains 90 ml of DMEM medium + 10 ml of FBS)) for 7 days. The cells were stained with fluorescein diacetate on the 1st, 4th, and 7th days after inoculation, and the living cells were observed by a light microscope. The results are shown in FIG. 13 . It can be seen from Figure 13 that on the 1st day after seeding (Panel A), cells can adhere to the surface of the microcarriers and spread, and then the number of cells was observed on the 4th day (Panel B) and the 7th day (Panel C). The culture time was prolonged and increased, indicating that the use of mushroom-derived chitosan as the surface modification material can support the adhesion and growth of cells on the surface of the microcarriers.

The microcarriers were used for cell culture, and the cells adhered and grew well.

The above examples are intended to illustrate the disclosed embodiments of the present invention, and should not be construed as limiting the present invention. Furthermore, various modifications set forth herein and variations in the methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in detail in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. Indeed, various modifications as described above which are obvious to those skilled in the art to obtain the invention are intended to be included within the scope of the present invention.

Claims (14)

1. A microcarrier for three-dimensional cell culture, which is obtained by forming a mixed solution containing an edible plant-derived ionomer-type polysaccharide and an edible non-animal-derived protein into droplets and performing a crosslinking and/or immobilization reaction.

2. The microcarrier for three-dimensional cell culture according to claim 1, comprising any one or more of the following 1) to 3):

1) the edible plant-derived ionic crosslinked gel type polysaccharide is selected from one or more of sodium alginate, gellan gum, carrageenan, pectin and polymannuronic acid;

2) the edible protein of non-animal origin is selected from one or more of vegetable protein and microbial protein;

3) the mass ratio of the edible plant-derived ionic crosslinked gel-type polysaccharide to the edible plant-derived protein is 1-50: 1-10.

3. The microcarrier for three-dimensional cell culture according to claim 1, comprising any one or more of the following 1) to 3):

1) the vegetable protein is selected from the group consisting of Glycine, Pisum, Zea, Solanum, Oryza, Arachis, Avena, Rosaceae, vigna, Cicer, Mentha, Chenopodium, Vicia, lablab, Cannabis, Juglans, Corylus, Sesamum, and Anacardium; preferably, the vegetable protein is selected from one or more of isolated soy protein, glutenin, pea protein, zein, potato protein, rice protein, peanut protein, oat protein, almond protein, mung bean protein, chickpea protein, chia seed protein, quinoa protein, broad bean protein, hyacinth bean protein, green soy bean protein, hemp seed protein, walnut protein, hazelnut protein, sesame protein and cashew protein;

2) the microbial protein is selected from one or more of yeast protein, mushroom protein, fungal protein and algae protein;

3) the microcarrier is also surface modified by a surface modifier.

4. The microcarrier of claim 3, wherein the surface modifier is selected from the group consisting of one or more of a carbohydrate, a protein, a polypeptide, a polysaccharide, a synthetic polymer, and an extracellular matrix component.

5. A method for preparing microcarriers for three-dimensional culture of cells, comprising the following steps:

(1) preparing a mixed solution containing an ionomer gel-type polysaccharide and a protein;

(2) generating protein-polysaccharide composite liquid drops by adopting a liquid drop generation method;

(3) collecting the liquid drops into a solution containing a cross-linking agent and/or a fixing agent, and carrying out a cross-linking reaction and/or a fixing reaction to obtain the microcarrier for three-dimensional cell culture.

6. The method of claim 5, comprising any one or more of the following 1) to 4):

1) the method for generating the liquid drops is any one of a spray-freezing method, a spray-drying method, an electrostatic spraying method, a coaxial airflow atomization method, an emulsification method, a micro-fluidic method and a microcapsule granulation method;

2) the particle size of the liquid drops is 50-2000 mu m;

3) the cross-linking agent is selected from one or more of calcium ions, potassium ions and magnesium ions;

4) the step (3) also comprises a step of collecting the liquid drops into a solution containing a fixing agent before or after crosslinking to carry out a fixing reaction; the fixing agent is one or more selected from transglutaminase, glucose oxidase, polyphenol oxidase, catalase, horseradish peroxidase, lactoperoxidase, phenolic compounds and genipin.

7. The method according to claim 6, wherein the method comprises any one or more of the following 1) to 6):

1) in the crosslinking reaction, the mass volume percentage concentration (g/mL) of the crosslinking agent is 0.2-15%;

2) in the fixing reaction, the mass volume percentage concentration (g/mL) of the fixing agent is 0.2-5%;

3) the temperature of the crosslinking reaction is 2-55 ℃;

4) the temperature of the immobilization reaction is 2-55 ℃;

5) the time of the crosslinking reaction is 5-500 min;

6) the time of the fixed reaction is 5-500 min.

8. The method of claim 5, wherein the microcarrier is further surface-modified with a surface modifier selected from the group consisting of one or more of a carbohydrate, a protein, a polypeptide, a polysaccharide, a synthetic polymer, and an extracellular matrix component.

9. The method of claim 8, comprising any one or more of the following 1) to 7):

1) in the surface modifier, the protein is selected from collagen, laminin, fibronectin, vitronectin, elastin, chondronectin and gelatin;

2) in the surface modifier, the polypeptide is selected from polylysine, polyornithine, RGD, YIGSR and IKVAV;

3) in the surface modifier, the polysaccharide is selected from chitosan, quaternized chitosan, lactose, N-acetylglucosamine and alginate;

4) in the surface modifier, the synthetic polymer compound is selected from polyurethane, polyacrylate and polyetherimide;

5) the mass volume percentage concentration (g/mL) of the surface modifier is 0.01-20%;

6) the temperature of the surface modification is 10-80 ℃;

7) the surface modification time is 5-30 min.

10. A microcarrier for three-dimensional culture of cells prepared by the method of any one of claims 5-9.

11. Use of the microcarrier according to any one of claims 1-5 or 10 for three-dimensional cell culture for culturing cells, culturing cell-microcarrier pellets, culturing meat.

12. A method for culturing cells using the microcarrier for three-dimensional cell culture according to any one of claims 1 to 5 or 10, which comprises:

(1) inoculating the microcarrier into cells and culturing for a certain time;

(2) harvesting the cells;

or, the microcarrier inoculated with the cells spontaneously aggregates to form a cell-microcarrier mass, and the cell-microcarrier mass is obtained after harvesting;

or, spontaneously agglomerating the microcarrier inoculated with the cells to form a cell-microcarrier agglomerate, and bonding and extruding the cell-microcarrier agglomerate to obtain the cell culture meat product.

13. A product obtained by culturing cells seeded on the microcarrier for three-dimensional culture of cells according to any one of claims 1-5 or 10.

14. The method of culturing cells according to claim 12 or the product according to claim 13, wherein the cells are selected from the group consisting of blood matrix proteins, hepatocytes, muscle cells, osteoblasts, fibroblasts, adipoblasts, odontoblasts, adult neuronal progenitor cells, neural stem cells, multipotent stem cells from the lower anterior brain region of the cerebral ventricle, ependymal neural stem cells, hematopoietic stem cells, hepatic hematopoietic stem cells, bone marrow stem cells, adipose fibroblasts, adipose stem cells, mesenchymal stem cells, embryonic stem cells, bone marrow stromal cells, islet cell-producing stem cells, pancreatic multipotent islet stem cells, muscle side population cells, bone marrow-derived recovered cells, blood-derived mesenchymal precursor cells, bone marrow-derived side population cells, muscle precursor cells, circulating bone stem cells, neural progenitor cells, pancreatic islet cells, pancreatic side population cells, bone marrow-derived recovered cells, blood-derived mesenchymal precursor cells, bone marrow-derived side population cells, muscle precursor cells, circulating skeletal stem cells, neural progenitor cells, pancreatic islet cells, pancreatic progenitor cells, pancreatic islet cells, pancreatic progenitor cells, and methods of using the method of producing pancreatic islet cells, Any one or more of multipotent adult progenitor cells, mesodermal progenitor cells, spinal cord progenitor cells and spore-like cells.

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