Microcarrier, preparation method therefor and use thereof, and method for cell adherent culture and recovery

Abstract

A microcarrier, a preparation method therefor and the use thereof, and a method for cell adherent culture and recovery. The prepared microcarrier can be used for cell culture, can maintain the survival rate of cells and can be quickly and completely dissolved during cell culture, and has a modified surface. Further provided in the present invention are the preparation of the microcarrier and the use of the microcarrier in cell adherent culture. Compared with an existing microcarrier, by means of using the microcarrier and method of the present application, the cell adherent culture can have a cell recovery efficiency of 95% or higher. In addition, by means of using a polysaccharide raw material, the introduction of an animal-derived pathogen into a cell product or a cell-derived biological product is prevented, and the manufacturing cost is significantly lower than that of raw materials such as recombinant gelatin and recombinant collagen.

Description

A microcarrier, preparation method and application thereof, and cell adherence culture and recovery method Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a microcarrier, a preparation method and application thereof, and a cell adherence culture and recovery method. Background Art

[0002] Cell culture is divided into two major categories: adherent culture, also known as monolayer or flat culture, and suspension culture. Cell culture can be used to produce cell therapy products such as stem cells, immune cells, and gene-modified cells for treating diseases, as well as cell-derived biologics such as viral vectors, vaccines, antibodies, peptide drugs, exosomes, and microvesicles.

[0003] Many cells from species such as humans, mammals, and insects require attachment to a surface to grow and proliferate. These cells are referred to as adherent cells. Containers such as tissue culture dishes, tissue culture flasks, and cell factories allow adherent cells to attach to flat surfaces like polystyrene and multiply. However, these culture vessels only provide a very limited surface area for adherent cells to grow, limiting the number of cells that can be expanded and cultured. Alternatively, microcarriers made from natural and / or synthetic polymers can provide a growth surface for adherent cells. Microcarriers used for cell culture are typically microspheres or nearly spherical microparticles with a diameter of 100-300 microns, and can be solid or porous.

[0004] Christian Weber reported that human mesenchymal stem cell lines were cultured on various microcarriers and then digested and recovered using trypsin, accutase, collagenase, or a mixture of these. The results showed that cell recovery efficiency from the dextran microcarriers Cytodex 1 and Cytodex 3 was extremely low, with only 5%-25% of cells being recovered from the microcarrier surface. We also found that it was difficult to separate mesenchymal stem cells using these proteases; even with extended enzymatic treatment times, cell recovery rates remained below 50%. Technical issues

[0005] In summary, although the various microcarriers currently on the market can support the expansion of adherent cells, they have one or more of the following problems: (1) The efficiency of recovering cells from microcarriers after cell expansion is low; (2) Residual substances on microcarriers and animal-derived substances in microcarriers increase the safety risks of cell products or biological products produced by cells; (3) The large-scale use of high-priced raw materials such as recombinant proteins or peptides makes the manufacturing cost of microcarriers high. Technical Solutions

[0006] In order to address the defects and shortcomings of the existing technology, the present invention provides a microcarrier and its preparation method and application, as well as a cell adherent culture and recovery method, which promotes the expansion and efficient recovery of adherent cells, does not contain animal-derived substances, and has low production costs.

[0007] The technical solution adopted by the present invention is: the present invention provides a microcarrier, which is a cross-linked microparticle obtained by cross-linking polysaccharide molecules. The microcarrier can maintain the structural stability of the polysaccharide microcarrier in a cell culture environment, and the microcarrier can be completely dissolved under the action of an enzyme.

[0008] In a preferred embodiment of the present invention, the microcarriers are completely dissolved under the action of the enzyme within 2 hours.

[0009] In a preferred embodiment of the present invention, the cross-linking degree of the polysaccharide molecules in the microcarrier is 13.1%-44.2%.

[0010] In another preferred embodiment of the present invention, the cross-linked microparticles are cross-linked microspheres or approximately spherical cross-linked microparticles.

[0011] In another preferred embodiment of the present invention, the microcarrier can maintain the microsphere or nearly spherical structure of the polysaccharide microcarrier stably for more than three days under a conventional cell culture environment.

[0012] In another preferred embodiment of the present invention, the microcarrier can maintain the microsphere or nearly spherical structure of the polysaccharide microcarrier stably for more than three days in a cell culture environment at 25-41°C.

[0013] In another preferred embodiment of the present invention, the degree of cross-linking of the polysaccharide molecules of the microcarrier enables the microcarrier to maintain the structure of the polysaccharide microcarrier in a cell culture environment and to be completely dissolved under the action of an enzyme. The degree of cross-linking is the ratio of the number of monosaccharide residues that undergo cross-linking reactions with the cross-linking agent or between monosaccharide residues among the monosaccharide residues constituting the polysaccharide to the total number of monosaccharide residues.

[0014] In another preferred embodiment of the present invention, the microcarriers can be completely dissolved under the action of the enzyme within 30 minutes.

[0015] In another preferred embodiment of the present invention, the cross-linking degree of the polysaccharide molecules in the microcarrier is 13.1%-38.7%.

[0016] In another preferred embodiment of the present invention, the cell culture environment is capable of maintaining a cell survival rate of more than 90%.

[0017] In another preferred embodiment of the present invention, the microcarriers can be completely dissolved by enzymes under the conditions of 10-39° C. and pH 6.5-8.0.

[0018] In another preferred embodiment of the present invention, the enzyme is polysaccharidase.

[0019] In another preferred embodiment of the present invention, the polysaccharidase comprises one or more of amylase, cellulase, glucanase, alginate, chitosanase, hyaluronidase, agarase, and pectinase.

[0020] In another preferred embodiment of the present invention, the surface of the microcarrier is smooth or porous.

[0021] In another preferred embodiment of the present invention, the pore size of the porous structure is greater than 1µm and less than 100µm.

[0022] In another preferred embodiment of the present invention, the pore size of the porous structure is controlled by conditions including but not limited to freezing temperature and freezing speed.

[0023] In another preferred embodiment of the present invention, the surface of the microcarrier is modified.

[0024] In another preferred embodiment of the present invention, the cross-linking degree of the polysaccharide molecules in the surface-modified microcarriers is 13.6-44.3%.

[0025] In another preferred embodiment of the present invention, the surface modification of the microcarrier is one or more of positively charged groups or molecules chemically bonded to the surface, or physically adsorbed positively charged groups or molecules, or proteins, polypeptides or polysaccharides that promote cell attachment through chemical bonding or physical adsorption.

[0026] In another preferred embodiment of the present invention, the positively charged groups or molecules bonded to the surface via chemical bonds include but are not limited to primary amines, secondary amines, tertiary amines, and quaternary ammonium salts.

[0027] In another preferred embodiment of the present invention, the physically adsorbed positively charged groups or molecules include, but are not limited to, cationic polymers selected from diethylaminoethyl dextran, poly-L-ornithine (PLO), poly-D-ornithine (PDO), poly-DL-ornithine, poly-D-lysine (PDL), poly-L-lysine (PLL), poly-DL-lysine, poly-L-arginine (PLA), poly-D-arginine (PDA), poly-DL-arginine, poly-L-homoarginine (PLHA), poly-D-homoarginine (PDHA), poly-DL-homoarginine, poly-L-histidine (PLH), poly-D-histidine (PDH), poly-DL-histidine, polymethylene-CO-guanidine (PMCG), polyallylamine (PAA), polyvinylamine (PVA), polyethyleneimine, allylamine-diallylamine copolymer, and allylamine-maleic acid copolymer.

[0028] In another preferred embodiment of the present invention, the cell attachment promoting proteins or polypeptides include but are not limited to collagen, gelatin, laminin, fibronectin, vitronectin, fragments of collagen, gelatin, laminin, fibronectin or vitronectin that promote cell attachment, and polypeptides containing arginine aspartic acid peptides.

[0029] In another preferred embodiment of the present invention, the polysaccharide comprises one or more of the following substances: starch, cellulose, dextran, sodium alginate, chitosan, hyaluronic acid, agarose, polygalacturonic acid and pectin, and derivatives of starch, cellulose, dextran, sodium alginate, chitosan, hyaluronic acid, agarose, polygalacturonic acid and pectin.

[0030] In another preferred embodiment of the present invention, the cross-linking method includes one or more of the following methods: chemical cross-linking, photocross-linking, dehydrogenation thermal cross-linking and ionic cross-linking.

[0031] In another preferred embodiment of the present invention, the chemical cross-linking utilizes cross-linking agents including but not limited to the following to cross-link or fix the chain structure of the polysaccharide molecules: 1-3-dichloropropanol, N,N′-methylenebisacrylamide, 2,3-dibromo-1-propanol, 1,2,7,8-diepoxyoctane, divinyl sulfone, glutaraldehyde, 1,4-butanediol diglycidyl ether, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, epichlorohydrin, tannic acid, and genipin.

[0032] In another preferred embodiment of the present invention, the photocrosslinking uses a light source that can trigger a reaction between the crosslinking agent and the polysaccharide molecules.

[0033] In another preferred embodiment of the present invention, the light source is ultraviolet light.

[0034] In another preferred embodiment of the present invention, the dehydrogenation thermal crosslinking is carried out in a near vacuum environment and at high temperature.

[0035] The present invention also provides an application of a microcarrier in culturing adherent cells.

[0036] In a preferred embodiment of the present invention, the cultured cells include but are not limited to insect cells, mammalian cells, avian cells, and human cells.

[0037] In another preferred embodiment of the present invention, the culture temperature of the mammalian cells or human cells is 36-38 degrees and the pH is 6.8-8.2.

[0038] In another preferred embodiment of the present invention, the method comprises:

[0039] The polysaccharide solution, the organic solvent immiscible with water and the emulsifier liquid are processed to form a water-in-oil emulsion, and a cross-linking agent is added to cause a cross-linking reaction of the polysaccharide;

[0040] Alternatively, a cross-linking agent is first added to the polysaccharide solution to cause a cross-linking reaction of the polysaccharide, and then the cross-linked polysaccharide solution, an organic solvent immiscible with water, and an emulsifier are treated to form a water-in-oil emulsion.

[0041] In another preferred embodiment of the present invention, the method of forming the water-in-oil emulsion includes but is not limited to mechanical stirring, homogenization emulsification, membrane emulsification, spraying, ultrasonic emulsification, ultrasonic spraying, and microfluidics.

[0042] In another preferred embodiment of the present invention, the method further comprises washing to remove impurities other than the organic solvent and the microcarriers.

[0043] In another preferred embodiment of the present invention, the concentration of polysaccharide in the polysaccharide solution is 10%-50% w / w.

[0044] In another preferred embodiment of the present invention, the method further comprises, after or simultaneously with the addition of the cross-linking agent, adding a positively charged substance or a substance that promotes cell attachment, so that the surface of the polysaccharide microspheres is provided with positive charges or cell attachment molecules through physical adsorption or chemical reaction.

[0045] In another preferred embodiment of the present invention, the organic solvent immiscible with water is vegetable oil.

[0046] In another preferred embodiment of the present invention, the emulsifier includes but is not limited to lecithin, carrageenan, guar gum, xanthan gum, polysorbate, cellulose, fatty acid monoglycerides and diglycerides, sucrose esters and sucrose glycerides, fatty acid polyglycerol esters, polyglycerol polyricinoleate, stearoyl lactylate, and sorbitan ester.

[0047] In another preferred embodiment of the present invention, the volume fraction of the polyglycerol polyricinoleate in the organic solvent is 1-10%.

[0048] In another preferred embodiment of the present invention, the cross-linking agent is epichlorohydrin.

[0049] In another preferred embodiment of the present invention, the molar ratio of epichlorohydrin to monosaccharide residues in the polysaccharide is 0.23-0.53:1.

[0050] In another preferred embodiment of the present invention, the cross-linking reaction time is greater than 20 hours.

[0051] The present invention also provides an application of a microcarrier as an adherent cell culture material.

[0052] The present invention also provides a method for cell adherence culture and cell recovery on a microcarrier, wherein the microcarriers, culture medium and adherent cells are mixed and cultured, and then the microcarriers are washed, and polysaccharidase and enzymes for decomposing proteins or polypeptides are added simultaneously or stepwise to dissolve the microcarriers, confirm that the microcarriers are completely dissolved, and then the adherent cells are recovered.

[0053] The present invention also provides a method for dissolving microcarriers, wherein the microcarriers whose surfaces are modified with proteins or polypeptides that promote cell attachment are dissolved by using polysaccharidase and enzymes that decompose proteins or polypeptides simultaneously or stepwise. Beneficial effects

[0054] The beneficial effects of the present invention are: the present invention provides a microcarrier and a preparation method and application as well as a cell adhesion culture and recovery method. The prepared microcarrier can be completely dissolved. Compared with the existing microcarriers, the cell adhesion culture and cell recovery efficiency of the microcarrier and method of the present application can reach more than 95%, and the use of polysaccharide raw materials avoids the introduction of animal-derived pathogens into cell products or cell-derived biological products. At the same time, the manufacturing cost is significantly lower than that of raw materials such as recombinant gelatin and recombinant collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 shows various microcarriers before and after enzyme treatment for half an hour.

[0056] Figure 2 shows cell attachment and live / dead staining (left: phase contrast micrograph; center: live cells stained green; right: dead cells stained red).

[0057] Figure 3 shows cell expansion using microcarriers.

[0058] Figure 4 shows microcarriers and cells before (left) and after (right) enzyme treatment.

[0059] Figure 5 shows the staining of cell attachment (left), live cells (center), and dead cells (right) on various microcarriers.

[0060] Figure 6 shows positively charged modified microcarriers and cells before (left) and after (right) enzyme treatment.

[0061] FIG7 shows a microcarrier with a porous surface structure. Modes for Carrying Out the Invention

[0062] The present invention is further described below with reference to the accompanying drawings and the following embodiments. It should be understood that the accompanying drawings and the following embodiments are only used to illustrate the present invention, rather than to limit the present invention.

[0063] Polysaccharides can be extracted from plants or produced by microbial fermentation. They are widely available, have low production costs, and exhibit excellent biocompatibility, making them suitable for large-scale preparation of cell culture microcarriers. The hydroxyl, amino, and carboxyl groups present in polysaccharide molecules can be used to immobilize the polysaccharide molecules within polysaccharide microspheres through the use of chemical crosslinkers or physical crosslinking methods, preventing dissolution of the polysaccharide microcarriers during cell culture. By controlling the type, content, temperature, and time of the crosslinking reaction, the degree of crosslinking of the polysaccharide in the microcarriers can be controlled. This not only maintains the structure of the polysaccharide microcarriers in the cell culture environment, but also allows for complete dissolution of the microcarriers by polysaccharidase after cell expansion and culture, enabling efficient cell recovery.

[0064] Polysaccharides suitable for preparing microcarriers include starch, cellulose, dextran, sodium alginate, chitosan, hyaluronic acid, agarose, polygalacturonic acid and pectin and its derivatives.

[0065] To prepare microcarriers, it is necessary to form droplets of a polysaccharide solution to control the size and shape of the microcarriers. Methods for forming polysaccharide solution droplets or water-in-oil emulsions include, but are not limited to, mechanical stirring, homogenous emulsification, membrane emulsification, spraying, ultrasonic emulsification, ultrasonic spraying, and microfluidics. In the emulsification method, the polysaccharide solution droplets are formed in a water-immiscible organic solvent. Water-immiscible organic solvents include, but are not limited to, toluene, mineral oil, paraffin oil, rapeseed oil, corn oil, cottonseed oil, safflower oil, soybean oil, extra virgin olive oil, sunflower oil, palm oil, MCT oil, and triolein. To maintain the stability of the resulting emulsion, an emulsifier is added during the emulsification process. These include, but are not limited to, lecithin, carrageenan, guar gum, xanthan gum, polysorbates, cellulose (including carboxymethyl cellulose), mono- and diglycerides of fatty acids, sucrose esters and sucrose glycerides, polyglycerol esters of fatty acids, polyglycerol polyricinoleate, stearoyl lactylate, and sorbitan esters.

[0066] Methods for cross-linking polysaccharides include chemical crosslinking, photo-crosslinking, dehydrothermal crosslinking, and ionic crosslinking. Chemical cross-linking uses cross-linking agents such as glutaraldehyde, 1,4-Butanediol diglycidyl ether, 1-Ethyl-3-[3-dimethylaminopropyl] Carbodiimide Hydrochloride (EDC), N-hydroxy-succinimide (NHS), epichlorohydrin, tannic acid, and genipin to cross-link or fix the chain structure of polysaccharide molecules. Photocross-linking uses a light source such as ultraviolet light to trigger a reaction between the cross-linker and the polysaccharide molecules. Dehydrogenative thermal cross-linking uses a near-vacuum environment and high temperature to react the carboxyl, hydroxyl, and amino groups on the polymer, thereby cross-linking the polymer.

[0067] The degree of cross-linking of polysaccharides in microcarriers can be defined as the ratio of the number of monosaccharide residues that undergo cross-linking reactions with a cross-linking agent or between functional groups of the monosaccharide residues to the total number of monosaccharide residues in the polysaccharide. Functional groups such as hydroxyl and carboxyl groups in the monosaccharide residues can form chemical bonds with cross-linking agents, and under certain conditions, chemical bonds can also form between the functional groups of the monosaccharide residues. If the monosaccharide residues that make up the polysaccharide do not undergo chemical reactions, they can be oxidized with sodium periodate to produce formic acid. By determining the amount of formic acid formed after the oxidation reaction through acid-base titration, the amount of monosaccharide residues that did not undergo chemical reactions can be determined, thereby calculating the amount of monosaccharide residues that did undergo chemical reactions and the degree of cross-linking of the polysaccharide. Assuming the mass of the polysaccharide is m (g), and the formic acid produced after oxidation is n (mol), we can infer that n (mol) of monosaccharide residues have not undergone chemical reactions, and their ratio to all monosaccharide residues in the polysaccharide (where the molecular weight of the monosaccharide residue is A) is n*A / m. The ratio of monosaccharide residues that have undergone chemical reactions is calculated as 1-n*A / m, which is the degree of polysaccharide cross-linking defined here. Furthermore, methods for measuring the degree of polysaccharide cross-linking include, but are not limited to, nuclear magnetic resonance (NMR) and total ion chromatography (TIC).

[0068] In order to promote cell attachment to the surface of the microcarrier, the surface of the microcarrier can be made to carry positive charges or molecules such as proteins and polypeptides that promote cell attachment. Positively charged groups include primary amines, secondary amines, tertiary amines and quaternary ammonium salts, which can be connected to the surface of the microcarrier through chemical reactions; in addition, by adsorbing polymers with positively charged groups, such as diethylaminoethyl dextran, poly-L-ornithine (PLO), poly-D-ornithine (PDO), poly-DL-ornithine, poly-D-lysine (PDL), poly-L-lysine (PLL), poly-DL-lysine, poly-L-arginine (PLA), poly-D-arginine (PDA), poly-DL-arginine Cationic polymers such as poly-L-homoarginine (PLHA), poly-D-homoarginine (PDHA), poly-DL-homoarginine, poly-L-histidine (PLH), poly-D-histidine (PDH), poly-DL-histidine, polymethylene-CO-guanidine (PMCG), polyallylamine (PAA), polyvinylamine (PVA), polyethyleneimine, allylamine-diallylamine copolymer, and allylamine-maleic acid copolymer can also enable cells with negatively charged surfaces to attach to microcarriers. Proteins and peptides that promote cell attachment can be extracted from animal tissues or obtained through recombinant protein technology; they include but are not limited to collagen, gelatin, laminin, fibronectin, vitronectin, or fragments of any of the aforementioned proteins that promote cell attachment, as well as peptides containing arginylglycylaspartic acid (RGD).

[0069] The culture environment for adherent cells using microcarriers varies depending on the type of adherent cells. Generally, the ideal culture temperature for insect cells is 26-30°C, with a pH of 6.0-6.4. The ideal culture temperature for mammalian cells is 36-38°C, with a pH of 6.8-8.2. The ideal culture temperature for avian cells is 39-41°C, with a pH of 7.0-8.5. For example, the optimal temperature for culturing chicken satellite cells is 41°C. Chicken embryonic cells can be subcultured long-term in an environment with a pH of 8.1-8.5. Insect cell growth slows below 26°C, while survival rates decrease above 30°C. Cell viability is the percentage of live cells in a sample. This can be determined by staining for dead and / or live cells and counting with a hemocytometer or automated cell counter.

[0070] Depending on the type of polysaccharide in the microcarriers, the polysaccharidase used to dissolve the microcarriers can be amylase, cellulase, glucanase, alginase, chitosanase, hyaluronidase, agarase, pectinase, or a combination thereof. If the polysaccharide microcarriers are surface-modified with proteins or peptides that promote cell attachment, polysaccharidase and protein- or peptide-dissolving enzymes such as collagenase, trypsin, papain, or TrypLE enzyme can be used simultaneously or sequentially to dissolve the microcarriers. For example, a protein- or peptide-dissolving enzyme can be used to dissolve the protein or peptide in the microcarriers first, followed by a polysaccharidase to dissolve the cross-linked polysaccharide; or alternatively, a polysaccharidase can be used to dissolve the cross-linked polysaccharide first, followed by a protein- or peptide-dissolving enzyme. Enzymatic dissolution of polysaccharide microcarriers should maintain cell viability, such as by ensuring that temperature and pH do not affect cell viability. For example, after culturing human mesenchymal stem cells on microcarriers, the polysaccharide microcarriers can be dissolved at 10-39°C and a pH of 6.5-8. Temperatures above 39°C, pH values ​​below 6.5, or pH values ​​above 8 can easily cause cell death.

[0071] Microscope observation can be used to detect whether the polysaccharide microcarriers are completely dissolved. Complete dissolution here means that no spherical or spherical-like microcarriers can be observed under a conventional optical microscope, as well as no granular matter remaining after the microcarriers are partially dissolved. A conventional optical microscope refers to an optical microscope that uses a 4-20x objective lens and a 10x eyepiece for observation. For microcarrier samples without cultured cells, the microcarriers can be directly treated with polysaccharidase, and after the microcarriers are dissolved, a conventional optical microscope is used to observe whether there are any microcarrier residues. After culturing cells with microcarriers and dissolving the microcarriers with polysaccharidase, cell lysis solution can be used to dissolve the cells, and then a conventional optical microscope can be used to observe whether there are any microcarrier residues.

[0072] The surface of the microcarrier can be smooth or porous. The smooth surface here means that there are no obvious concave and convex structures on the surface of the microcarrier when observed under a conventional optical microscope. This smooth surface allows adherent cells to spread on the surface of the microcarrier and proliferate. The surface of the microcarrier can also be porous, allowing adherent cells to grow inside the pores. The pore size of the microcarrier surface can be 2-100µm, or 10-80µm, or 15-50µm. The pore size of the microcarrier can be controlled by conditions such as the freezing temperature and freezing speed during material preparation. Generally speaking, within the range of 0 to liquid nitrogen temperature, the lower the freezing temperature, the smaller the pore size of the microcarrier material formed.

[0073] Example 1 Preparation of microcarriers

[0074] Dextran was dissolved in 6 mL of 5 M sodium hydroxide solution to obtain a 40% (w / w) dextran solution. Polyglycerol polyricinoleate (PGPR) was dissolved in soybean oil to obtain oil phases with 0.5%, 1%, 2%, 3%, 5%, and 10% (v / v) PGPR. Six 1g portions of dextran solution were added with 1 mL of each of the six oil phases, followed by 50 µL of epichlorohydrin crosslinker. Each mixture was mixed using a high-speed homogenizer at 1000 rpm for 3 minutes. The results showed that oil phases containing 1-10% (v / v) PGPR formed stable water-in-oil emulsions.

[0075] Dextran was dissolved in 6 mL of 5 M sodium hydroxide solution to obtain a 40% (w / w) dextran solution. According to Table 1, four aliquots (1 g) of dextran solution were prepared, and 1 mL of soybean oil and 20 µL of polyglycerol polyricinoleate (PGPR) were added to each aliquot. Then, 100 µL, 75 µL, 50 µL, or 25 µL of epichlorohydrin crosslinker were added. Each of the four mixtures was mixed using a high-speed homogenizer at 1000 rpm for 3 minutes. The resulting emulsions were then transferred to 2 mL centrifuge tubes. Each centrifuge tube was placed in a 50°C oven for cross-linking and removed after 20 hours. The emulsions were transferred to 50 mL centrifuge tubes and washed with 10 mL of acetone for stirring. After stabilization, the supernatant was removed and washed with 10 mL of anhydrous ethanol. The cross-linked dextran microspheres were then washed six times with pure water. Sample 4 partially dissolved during the washing process. 0.04 mL of 0.5 g / mL DEAE-dextran solution was added to the remaining three samples and mixed at room temperature for 2 hours using a rotary mixer to cover the surface of the dextran microspheres with DEAE-dextran molecules to obtain microcarriers. The microcarriers were then washed six times with pure water and twice with phosphate buffered saline (PBS).

[0076] The microcarriers of samples 1 to 3 were shaken in PBS, and 0.1 mL was added to a 96-well plate. The 96-well plate was placed in a 37°C cell culture incubator for 3 days. Using an inverted optical microscope with 4x and 10x magnification objectives, the microcarriers maintained their structural integrity and did not dissolve. A dextranase solution (1000 units / mL) was added to the microcarriers in the 96-well plate and mixed thoroughly. The 96-well plate was then placed in a 37°C cell culture incubator for 30 minutes. The 96-well plate was then removed and observed using an inverted optical microscope with 4x and 10x magnification objectives. The results showed that the microcarriers in sample 1 were only partially dissolved, while the microcarriers in samples 2 and 3 were completely dissolved, with no residual microcarriers or particulate matter observed.

[0077] Table 1. Raw material composition of polysaccharide microcarriers and dissolution results after enzyme treatment

[0078] Sample No. Sample 1 Sample 2 Sample 3 Sample 4 Dextran solution (g) 1111 Soybean oil (mL) 1111 PGPR (µL) 2202020 Cross-linking agent (µL) 100755025 Can be dissolved by enzyme Partially soluble Can be completely soluble Can be completely soluble -

[0079] Example 2 Preparation of microcarriers

[0080] Dextran was dissolved in 6M sodium hydroxide solution to obtain a 30% (w / w) dextran solution. According to Table 2, three aliquots of 1 g of dextran solution were added to each, with 50 µL, 44 µL, or 37.5 µL of epichlorohydrin crosslinker added. 1 mL of soybean oil and 20 µL of polyglycerol polyricinoleate (PGPR) were then added to each aliquot. Each of the four mixtures was mixed using a high-speed homogenizer at 500 rpm for 3 minutes. The resulting emulsions were then added to 2 mL centrifuge tubes. Each centrifuge tube was placed in a 50°C oven for crosslinking and removed after 24 hours. The emulsions were transferred to 50 mL centrifuge tubes and washed with 10 mL of acetone for stirring. After stabilization, the supernatant was removed and washed with 10 mL of anhydrous ethanol. The cross-linked dextran microspheres were then washed six times with pure water. Sample 6 partially dissolved during the washing process. 0.04 mL of 0.5 g / mL DEAE-dextran solution was added to the remaining two samples and mixed at room temperature for 2 hours using a rotary mixer to cover the surface of the dextran microspheres with DEAE-dextran to obtain microcarriers. The microcarriers were then washed six times with pure water and twice with PBS.

[0081] After shaking, 0.1 mL of the microcarriers of Sample 4 and Sample 5 was added to a 96-well plate. The 96-well plate was placed in a 37-degree cell culture incubator for 3 days. Using the 4x and 10x objectives of an inverted optical microscope, it was observed that the microcarriers maintained their structural integrity and did not dissolve. Glucanase solution (1000 u / mL) was added to the microcarriers in the 96-well plate and mixed, and the 96-well plate was placed in a 37-degree cell culture incubator for 30 minutes. The 96-well plate was then removed and observed using the 4x and 10x objectives of an inverted optical microscope. The results showed that the microcarriers of Sample 4 and Sample 5 were completely dissolved, and no residual microcarriers or particles were observed.

[0082] Table 2. Raw material composition of polysaccharide microcarriers and dissolution results after enzyme treatment

[0083] Sample No. Sample 4 Sample 5 Sample 6 Dextran solution (g) 111 Soybean oil (mL) 111 PGPR (µL) 20 20 20 Cross-linking agent (µL) 50 4 4 37.5 Can it be dissolved by enzyme Can be completely dissolved Can be completely dissolved -

[0084] Example 3 Preparation of microcarriers

[0085] Dissolve 40 g of dextran in 60 mL of 5 M sodium hydroxide solution. According to Table 3, eight aliquots (10 g) of the dextran solution were prepared, and 10 mL of soybean oil and 200 µL of polyglycerol polyricinoleate (PGPR) were added to each aliquot. Then, 400 µL, 500 µL, 600 µL, 700 µL, 800 µL, 900 µL, 1200 µL, or 1500 µL of epichlorohydrin crosslinker were added. The molar ratios of epichlorohydrin to monosaccharide residues in the polysaccharide were 0.23:1, 0.29:1, 0.35:1, 0.41:1, 0.47:1, 0.53:1, 0.70:1, and 0.88:1, respectively. Each of the eight aliquots was stirred at 200 rpm for 3 minutes using a mechanical stirrer. The resulting eight emulsions were then added to 50 mL centrifuge tubes. Place each centrifuge tube in a 50-degree oven for cross-linking reaction and take it out after 24 hours. Transfer the emulsion after the reaction to a 500mL beaker, add 50mL of acetone for stirring and washing, remove the supernatant after standing, and add 50mL of anhydrous ethanol for washing. Then wash it with pure water 6 times to obtain cross-linked dextran microspheres. Add 0.4mL of 0.5g / mL DEAE-dextran solution to each sample and use a rotary mixer to mix at room temperature for 2 hours to cover the surface of the dextran microspheres with DEAE-dextran to obtain microcarriers. Then wash the microcarriers with pure water 6 times and then wash them twice with PBS.

[0086] After shaking, 0.1 mL of each of the eight microcarriers was added to a 96-well plate. The 96-well plate was placed in a 37°C cell culture incubator for 3 days. Using an inverted optical microscope with 4x and 10x magnification objectives, the microcarriers maintained their structural integrity and did not dissolve. Dextranase solution was added to the microcarriers in the 96-well plate and mixed thoroughly. The 96-well plate was placed in a 37°C cell culture incubator for 30 minutes. The 96-well plate was then removed and observed using an inverted optical microscope with 4x and 10x magnification objectives.

[0087] After 30 minutes of enzyme treatment, the results showed that the Cr400, Cr500, Cr600, Cr700, and Cr800 microcarrier samples were completely dissolved by the enzyme, with no residual microcarriers or particles observed. Cr900 was only partially dissolved, and the overall size of the microcarriers decreased. The size and shape of the Cr1200 and Cr1500 microcarriers remained unchanged, remaining spherical or nearly spherical (Figure 1). Extending the enzyme treatment time to 2 hours gradually dissolved and disappeared the Cr900 microcarriers, while the size and shape of the Cr1200 and Cr1500 microcarriers remained unchanged (Table 3). Samples that were solubilized within 2 hours corresponded to a molar ratio of epichlorohydrin to monosaccharide residues in the polysaccharide during preparation of 0.23 to 0.53:1.

[0088] Similarly, commercially available Cytodex1 microcarriers (Cytiva) were treated with enzymes for 30 minutes. Under an optical microscope, it was observed that the microcarriers were partially dissolved, leaving behind many granular residues ranging from several micrometers to tens of micrometers, and a small amount of spherical microcarriers (Figure 1). When the enzyme treatment time was extended to 2 hours, the microcarrier residues did not disappear.

[0089] Table 3. Raw material composition of polysaccharide microcarriers and dissolution results after enzyme treatment

[0090] Sample name Cr400Cr500Cr600Cr700Cr800Cr900Cr1200Cr1500 Dextran solution (g) 101010101010101010 Soybean oil (mL) 1010101010101010 PGPR (µL) 200200200200200200200200 Cross-linking agent (µL) 40050060070080090012001500 Can be dissolved by enzyme? Can be completely dissolved? Can be completely dissolved? Can be completely dissolved? Can be completely dissolved? Can be completely dissolved? Can be completely dissolved? Can be completely dissolved? Can be completely dissolved? Can be completely dissolved? Can be completely dissolved? Can be completely dissolved? Can be completely dissolved?

[0091] Example 4 Measurement of cross-linking degree of microcarriers

[0092] Place 40 mL of each of the various microcarriers obtained in Example 3 into a 50 mL beaker. Separately, place 0.3 g of Cytodex 1 microcarriers and 0.3 g of dextran into a 50 mL beaker. Dry the beakers containing the various samples in a forced-air dryer at 70°C for at least 24 hours, until the sample weight changes by less than 0.002 g. Then, transfer 0.200 g of each dried sample to a 100 mL glass bottle.

[0093] Dissolve 5.35g of sodium periodate in 500mL of pure water. Add 50mL of the sodium periodate solution to the dried sample in each glass bottle. Place a magnetic stir bar, cap the bottle, and wrap it with aluminum foil to block light. Place each reaction vessel at 20°C and use magnetic stirring to mix and oxidize the solution. Measure the pH of the reaction solution with a pH meter every 3-6 hours until the pH change between 3 and 6 hours is less than 0.02, indicating the reaction is complete.

[0094] Transfer 10 mL of the supernatant from each 100 mL glass bottle to a beaker, add 1 mL of ethylene glycol, and allow to react at room temperature for at least 15 minutes. Then, perform an acid-base titration on the reacted solution using 0.01 mol / L NaOH solution. Terminate the titration when a pH jump between 6.5 and 8.0 appears. Measure the weight (M g) of NaOH solution consumed during the titration. The density of 0.01 mol / L NaOH solution is close to that of pure water (the difference is less than 1 / 1000), so the volume of NaOH solution consumed is also M mL. Calculate the degree of cross-linking (D) of the dextran microcarrier or dextran: D = 1 - (5 × M × 0.01 × 162 / (1000 × 0.2)).

[0095] The results are shown in Table 4. The calculated cross-linking degree of glucan without cross-linking was only 2.86%, very close to the proportion of α-(1-3) glycosidic bonds in the glucan molecule (approximately 3%). The degree of cross-linking gradually increased with increasing cross-linking agent usage. Within a certain range, the microcarriers were completely soluble after enzyme treatment. Beyond a certain range, the microcarriers were less susceptible to enzyme dissolution. Combined with the results of Example 3, microcarriers with a cross-linking degree between 13.1% and 38.7% were completely soluble by the enzyme in about half an hour, microcarriers with a cross-linking degree of 44.2% required 1-2 hours to be soluble by the enzyme, and microcarriers with a cross-linking degree of 55.1% or more were difficult to be soluble by the enzyme within 2 hours. The cross-linking degree of Cytodex1 microcarriers available on the market was 56.7%, making them only partially soluble by the enzyme.

[0096] Table 4. Degree of cross-linking between various dextran microcarriers and dextran

[0097] Sample name Dextran Cr400Cr500Cr600Cr700Cr800Cr900Cr1200Cr1500Cytodex 1 Cross-linking degree (D×100%) 2.86% 13.1% 19.6% 23.3% 28.9% 38.7% 44.2% 55.1% 66.4% 56.7%

[0098] Example 5: Using microcarriers to culture and recover cells

[0099] The dextran microcarriers (Cr500) from Example 3 were separated using a standard sieve to remove a fraction between 200 and 300 µm. A 0.4 mL aliquot was suspended in 5 mL of PBS and autoclaved at 120°C for 20 minutes. After sterilization, the microcarriers were washed with alpha MEM medium supplemented with 10% fetal bovine serum. 3×10^5 human umbilical cord-derived mesenchymal stem cells (passage P5) were seeded onto the microcarriers and cultured in a cell culture incubator at 37°C and 5% CO2. On the third day of culture, half of the culture medium was removed and replaced with an equal volume of fresh medium. After one, three, and six days, 0.5 mL of the microcarrier-cultured cells were sampled for cell viability staining (Elabscience® Calcein AM / PI Double Staining Kit) and microscopic observation. Cell nuclei were released using a solution containing 0.1% crystal violet and 0.1 mol / L citric acid, and cell counts were performed by counting nuclei. After six days, all microcarriers in the culture vessel were washed with PBS, and 5 mL of TrypLE and 1% dextranase were added. The cells were incubated at 37°C for 20-25 minutes, and the dissolution of the microcarriers was observed. The enzymatically treated cells were counted using trypan blue staining and a hemocytometer to calculate cell viability and recovery efficiency. The cell recovery efficiency is the ratio of the number of recovered cells to the number of cells calculated from the pre-recovery cell sampling. Similarly, the Cytodex1 microcarriers were sterilized and cultured, and cell proliferation and recovery efficiency were compared.

[0100] Figure 3 shows that mesenchymal stem cells adhered well to the Cr500 microcarriers and maintained a high survival rate. After 6 days of culture, the cells expanded approximately 14-fold. Figure 4 shows that after enzyme treatment, the microcarriers were completely dissolved and the cells on their surfaces were isolated. The cell recovery efficiency reached 99%, and the cell survival rate was 98%.

[0101] After 6 days of culture using Cytodex 1, the cells expanded 7.8-fold, and the recovery efficiency of cells from Cytodex 1 microcarriers was only 36%.

[0102] Example 6 Positive Charge Modification of Microcarrier Surface

[0103] Cross-linked dextran microspheres were prepared using the raw materials of sample Cr500 according to the steps of Example 3 and washed six times with pure water. After screening, six 10 mL portions of cross-linked dextran microspheres were collected. 3 mL of 6 M NaOH solution and 0.1 mL, 0.5 mL, 1 mL, 1.5 mL, 1.75 mL, or 2 mL of 1 M 2-dimethylaminoethyl chloride hydrochloride (DEAE) aqueous solution were added to each portion. After mixing, the mixture was stirred in a 60°C water bath for 1 hour. The mixture was then washed six times with pure water and twice with PBS to obtain microcarriers with positively charged surfaces. The microcarriers were dried and the degree of cross-linking was measured using the method described in Example 4. The results showed that the degree of cross-linking of the modified microcarriers ranged from 19% to 22%.

[0104] Table 5. Six microcarriers with positively charged surfaces

[0105] Sample name 0.1D 0.5D 1D 1.5D 1.75D 2D DEAE addition amount (mL) 0.1 0.5 1 1.5 1.75 2

[0106] From each of the six microcarriers, 0.2 mL was suspended in 5 mL of PBS and autoclaved. The microcarriers were then washed twice with the aforementioned cell culture medium and 5 mL of culture medium was added. Each microcarrier was seeded with 3 × 10^5 human umbilical cord-derived mesenchymal stem cells (passage P5) and cultured in a cell culture incubator at 37°C and 5% CO2. After one and six days, 0.5 mL of the microcarrier culture medium was sampled for cell viability staining, microscopic observation, and cell counting. Then, 2 mL of TrypLE and 1% dextranase were added and incubated at 37°C for 20–25 minutes. The microcarriers were then observed for dissolution.

[0107] Figure 5 shows that when the positive charge on the surface of the microcarriers (0.5D) is too low, cells are less likely to attach to the microcarriers. As the positive charge increases, cells more readily attach to the microcarriers (sample 1D). However, microcarriers with excessive positive charge (sample 2D) can reduce cell survival. Figure 6 shows that the positively charged microcarriers can be completely dissolved by enzymes, releasing cells from their surfaces. The recovery efficiency of cells from the microcarriers is over 96%.

[0108] Example 7 Positively charged microcarriers with different cross-linking degrees

[0109] Six 10 mL portions of the unmodified, positively charged dextran microspheres (Cr400, Cr500, Cr600, Cr700, Cr800, and Cr900) obtained in Example 3 were screened and added to each portion. 3 mL of 6M NaOH solution and 1 mL of 1M 2-Dimethylaminoethyl chloride hydrochloride (DEAE) aqueous solution were added. The mixture was stirred in a 60°C water bath for 1 hour. The microspheres were then washed six times with pure water and twice with PBS to obtain microcarriers with varying degrees of cross-linking modified with positive charges. The microcarriers were dried and the degree of cross-linking was measured using the method described in Example 4. The results showed that the degree of cross-linking of the modified microcarriers ranged from 13.6% to 44.3%.

[0110] Cells were cultured and recovered using the cell culture and recovery methods described in Example 5 on positively charged microcarriers with varying degrees of cross-linking. During cell recovery, microcarriers corresponding to Cr400, Cr500, Cr600, Cr700, and Cr800 were enzymatically dissolved within 30 minutes, while microcarriers corresponding to Cr900 were enzymatically dissolved within 1-2 hours. The cell recovery efficiency was consistently above 95%.

[0111] Example 8 Microcarriers with Surface Modified Cell Attachment Proteins

[0112] According to the steps of Example 3, cross-linked dextran microspheres were prepared using the raw materials of sample Cr500 and washed 6 times with pure water. Take two 2mL portions of cross-linked dextran microspheres, add 1mL of 2% mass fraction recombinant gelatin solution or 1mL of 25µg / mL recombinant Vitronectin, add 1mL of an aqueous solution containing 50mM EDC and 20mM NHS, and react by stirring at room temperature for 6 hours. Afterwards, wash with pure water 6 times to obtain two microcarriers whose surfaces are modified with cell attachment proteins. Use the method described in Example 4 to dry the microcarriers and measure the degree of cross-linking. The results showed that the degrees of cross-linking of the microcarriers modified with recombinant gelatin or recombinant Vitronectin were 19.7% and 20.1%, respectively.

[0113] Mesenchymal stem cells were cultured for 6 days using microcarriers containing at least 0.2 mL of culture medium according to the cell culture method described in Example 5. After adding 2 mL of TrypLE solution and a 1% volume fraction of dextranase solution and incubating at 37°C for 20 minutes, the microcarriers were completely dissolved. The cell recovery rates from the recombinant gelatin- or recombinant Vitronectin-modified microcarriers were 97% and 99%, respectively, and the cell viabilities were 98% and 99%, respectively.

[0114] Example 9 Preparation of Surface Porous Microcarriers

[0115] Dextran microcarriers were prepared according to the procedures of Example 3 using the raw material of sample Cr500. The microcarriers were rinsed with pure water and placed in a -80°C ultra-low temperature freezer for 12 hours. The frozen microcarriers were freeze-dried in a freeze dryer at -50°C, 20 Pa, for 48 hours. A small amount of the microcarriers was then soaked in PBS for observation.

[0116] Optical microscopy revealed a porous structure with pore sizes ranging from 10 to 80 µm on the microcarrier surface (Figure 7). The microcarriers were thoroughly dried and the degree of cross-linking was measured using the method described in Example 4. The results showed a cross-linking degree of 19.4% for the porous microcarrier surfaces.

[0117] Example 10 Preparation of pectin microcarriers

[0118] Pectin was dissolved in 30 mL of 6 mol / L sodium hydroxide solution to obtain a 15% mass fraction pectin solution. To 10 g of the pectin solution, 10 mL of soybean oil and 200 µL of polyglycerol polyricinoleate (PGPR) were added, followed by 500 µL of epichlorohydrin crosslinker. Emulsification, crosslinking, and cleaning were performed according to the procedures in Example 3 to obtain cross-linked pectin microcarriers. The microcarriers were dried and the degree of crosslinking was measured using the method described in Example 4. The degree of crosslinking (D) of the microcarriers was calculated using the following formula: D = 1 - (5 × M × 0.01 × 176 / (1000 × 0.2)), where M is the volume (mL) of 0.01 M NaOH consumed in the acid-base titration, 176 is the molecular weight of the monosaccharides that make up the pectin, and 0.2 is the mass (g) of the dry pectin microcarriers. The results showed that the degree of crosslinking of the pectin microcarriers was 21.6%.

[0119] Mesenchymal stem cells were cultured for 6 days using microcarriers (0.2 mL or more) according to the cell culture method described in Example 5. After addition of 2 mL of TrypLE solution and 1% pectinase solution, the cells were incubated at 37°C for 20 minutes. After this, the microcarriers were completely dissolved, resulting in a cell recovery rate of 95% and a cell viability of 96%.

[0120] Example 11 Cultivation of 293T cells using microcarriers

[0121] Dextran microcarriers (Cr600) were screened and sterilized according to Example 5. After sterilization, the microcarriers were washed with DMEM supplemented with 10% fetal bovine serum. 3×10^5 293T cells were seeded onto the microcarriers and cultured in a cell culture incubator at 37°C and 5% CO2. After one, three, and six days, 0.5 mL of the culture medium was removed from the microcarriers for viability staining and microscopic observation. On the third day of culture, half of the culture medium was removed and an equal amount of fresh medium was added. After six days of culture, cells were sampled and counted. The microcarriers were then solubilized using TrypLE and 1% dextranase, and the cells were recovered. Cell survival and recovery efficiency were calculated. Microscopic observation showed that the 293T cells adhered well to the microcarriers, with a cell survival rate exceeding 90%. After enzymatic solubilization of the microcarriers, the cell recovery rate was 95%.

[0122] Technical personnel should note: Although the present invention has been described according to the above specific implementation methods, the inventive concept of the present invention is not limited to this invention. Any modification using the inventive concept will be included in the scope of protection of this patent.

[0123] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiment. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A microcarrier, characterized in that The microcarrier is a cross-linked microparticle obtained by cross-linking polysaccharide molecules. The microcarrier can maintain the structural stability of the polysaccharide microcarrier in a cell culture environment, and the microcarrier can be completely dissolved under the action of an enzyme.

2. The microcarrier according to claim 1, characterized in that The time for the microcarrier to completely dissolve under the action of the enzyme is within 2 hours.

3. The microcarrier according to claim 1, characterized in that The cross-linking degree of the polysaccharide molecules in the microcarrier is 13.1%-44.2%.

4. The microcarrier according to claim 1, characterized in that The cross-linked microparticles are cross-linked microspheres or cross-linked microparticles that are approximately spherical.

5. The microcarrier according to claim 4, characterized in that The microcarrier can maintain the microsphere or nearly spherical structure of the polysaccharide microcarrier stably for more than three days under a conventional cell culture environment.

6. The microcarrier according to claim 5, characterized in that The microcarrier can maintain the microsphere or nearly spherical structure of the polysaccharide microcarrier stably for more than three days in a cell culture environment of 25-41°C.

7. The microcarrier according to claim 3, characterized in that The cross-linking degree is the ratio of the number of monosaccharide residues that undergo cross-linking reactions with the cross-linking agent or between monosaccharide residues to the number of all monosaccharide residues in the monosaccharide residues constituting the polysaccharide.

8. The microcarrier according to claim 2, characterized in that The time for the microcarrier to be completely dissolved under the action of the enzyme is within 30 minutes.

9. The microcarrier according to claim 8, characterized in that The cross-linking degree of the polysaccharide molecules in the microcarrier is 13.1%-38.7%.

10. The microcarrier according to claim 1, characterized in that The surface of the microcarrier is modified.

11. The microcarrier according to claim 10, characterized in that The surface modification of the microcarrier is a positively charged group or molecule bonded to the surface by chemical bonds, or a positively charged group or molecule physically adsorbed, or one or more of proteins, polypeptides or polysaccharides that promote cell attachment by chemical bonds or physical adsorption.

12. The microcarrier according to claim 10, characterized in that The cross-linking degree of the polysaccharide molecules in the microcarrier with modified surface is 13.6-44.3%.

13. The microcarrier according to claim 11, characterized in that The physically adsorbed positively charged groups or molecules include, but are not limited to, cationic polymers in diethylaminoethyl dextran, poly-L-ornithine (PLO), poly-D-ornithine (PDO), poly-DL-ornithine, poly-D-lysine (PDL), poly-L-lysine (PLL), poly-DL-lysine, poly-L-arginine (PLA), poly-D-arginine (PDA), poly-DL-arginine, poly-L-homoarginine (PLHA), poly-D-homoarginine (PDHA), poly-DL-homoarginine, poly-L-histidine (PLH), poly-D-histidine (PDH), poly-DL-histidine, polymethylene-CO-guanidine (PMCG), polyallylamine (PAA), polyvinylamine (PVA), polyethyleneimine, allylamine-diallylamine copolymer and allylamine-maleic acid copolymer.

14. The microcarrier according to claim 11, characterized in that The cell attachment-promoting proteins or polypeptides include, but are not limited to, collagen, gelatin, laminin, fibronectin, vitronectin, fragments of collagen, gelatin, laminin, fibronectin or vitronectin that promote cell attachment, and polypeptides containing arginine aspartate peptides.

15. The microcarrier according to claim 1, characterized in that The polysaccharide includes one or more of the following substances: starch, cellulose, dextran, sodium alginate, chitosan, hyaluronic acid, agarose, polygalacturonic acid and pectin and derivatives of starch, cellulose, dextran, sodium alginate, chitosan, hyaluronic acid, agarose, polygalacturonic acid and pectin.

16. The microcarrier according to claim 1, characterized in that The cross-linking method is one or more of chemical cross-linking, photo cross-linking, dehydrogenation thermal cross-linking, and ion cross-linking.

17. The microcarrier according to claim 16, characterized in that The chemical cross-linking uses cross-linking agents including but not limited to the following to cross-link or fix the chain structure of the polysaccharide molecules: 1-3-dichloropropanol, N,N′-methylenebisacrylamide, 2,3-dibromo-1-propanol, 1,2,7,8-diepoxyoctane, divinyl sulfone, glutaraldehyde, 1,4-butanediol diglycidyl ether, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, epichlorohydrin, tannic acid, and genipin.

18. The microcarrier according to claim 16, characterized in that The photo-crosslinking uses a light source that can trigger a reaction between the crosslinking agent and the polysaccharide molecules.

19. The microcarrier according to claim 16, characterized in that The dehydrogenation thermal crosslinking is carried out in a near vacuum environment and at high temperature.

20. The microcarrier according to claim 1, characterized in that The surface of the microcarrier is smooth or porous.

21. The microcarrier according to claim 20, characterized in that The pore size of the porous structure is 1-100 μm.

22. The microcarrier according to claim 21, characterized in that The pore size of the porous structure is controlled by conditions including but not limited to freezing temperature and freezing speed.

23. A method for preparing the microcarrier according to claim 1, characterized in that: The following steps are involved: The polysaccharide solution, the organic solvent immiscible with water and the liquid of the emulsifier are processed to form a water-in-oil emulsion, and a cross-linking agent is added to cause a cross-linking reaction of the polysaccharide; Alternatively, a cross-linking agent is first added to the polysaccharide solution to cause a cross-linking reaction of the polysaccharide, and then the cross-linked polysaccharide solution, an organic solvent immiscible with water and an emulsifier are treated to form an oil-in-water emulsion.

24. The preparation method according to claim 23, characterized in that: The concentration of polysaccharide in the polysaccharide solution is 10%-50% w / w.

25. The preparation method according to claim 23, characterized in that: The method also includes adding positively charged substances or substances that promote cell adhesion after or while adding the crosslinking agent, so that the surface of the polysaccharide microspheres has positive charges or cell adhesion molecules through physical adsorption or chemical reaction.

26. The preparation method according to claim 23, characterized in that: The organic solvent that is immiscible with water is vegetable oil.

27. The preparation method according to claim 23, characterized in that: The emulsifier includes but is not limited to lecithin, carrageenan, guar gum, xanthan gum, polysorbate, cellulose, fatty acid monoglycerides and diglycerides, sucrose esters and sucrose glycerides, fatty acid polyglycerol esters, polyglycerol polyricinoleate, stearoyl lactylate, and sorbitan ester.

28. The preparation method according to claim 27, characterized in that: The volume fraction of the polyglycerol polyricinoleate in the organic solvent is 1-10%.

29. The preparation method according to claim 23, characterized in that: The cross-linking agent is epichlorohydrin.

30. The preparation method according to claim 29, characterized in that: The molar ratio of epichlorohydrin to monosaccharide residue in polysaccharide is 0.23-0.53:

1.

31. The preparation method according to claim 23, characterized in that: The cross-linking reaction time is greater than 20 hours.

32. Use of the microcarrier according to any one of claims 1 to 22 as a culture material for adherent cells.

33. The use according to claim 32, characterized in that The microcarrier can be completely dissolved by enzyme under the conditions of 10-39°C and pH 6.5-8.

0.

34. A method for cell adherence culture and cell recovery using the microcarrier according to any one of claims 1 to 22, characterized in that: The method comprises the following steps: mixing microcarriers, culture medium and adherent cells, carrying out cell culture, then washing the microcarriers, simultaneously or stepwise adding polysaccharide enzymes and enzymes for decomposing proteins or polypeptides to dissolve the microcarriers, confirming that the microcarriers are completely dissolved, and recovering the adherent cells.

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