Microcarriers, methods of making and using, and methods of adherent cell culture and recovery

Abstract

The application relates to a kind of microcarriers and preparation method and application and cell adherent culture and recovery method, the microcarrier prepared can be used for cell culture, while cell culture, the survival rate of cell can be maintained, can be quickly and completely dissolved, surface has modification, the application also provides the preparation of the microcarrier and the use of the microcarrier in cell adherent culture, compared with the existing microcarrier, cell adherent culture is carried out using the microcarrier and method of the application, and the recovery efficiency of cell can reach more than 95%, and polysaccharide raw material is used to avoid introducing animal source pathogen into cell product or cell source biological product, and the manufacturing cost is significantly lower than that of recombinant gelatin, recombinant collagen and other raw materials.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a microcarrier, its preparation method and application, and a method for cell adhesion culture and recovery. Background Technology

[0002] Cell culture is divided into two main categories: adherent culture, also known as monolayer culture or planar culture, and suspension culture. Through cell culture, cell therapy products such as stem cells, immune cells, and genetically modified cells can be produced to treat diseases, as well as cell-derived biological products such as viral vectors, vaccines, antibodies, peptide drugs, exosomes, and microvesicles.

[0003] Many cells derived from species such as humans, mammals, and insects require attachment to the surface of materials to grow and proliferate; these cells are called adherent cells. Containers such as tissue culture dishes, tissue culture flasks, and cell factories allow adherent cells to attach to the surface of flat materials like polystyrene and proliferate. However, these culture containers only provide a very limited growth surface for adherent cells, limiting the number of cells after expansion culture. Alternatively, microcarriers prepared from natural and / or synthetic polymers can provide a growth surface for adherent cells. Microcarriers used for cell culture are generally microspheres or near-spherical microparticles with a diameter of 100-300 micrometers; they can be solid or porous.

[0004] Christian Weber reported on the digestion and recovery of human mesenchymal stem cell lines cultured using various microcarriers, followed by digestion and recovery using trypsin, accutase, collagenase, or mixtures thereof. The results showed extremely low cell recovery efficiency from dextran microcarriers Cytodex 1 and Cytodex 3, with only 5%–25% of cells recoverable from the microcarrier surface. We also found that it was difficult to separate mesenchymal stem cells using these proteases; even with prolonged enzyme treatment, cell recovery rates remained below 50%.

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

[0006] To address the shortcomings and deficiencies of existing technologies, this invention provides a microcarrier, its preparation method and application, as well as a method for cell adherence culture and recovery, which promotes adherent cell expansion and efficient recovery, and is free of animal-derived substances and has low production costs.

[0007] The technical solution adopted in this invention is as follows: This invention provides a microcarrier, wherein 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 enzymes.

[0008] In a preferred embodiment of the present invention, the microcarrier is completely dissolved within 2 hours under the action of enzymes.

[0009] In a preferred embodiment of the present invention, the degree of cross-linking of 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 stability of the microsphere or near-spherical structure of the polysaccharide microcarrier for more than three days under conventional cell culture conditions.

[0012] In another preferred embodiment of the present invention, the microcarrier can maintain the stability of the microsphere or near-spherical structure of the polysaccharide microcarrier 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 its structure under cell culture conditions and to be completely dissolved under the action of enzymes. The degree of cross-linking is the proportion of monosaccharide residues that undergo cross-linking reactions with cross-linking agents or with each other among the monosaccharide residues that make up the polysaccharide to the total number of monosaccharide residues.

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

[0015] In another preferred embodiment of the present invention, the degree of cross-linking of 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 cell survival.

[0017] The success rate is over 90%.

[0018] In another preferred embodiment of the present invention, the microcarrier can be completely dissolved by an enzyme under conditions of 10-39°C and pH 6.5-8.0.

[0019] In another preferred embodiment of the present invention, the enzyme is a polysaccharide enzyme.

[0020] In another preferred embodiment of the present invention, the polysaccharide enzyme includes amylase, cellulase, and dextran.

[0021] One or more of the following: glycosylase, alginate, chitosanase, hyaluronidase, agarosease, and pectinase.

[0022] In another preferred embodiment of the present invention, the surface of the microcarrier is smooth or porous.

[0023] In another preferred embodiment of the present invention, the pore size of the porous structure is greater than 1µm and less than 1µm.

[0024] 100µm.

[0025] In another preferred embodiment of the present invention, the pore size of the porous structure is determined by means including but not limited to cold...

[0026] The freezing temperature and freezing speed are controlled.

[0027] In another preferred embodiment of the present invention, the surface of the microcarrier is modified.

[0028] In another preferred embodiment of the present invention, the degree of cross-linking of polysaccharide molecules in the surface-modified microcarrier is 13.6-44.3%.

[0029] In another preferred embodiment of the present invention, the surface modification of the microcarrier is one or more of the following: positively charged groups or molecules bonded to the surface by chemical bonds, or positively charged groups or molecules physically adsorbed, or proteins, polypeptides or polysaccharides that promote cell attachment through chemical bonding or physical adsorption.

[0030] In another preferred embodiment of the present invention, the positively charged groups or molecules bonded to the surface by chemical bonds include, but are not limited to, primary amines, secondary amines, tertiary amines, and quaternary ammonium salts.

[0031] In another preferred embodiment of the present invention, the positively charged groups or molecules of the physically adsorbed substances include, but are not limited to, cationic polymers among 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-homogeneous arginine (PLHA), poly-D-homogeneous arginine (PDHA), poly-DL-homogeneous arginine, poly-L-histidine (PLH), poly-D-histidine (PDH), poly-DL-histidine, polymethylene-CO-guanidine (PMCG), polyallylamine (PAA), polyvinylamine (PVA), polyethyleneimine, allylamine-diallylamine copolymers, and allylamine-maleic acid copolymers.

[0032] In another preferred embodiment of the present invention, the cell adhesion promoting protein or polypeptide includes, but is not limited to, collagen, gelatin, laminin, fibronectin, vitrin, fragments of collagen, gelatin, laminin, fibronectin or vitrin that promote cell adhesion, and polypeptides containing arginine aspartic peptide.

[0033] 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, as well as derivatives of starch, cellulose, dextran, sodium alginate, chitosan, hyaluronic acid, agarose, polygalacturonic acid and pectin.

[0034] In another preferred embodiment of the present invention, the crosslinking method includes one or more of the following methods: chemical crosslinking, photocrosslinking, dehydrogenation thermal crosslinking, and ionic crosslinking.

[0035] In another preferred embodiment of the present invention, the chemical crosslinking utilizes crosslinking agents including but not limited to the following to crosslink 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.

[0036] In another preferred embodiment of the invention, the photocrosslinking uses a light source capable of initiating a reaction between the crosslinking agent and the polysaccharide molecules.

[0037] In another preferred embodiment of the present invention, the light source is ultraviolet light.

[0038] In another preferred embodiment of the present invention, the dehydrogenation thermal crosslinking is carried out in a near-vacuum environment and at high temperature.

[0039] The present invention also provides an application of microcarriers in the culture of adherent cells.

[0040] In a preferred embodiment of the present invention, the cultured cells include, but are not limited to, insect cells, mammalian cells, bird cells, and human cells.

[0041] In another preferred embodiment of the present invention, the culture temperature of mammalian cells or human cells is 36-38 degrees Celsius and the pH is 6.8-8.2.

[0042] In another preferred embodiment of the present invention, the method comprises:

[0043] A water-in-oil emulsion is formed by treating a polysaccharide solution, an organic solvent that is immiscible with water, and an emulsifier. A cross-linking agent is then added to induce a cross-linking reaction in the polysaccharide.

[0044] Alternatively, a cross-linking agent can be added to the polysaccharide solution to induce a cross-linking reaction in the polysaccharide, and then the cross-linked polysaccharide solution, an organic solvent that is immiscible with water, and an emulsifier can be processed to form a water-in-oil emulsion.

[0045] In another preferred embodiment of the present invention, the method for forming the water-in-oil emulsion includes, but is not limited to, mechanical stirring, homogenization emulsification, membrane emulsification, spraying, ultrasonic emulsification, ultrasonic spraying, and microfluidic methods.

[0046] In another preferred embodiment of the invention, the method further includes washing to remove impurities other than organic solvents and microcarriers.

[0047] In another preferred embodiment of the present invention, the concentration of polysaccharide in the polysaccharide solution is 10%-50% w / w.

[0048] In another preferred embodiment of the present invention, the method further includes adding a positively charged substance or a substance that promotes cell adhesion after adding the crosslinking agent or at the same time as adding the crosslinking agent, so that the surface of the polysaccharide microspheres is positively charged or contains cell adhesion molecules through physical adsorption or chemical reaction.

[0049] In another preferred embodiment of the present invention, the organic solvent that is not miscible with water is vegetable oil.

[0050] 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 polyglycerides, polyglycerol polyricinoleate, stearoyl lactate, and dehydrated sorbitol ester.

[0051] In another preferred embodiment of the present invention, the polyglycerol polyricinoleate has a volume fraction of 1-10% in the organic solvent.

[0052] In another preferred embodiment of the present invention, the crosslinking agent is epichlorohydrin.

[0053] In another preferred embodiment of the present invention, the molar ratio of epichlorohydrin to monosaccharide residues in the polysaccharide is 0.23 to 0.53:1.

[0054] In another preferred embodiment of the present invention, the crosslinking reaction takes more than 20 hours.

[0055] The present invention also provides an application of microcarriers as adherent cell culture materials.

[0056] The present invention also provides a method for cell adhesion culture and cell recovery using microcarriers. The method involves mixing microcarriers, culture medium, and adherent cells, culturing the cells, washing the microcarriers, and simultaneously or stepwise adding polysaccharide enzymes and enzymes that decompose proteins or peptides to dissolve the microcarriers. After confirming that the microcarriers are completely dissolved, the adherent cells are recovered.

[0057] The present invention also provides a method for dissolving microcarriers, wherein for microcarriers whose surfaces are modified with proteins or peptides that promote cell adhesion, the method involves simultaneously or stepwise using a polysaccharide enzyme and an enzyme that breaks down the protein or peptide to dissolve the microcarriers.

[0058] The beneficial effects of this invention are as follows: This invention provides a microcarrier, its preparation method and application, as well as a method for cell adherence culture and recovery. The prepared microcarrier can be completely dissolved. Compared with existing microcarriers, the cell recovery efficiency of cell adherence culture using the microcarrier and method of this application can reach more than 95%. Furthermore, 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. Attached Figure Description

[0059] Figure 1 Various microcarriers were used before and half an hour after enzyme treatment.

[0060] Figure 2 Cell adhesion and staining for cell viability and death; (Left: Phase contrast microscopy image; Middle: Live cells stained green; Right: Dead cells stained red).

[0061] Figure 3 For cell expansion using microcarriers.

[0062] Figure 4 Microcarriers and cells before (left) and after (right) enzyme treatment.

[0063] Figure 5 Staining of cells attached to various microcarriers (left), live cells (middle), and dead cells (right).

[0064] Figure 6 Positively charged modified microcarriers and cells before (left) and after (right) enzyme treatment.

[0065] Figure 7 It is a microcarrier with a porous surface. Detailed Implementation

[0066] The present invention will be 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 for illustrative purposes only and are not intended to limit the present invention.

[0067] Polysaccharides, produced from plant extracts or microbial fermentation, are widely available, low-cost, and biocompatible, making them ideal for large-scale preparation of cell culture microcarriers. The hydroxyl, amino, and carboxyl groups of polysaccharide molecules can be used to immobilize polysaccharide molecules within polysaccharide microspheres using chemical crosslinkers or physical crosslinking methods, preventing dissolution of the microcarriers during cell culture. By controlling the type and amount of chemical crosslinking agent, reaction temperature, and reaction time, the degree of polysaccharide crosslinking 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 after cell expansion culture under the action of polysaccharide enzymes, achieving efficient cell recovery.

[0068] Polysaccharides suitable for preparing microcarriers include starch, cellulose, dextran, sodium alginate, chitosan, hyaluronic acid, agarose, polygalacturonic acid, and pectin and its derivatives.

[0069] To prepare microcarriers, the size and shape of the microcarriers need to be controlled by the droplets forming the polysaccharide solution. Methods for forming polysaccharide solution droplets or water-in-oil emulsions include, but are not limited to, mechanical stirring, homogenization emulsification, membrane emulsification, spraying, ultrasonic emulsification, ultrasonic spraying, and microfluidic methods. In emulsification, the polysaccharide solution droplets need to be formed in an organic solvent that is immiscible with water. Organic solvents that are immiscible with water 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 trioleic acid oil. To maintain the stability of the emulsion obtained after emulsification, emulsifiers need to be added during the emulsification process, including but not limited to lecithin, carrageenan, guar gum, xanthan gum, polysorbate, cellulose (including carboxymethyl cellulose), fatty acid monoglycerides and diglycerides, sucrose esters and sucrose glycerides, fatty acid polyglycerides, polyglycerol polyricinoleate, stearoyl lactate, and dehydrated sorbitol esters.

[0070] Methods for crosslinking polysaccharides include chemical crosslinking, photo-crosslinking, dehydrothermal crosslinking, and ionic crosslinking. Chemical cross-linking methods utilize 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 can be initiated by light sources such as ultraviolet light to cause a reaction between the cross-linking agent and the polysaccharide molecules. Dehydrogenation thermal cross-linking refers to using a near-vacuum environment and high temperature to react the carboxyl, hydroxyl, and amino groups on the polymer, thereby cross-linking the polymer.

[0071] The degree of cross-linking of polysaccharides in microcarriers can be defined as the proportion of monosaccharide residues that undergo cross-linking reactions with cross-linking agents or between functional groups of monosaccharide residues. The hydroxyl and carboxyl functional groups of the monosaccharide residues can form chemical bonds with cross-linking agents, and the functional groups of the monosaccharide residues can also form chemical bonds with each other under certain conditions. If the monosaccharide residues do not undergo chemical reactions, formic acid can be generated under the oxidation of sodium periodate. 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 (in grams), and the formic acid produced after oxidation is n (in mol), the number of monosaccharide residues that did not undergo the chemical reaction can be calculated to be n (in mol). The proportion of these unreacted monosaccharide residues to all monosaccharide residues (with a molecular weight of A) is n*A / m. The proportion of monosaccharide residues that underwent the chemical reaction is calculated to be 1-n*A / m, which is the degree of polysaccharide crosslinking as defined here. Furthermore, methods for detecting the degree of polysaccharide crosslinking include, but are not limited to, nuclear magnetic resonance (NMR) and total ion chromatography (TIC).

[0072] To promote cell adhesion to the surface of microcarriers, the surface of the microcarriers can be positively charged or contain proteins, peptides, or other molecules that promote cell adhesion. Positively charged groups include primary amines, secondary amines, tertiary amines, and quaternary ammonium salts, which can be chemically attached to the surface of the microcarriers. Alternatively, positively charged polymers 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), and poly-DL-arginine can be adsorbed onto the surface. Cationic polymers such as poly-L-arginine (PLHA), poly-D-arginine (PDHA), poly-DL-arginine, poly-L-histidine (PLH), poly-D-histidine (PDH), poly-DL-histidine, polymethylene-CO-guanidine (PMCG), polyallylamine (PAA), polyvinylamine (PVA), polyethyleneimine, allylamine-diallylamine copolymers, and allylamine-maleic acid copolymers can also enable negatively charged cells to attach to microcarriers. Proteins and peptides that promote cell attachment can be obtained from animal tissues or through recombinant protein technology; these include, but are not limited to, collagen, gelatin, laminin, fibronectin, vitronectin, or fragments of the aforementioned proteins that promote cell attachment, as well as peptides containing arginyl glycylaspartic acid (RGD).

[0073] The culture environment for adherent cells using microcarriers varies depending on the type of adherent cell. Generally, the suitable culture temperature for insect cells is 26-30°C, and the pH is 6.0-6.4. The suitable culture temperature for mammalian cells is 36-38°C, and the pH is 6.8-8.2. The suitable culture temperature for avian cells is 39-41°C, and the pH is 7.0-8.5. For example, the optimal temperature for culturing chicken satellite cells is 41°C. Chicken embryonic cells can be passaged long-term in an environment with a pH of 8.1-8.5. Insect cells grow slowly below 26°C, and their survival rate decreases above 30°C. Cell viability is the proportion of live cells in a cell sample, which can be determined by staining dead and / or live cells, as well as by using a hemocytometer or automated cell counter.

[0074] Depending on the type of polysaccharide in the microcarrier, the polysaccharide-dissolving enzyme can be amylase, cellulase, glucanase, alginate, chitosanase, hyaluronidase, agarosease, pectinase, or a combination thereof. If the surface of the polysaccharide microcarrier is modified with proteins or peptides that promote cell adhesion, the microcarrier can be dissolved simultaneously or stepwise using enzymes that dissolve proteins or peptides, such as collagenase, trypsin, papain, or TrypLE. For example, the protein or peptide in the microcarrier can be dissolved first using an enzyme that breaks down proteins or peptides, and then the cross-linked polysaccharide can be dissolved using a polysaccharide-dissolving enzyme; or the cross-linked polysaccharide can be dissolved first using a polysaccharide-dissolving enzyme, and then the protein or peptide incubator can be used. The conditions for dissolving polysaccharide microcarriers with enzymes should maintain cell viability; for example, temperature and pH should not affect cell viability. For example, after culturing human mesenchymal stem cells using microcarriers, the conditions for dissolving polysaccharide microcarriers can be 10-39℃ and pH 6.5-8. If the temperature is higher than 39℃, the pH is lower than 6.5, or the pH is higher than 8, cell death is likely to occur.

[0075] Microscopic observation can be used to determine whether polysaccharide microcarriers are completely dissolved. Complete dissolution here means that no spherical or near-spherical microcarriers are visible under a conventional optical microscope, nor are there any granular residues remaining after partial dissolution. A conventional optical microscope uses an objective lens of 4-20x and an eyepiece of 10x. For microcarrier samples without cultured cells, the microcarriers can be directly treated with polysaccharide enzymes. After dissolution, the presence of microcarrier residues can be observed using a conventional optical microscope. Alternatively, after culturing cells with microcarriers and dissolving the microcarriers with polysaccharide enzymes, cell lysis buffer can be used to dissolve the cells, and then the presence of microcarrier residues can be observed using a conventional optical microscope.

[0076] The surface of a microcarrier can be smooth or porous. A smooth surface, as defined here, refers to a surface without any noticeable unevenness or depressions when observed under a conventional optical microscope. This smooth surface allows adherent cells to extend and proliferate on the microcarrier surface. Alternatively, the surface of a microcarrier can be porous, allowing adherent cells to grow within the pores. The pore size of the microcarrier surface can range from 2-100 µm, 10-80 µm, or 15-50 µm. The pore size of the microcarrier can be controlled by factors such as freezing temperature and freezing rate during material preparation. Generally, within the range of 0 to liquid nitrogen, lower freezing temperatures result in smaller pore sizes in the formed microcarrier material.

[0077] Example 1: Preparation of Microcarriers

[0078] 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% PGPR (v / v). Six 1 g dextran solutions were taken, and 1 mL of each of the six oil phases was added, followed by 50 µL of epichlorohydrin crosslinking agent. Each mixture was homogenized at 1000 rpm for 3 minutes using a high-speed homogenizer. The results showed that the oil phases containing 1-10% (v / v) PGPR could form stable water-in-oil emulsions.

[0079] 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 1 g dextran solutions were taken, and 1 mL of soybean oil and 20 µL of polyglycerol polyricinoleate (PGPR) were added to each. Then, 100 µL, 75 µL, 50 µL, or 25 µL of epichlorohydrin crosslinking agent were added respectively. Using a high-speed homogenizer, the four mixtures were mixed at 1000 rpm for 3 minutes, and the resulting four emulsions were added to 2 mL centrifuge tubes. Each centrifuge tube was placed in a 50°C oven for crosslinking reaction, and removed after 20 hours. The reacted emulsions were transferred to 50 mL centrifuge tubes, and 10 mL of acetone was added for stirring and washing. After standing, the supernatant was removed, and 10 mL of anhydrous ethanol was added for washing. The crosslinked dextran microspheres were then washed six times with pure water. Sample 4 partially dissolved during the washing process. Add 0.04 mL of 0.5 g / mL DEAE-dextran solution to the remaining three samples and mix for 2 hours at room temperature using a rotary mixer to coat the surface of the dextran microspheres with DEAE-dextran molecules to obtain microcarriers. Then wash the microcarriers 6 times with pure water and 2 times with phosphate buffered saline (PBS).

[0080] After mixing the microcarriers of samples 1-3 in PBS, 0.1 mL was added to each well of a 96-well plate. The 96-well plates were placed in a 37°C cell culture incubator for 3 days. Using a 4x and 10x objective lens of an inverted optical microscope, it was observed that the microcarriers maintained their structural integrity and did not dissolve. Dextranase solution (1000 u / mL) was added to the microcarriers in the 96-well plates and mixed well. The 96-well plates were then placed in a 37°C cell culture incubator for 30 minutes. Afterward, the 96-well plates were removed and observed using a 4x and 10x objective lens of an inverted optical microscope. The results showed that the microcarriers of sample 1 only partially dissolved, while the microcarriers of samples 2 and 3 completely dissolved, with no residual microcarriers or particulate matter observed.

[0081] Table 1. Raw material composition and dissolution results of polysaccharide microcarriers after enzyme treatment

[0082]

[0083] Example 2: Preparation of microcarriers

[0084] Dextran was dissolved in a 6M sodium hydroxide solution to obtain a 30% (w / w) dextran solution. According to Table 2, three 1 g dextran solutions were taken, and 50 µL, 44 µL, or 37.5 µL of epichlorohydrin crosslinking agent were added to each. Then, 1 mL of soybean oil and 20 µL of polyglycerol polyricinoleate (PGPR) were added to each. Using a high-speed homogenizer, the four mixtures were mixed at 500 rpm for 3 minutes, respectively. The resulting three emulsions were added to 2 mL centrifuge tubes. The centrifuge tubes were placed in a 50°C oven for the crosslinking reaction, and removed after 24 hours. The reacted emulsions were transferred to 50 mL centrifuge tubes, and 10 mL of acetone was added for stirring and washing. After standing, the supernatant was removed, and 10 mL of anhydrous ethanol was added for washing. The crosslinked dextran microspheres were then washed six times with pure water. Sample 6 partially dissolved during the washing process. Add 0.04 mL of 0.5 g / mL DEAE-dextran solution to the remaining two samples and mix for 2 hours at room temperature using a rotary mixer to coat the surface of the dextran microspheres with DEAE-dextran, thus obtaining microcarriers. Then wash the microcarriers 6 times with pure water and 2 times with PBS.

[0085] After thorough mixing, 0.1 mL of the microcarriers from samples 4 and 5 were added to 96-well plates. The plates were then incubated at 37°C for 3 days. Using a 4x and 10x objective lens of an inverted optical microscope, the microcarriers maintained their structural integrity and showed no dissolution. Dextranase solution (1000 u / mL) was then added to the microcarriers in the 96-well plates and mixed well. The plates were then incubated at 37°C for 30 minutes. Afterward, the 96-well plates were removed and observed using a 4x and 10x objective lens of an inverted optical microscope. The results showed that the microcarriers from samples 4 and 5 were completely dissolved, with no residual microcarriers or particles observed.

[0086] Table 2. Raw material composition and dissolution results of polysaccharide microcarriers after enzyme treatment

[0087]

[0088] Example 3: Preparation of microcarriers

[0089] Dissolve 40g of dextran in 60mL of 5M sodium hydroxide solution. According to Table 3, take eight 10g dextran solutions, add 10mL of soybean oil and 200µL of polyglycerol polyricinoleate (PGPR) to each, then add 400µL, 500µL, 600µL, 700µL, 800µL, 900µL, 1200µL, or 1500µL of epichlorohydrin crosslinking agent. The molar ratio of epichlorohydrin to monosaccharide residues in the polysaccharide is 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. Using a mechanical stirrer, stir each of the eight mixtures at 200rpm for 3 minutes to obtain eight emulsions, which are then added to 50mL centrifuge tubes. Centrifuge tubes were placed in a 50°C oven for cross-linking reaction and removed after 24 hours. The resulting emulsion was transferred to a 500mL beaker, 50mL of acetone was added, and the mixture was stirred and washed. After standing, the supernatant was removed, and 50mL of anhydrous ethanol was added for washing. The mixture was then washed six times with pure water to obtain cross-linked dextran microspheres. 0.4mL of 0.5g / mL DEAE-dextran solution was added to each sample, and the mixture was stirred at room temperature for 2 hours using a rotary mixer to coat the surface of the dextran microspheres with DEAE-dextran, obtaining microcarriers. The microcarriers were then washed six times with pure water and twice with PBS.

[0090] Eight different microcarriers were mixed thoroughly, and 0.1 mL of each was added to a 96-well plate. The 96-well plates were placed in a 37°C cell culture incubator for 3 days. Using a 4x and 10x objective lens of an inverted optical microscope, it was observed that the microcarriers maintained their structural integrity and did not dissolve. Dextranase solution was then added to the microcarriers in the 96-well plates and mixed well. The 96-well plates were then placed in a 37°C cell culture incubator for 30 minutes. Afterward, the 96-well plates were removed and observed using a 4x and 10x objective lens of an inverted optical microscope.

[0091] After 30 minutes of enzyme treatment, the results showed that Cr400, Cr500, Cr600, Cr700, and Cr800 microcarrier samples were completely dissolved by the enzyme, with no residual microcarriers or particles observed. Cr900 only partially dissolved, and the overall size of the microcarriers decreased. The size and shape of Cr1200 and Cr1500 microcarriers did not change significantly, remaining spherical or nearly spherical. Figure 1 Extending the enzyme treatment time to 2 hours, the Cr900 microcarriers gradually dissolved and disappeared, while the size and shape of the Cr1200 and Cr1500 microcarriers showed no significant changes (Table 3). The samples that could be dissolved by the enzyme within 2 hours corresponded to a molar ratio of epichlorohydrin to monosaccharide residues in the polysaccharide during the preparation process of 0.23–0.53:1.

[0092] Similarly, when commercially available Cytodex1 microcarriers (Cytiva) were treated with enzymes for 30 minutes, partial dissolution of the microcarriers was observed under an optical microscope, leaving many particulate residues ranging from a few micrometers to tens of micrometers in size, along with a small number of spherical microcarriers. Figure 1 Extending the enzyme treatment time to 2 hours did not eliminate the residue of the microcarriers.

[0093] Table 3. Raw material composition and dissolution results of polysaccharide microcarriers after enzyme treatment

[0094]

[0095] Example 4: Measurement of the degree of crosslinking of microcarriers

[0096] Take 40 mL of each of the various microcarriers obtained in Example 3 and place them in a 50 mL beaker. Separately, take 0.3 g of Cytodex1 microcarrier and 0.3 g of dextran and place them in a 50 mL beaker. Place the beakers containing the different samples in a 70°C forced-air dryer and dry for at least 24 hours until the sample weight change is less than 0.002 g. Afterward, take 0.200 g of each dried sample and place it in a 100 mL glass bottle.

[0097] Dissolve 5.35 g of sodium periodate in 500 mL of pure water. Add 50 mL of the sodium periodate solution to each of the dry sample vials. Place a magnetic stir bar in each vial, cap the vials, and wrap them with aluminum foil to protect them from light. Place each reaction vessel at room temperature (20°C) and use magnetic stirring to mix and oxidize the solution. Measure the pH of the reaction solution every 3-6 hours using a pH meter. The reaction is considered complete when the pH change is less than 0.02 over 3-6 hours.

[0098] Take 10 mL of supernatant from each 100 mL glass bottle and transfer it to a beaker. Add 1 mL of ethylene glycol and react at room temperature for at least 15 minutes. Then, titrate the resulting solution with 0.01 mol / L NaOH solution. Stop the titration when the pH changes abruptly between 6.5 and 8.0. Measure the weight M (in grams) 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, where D = 1 - (5 × M × 0.01 × 16² / (1000 × 0.2)).

[0099] The results are shown in Table 4. The calculated degree of cross-linking of the uncross-linked dextran was only 2.86%, very close to the proportion of α-(1-3) glycosidic bonds in the dextran molecule (approximately 3%). The degree of cross-linking gradually increased with the increase in the amount of cross-linking agent used. Within a certain range of cross-linking degree, the microcarriers could be completely dissolved after enzyme treatment. Beyond a certain range, the microcarriers became less easily dissolved by enzymes. Based on the results of Example 3, microcarriers with a cross-linking degree between 13.1% and 38.7% could be completely dissolved by enzymes in about half an hour; microcarriers with a cross-linking degree of 44.2% required 1-2 hours to be dissolved; and microcarriers with a cross-linking degree above 55.1% were difficult to dissolve by enzymes within 2 hours. The commercially available Cytodex1 microcarriers had a cross-linking degree of 56.7%, and could only be partially dissolved by enzymes.

[0100] Table 4. Degree of cross-linking of various dextran microcarriers and dextran

[0101]

[0102] Example 5: Cell culture and recovery using microcarriers

[0103] The dextran microcarriers (Cr500) from Example 3 were used to separate the 200-300µm fraction using a standard sieve. 0.4ml of each fraction was suspended in 5mL 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, and then seeded with 3×10^5 human umbilical cord-derived mesenchymal stem cells (passage number P5). The microcarriers were cultured in a 37°C, 5% CO2 incubator. On the third day of culture, half of the culture medium was removed and an equal volume of fresh medium was added. After one, three, and six days, 0.5mL of the cell culture using the microcarriers was taken 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 0.1mol / L citric acid, and cell counts were performed by counting the number of nuclei. Six days later, all microcarriers in the culture container were washed with PBS, and 5 mL of TrypLE and 1% (v / v) dextranase were added. The mixture was then incubated at 37°C for 20-25 minutes, and the dissolution of the microcarriers was observed. Cells treated with the enzyme were counted using trypan blue staining and a hemocytometer to calculate cell viability and recovery efficiency. Cell recovery efficiency was defined as the ratio of the number of recovered cells to the number of cells calculated from the initial cell sampling. Cytodex1 microcarriers were similarly sterilized and cultured, and cell proliferation and recovery efficiency were compared.

[0104] Figure 3The results showed that mesenchymal stem cells adhered well to the Cr500 microcarriers and maintained a high survival rate. After 6 days of culture, the cell count increased approximately 14-fold. Figure 4 The results showed that the microcarriers were completely dissolved after enzyme treatment, and the cells on their surface were separated. The cell recovery efficiency was as high as 99%, and the cell viability was 98%.

[0105] After culturing with Cytodex 1 for 6 days, the cells expanded 7.8 times, but the recovery efficiency of cells recovered from Cytodex 1 microcarriers was only 36%.

[0106] Example 6: Positive charge modification of microcarrier surface

[0107] Following the steps of Example 3, cross-linked dextran microspheres were prepared using sample Cr500 as the raw material, and washed six times with pure water. After sieving, six 10 mL aliquots of cross-linked dextran microspheres were taken, and 3 mL of 6M NaOH solution and 0.1 mL, 0.5 mL, 1 mL, 1.5 mL, 1.75 mL, or 2 mL of 1M dimethylaminoethylchloride hydrochloride (DEAE) aqueous solution were added to each aliquot. The liquids were mixed and stirred in a 60°C water bath for 1 hour. Afterwards, the microspheres were 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%.

[0108] Table 5. Six types of microcarriers with positively charged surfaces

[0109]

[0110] Take 0.2 mL of each of the six microcarriers, suspend them in 5 mL of PBS, autoclave, and then wash them twice with the cell culture medium described above, adding 5 mL of medium each time. Seed 3 × 10^5 human umbilical cord-derived mesenchymal stem cells (cell passage number P5) into each microcarrier and culture them in a cell culture incubator at 37°C and 5% CO2. After one day and six days, take 0.5 mL of the cell culture using the microcarriers for cell viability staining, microscopic observation, and cell counting. Then add 2 mL of TrypLE and 1% (v / v) dextranase, and incubate at 37°C for 20-25 minutes, observing the dissolution of the microcarriers.

[0111] Figure 5The results showed that when the positive charge on the surface of the 0.5D microcarrier was too low, cells did not easily adhere to the microcarrier; as the positive charge increased, cells adhered to the microcarrier more easily (sample 1D); however, microcarriers with too much positive charge (sample 2D) could reduce cell survival. Figure 6 The results showed that the positively charged microcarriers could be completely dissolved by enzymes, releasing the cells on their surface. The cell recovery efficiency from the microcarriers was over 96%.

[0112] Example 7 Positively charged microcarriers with different degrees of crosslinking

[0113] Dextran microspheres (Cr400, Cr500, Cr600, Cr700, Cr800, and Cr900) with different degrees of cross-linking and no positive charge modification obtained in Example 3 were sieved. Six 10 mL aliquots of each type of cross-linked dextran microsphere were taken, and 3 mL of 6M NaOH solution and 1 mL of 1M 2-dimethylaminoethylchloride hydrochloride (DEAE) aqueous solution were added to each aliquot. The liquids were mixed and stirred in a 60°C water bath for 1 hour. The microspheres were then washed 6 times with pure water and twice with PBS to obtain microcarriers with different degrees of cross-linking and positive charge modification. 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%.

[0114] Following the cell culture and recovery method described in Example 5, cells were amplified on positively charged microcarriers with varying degrees of cross-linking. During cell recovery, the microcarriers corresponding to Cr400, Cr500, Cr600, Cr700, and Cr800 were dissolved by enzymes within 30 minutes, while the microcarrier corresponding to Cr900 was dissolved by enzymes within 1-2 hours. The cell recovery efficiency was consistently above 95%.

[0115] Example 8 Microcarriers with surface modified by cell adhesion proteins

[0116] Following the steps of Example 3, cross-linked dextran microspheres were prepared using sample Cr500 as the raw material, and washed six times with pure water. Two 2 mL portions of cross-linked dextran microspheres were taken, and 1 mL of either 2% (w / w) recombinant gelatin solution or 1 mL of 25 µg / mL recombinant vitronectin was added. 1 mL of an aqueous solution containing 50 mM EDC and 20 mM NHS was added, and the mixture was reacted at room temperature with stirring for 6 hours. Afterwards, the microcarriers were washed six times with pure water to obtain two types of microcarriers with surfaces modified by cell adhesion proteins. 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 for the microcarriers modified with recombinant gelatin and recombinant vitronectin was 19.7% and 20.1%, respectively.

[0117] According to the cell culture method described in Example 5, mesenchymal stem cells were cultured for 6 days using at least 0.2 mL of microcarriers. 2 mL of TrypLE solution and 1% (v / v) dextranase solution were added, and the mixture was incubated at 37°C for 20 minutes. After complete dissolution of the microcarriers, the cell recovery rates from the recombinant gelatin-modified or recombinant Vitronectin-modified microcarriers were 97% and 99%, respectively, with cell viability rates of 98% and 99%, respectively.

[0118] Example 9 Preparation of surface porous microcarriers

[0119] Following the steps in Example 3, dextran microcarriers were prepared using sample Cr500 as the raw material. The microcarriers were washed with pure water and then placed in a -80°C ultra-low temperature freezer for 12 hours. The frozen microcarriers were then freeze-dried at -50°C, 20 Pa for 48 hours using a freeze dryer. A small amount of the microcarriers was then immersed in PBS for observation.

[0120] The microcarrier surface was observed to have a pore structure with a diameter of 10-80 µm using an optical microscope. Figure 7 The method described in Example 4 was used to thoroughly dry the microcarriers and measure the degree of crosslinking. The results showed that the degree of crosslinking of the surface porous microcarriers was 19.4%.

[0121] Example 10: Preparation of pectin microcarriers

[0122] Pectin was dissolved in 30 mL of 6 mol / L sodium hydroxide solution to obtain a 15% pectin solution. 10 g of the pectin solution was taken, and 10 mL of soybean oil and 200 µL of polyglycerol polyricinoleate (PGPR) were added, followed by 500 µL of epichlorohydrin crosslinking agent. Emulsification, crosslinking, and material washing were performed according to the steps of Example 3 to obtain crosslinked 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 monosaccharide in the pectin, and 0.2 is the mass (g) of the dried pectin microcarriers. The results showed that the degree of crosslinking of the pectin microcarriers was 21.6%.

[0123] According to the cell culture method described in Example 5, mesenchymal stem cells were cultured for 6 days using at least 0.2 mL of microcarriers. 2 mL of TrypLE solution and 1% pectinase solution were added, and the mixture was incubated at 37 degrees Celsius for 20 minutes. After this process, the microcarriers were completely dissolved, the cell recovery rate was 95%, and the cell viability was 96%.

[0124] Example 11: Culture of 293T cells using microcarriers

[0125] The dextran microcarriers (Cr600) were screened and sterilized according to Example 5. After sterilization, the microcarriers were washed with DMEM medium supplemented with 10% fetal bovine serum, and then 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 cell culture using the microcarriers was taken for cell viability staining and microscopic observation. On the third day of culture, half of the medium was removed and an equal volume of fresh medium was added. After six days of culture, cells were sampled and counted, the microcarriers were dissolved using TrypLE and 1% (v / v) dextranase, and the cells were recovered. Cell viability and recovery efficiency were calculated. Microscopic observation showed that the 293T cells adhered well to the microcarriers, with a cell viability of over 90%, and the cell recovery rate after dissolving the microcarriers with enzymes was 95%.

[0126] Please note to all technical personnel: Although the present invention has been described according to the specific embodiments above, the inventive concept of the present invention is not limited to this invention. Any modifications that utilize the inventive concept will be included within the scope of protection of this patent.

[0127] 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 embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing 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 micro-particle obtained by cross-linking of polysaccharide molecules, the microcarrier can maintain the structure of the polysaccharide microcarrier stable in a cell culture environment, the microcarrier can be completely dissolved under the action of an enzyme, the microcarrier can be completely dissolved under the action of the enzyme within 30 minutes, the microcarrier can be completely dissolved by the enzyme under the conditions of 10-39℃ and pH 6.5-8.0, the cross-linking degree of the polysaccharide molecules in the microcarrier is 13.1%-38.7%, the cross-linking degree is the proportion of the number of monosaccharide residues involved in the cross-linking reaction between the cross-linking agent or between the monosaccharide residues to the total number of monosaccharide residues in the polysaccharide, and 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.

2. The microcarrier according to claim 1, characterized in that, The cross-linked micro-particle is a cross-linked micro-sphere or a nearly spherical cross-linked micro-particle.

3. The microcarrier according to claim 2, characterized in that, The microcarrier can maintain the micro-sphere or nearly spherical structure of the polysaccharide microcarrier stable for more than three days in a conventional cell culture environment.

4. The microcarrier according to claim 3, characterized in that, The microcarrier can maintain the micro-sphere or nearly spherical structure of the polysaccharide microcarrier stable for more than three days in a cell culture environment at 25-41℃.

5. The microcarrier according to claim 1, wherein, The surface of the microcarrier is modified.

6. The microcarrier according to claim 5, characterized in that, The surface modification of the microcarrier is a positively charged group or molecule combined with 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 adhesion by chemical bonds or physical adsorption.

7. The microcarrier according to claim 6, characterized in that, The physically adsorbed positively charged group or molecule includes cationic polymers in diethylaminoethyl dextran, poly-L-ornithine, poly-D-ornithine, poly-DL-ornithine, poly-D-lysine, poly-L-lysine, poly-DL-lysine, poly-L-arginine, poly-D-arginine, poly-DL-arginine, poly-L-homoarginine, poly-D-homoarginine, poly-DL-homoarginine, poly-L-histidine, poly-D-histidine, poly-DL-histidine, poly-methylene-CO-guanidine, polyallylamine, polyvinylamine, polyethyleneimine, allylamine-diallylamine copolymer and allylamine-maleic acid copolymer.

8. The microcarrier according to claim 6, wherein, The protein or polypeptide that promotes cell adhesion includes collagen, gelatin, laminin, fibronectin, vitronectin or fragments of collagen, gelatin, laminin, fibronectin or vitronectin that promote cell adhesion, or a polypeptide containing arginine aspartic acid peptide.

9. The microcarrier according to claim 1, wherein, The cross-linking method is one or more of chemical cross-linking, photo-crosslinking, dehydrogenation thermal cross-linking and ionic cross-linking.

10. The microcarrier according to claim 9, characterized in that, The chemical cross-linking uses a cross-linking agent to cross-link or fix the chain structure of the polysaccharide molecules, including 1-3-dichloropropanol, N,N'-methylene bisacrylamide, 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, genipin.

11. The microcarrier according to claim 9, characterized in that, The photo-cross-linking uses a light source to initiate the reaction between the cross-linking agent and the polysaccharide molecules.

12. The microcarrier according to claim 9, wherein, The dehydrogenation thermal cross-linking is performed in a near-vacuum environment and at a high temperature.

13. The microcarrier according to claim 1, wherein, The surface of the microcarriers is smooth or porous.

14. The microcarrier according to claim 13, characterized in that, The pore size of the porous structure is 1-100 µm.

15. The microcarrier according to claim 14, characterized in that, The pore size of the porous structure is controlled by conditions including freezing temperature and freezing speed.

16. The microcarrier of claim 1, wherein, The microcarriers are prepared by the following steps: The polysaccharide solution, the water-immiscible organic solvent, and the liquid emulsifier are processed to form a water-in-oil emulsion, and the cross-linking agent is added to cause the polysaccharide to undergo a cross-linking reaction. Alternatively, the cross-linking agent is first added to the polysaccharide solution to cause the polysaccharide to undergo a cross-linking reaction, and then the cross-linked polysaccharide solution, the water-immiscible organic solvent, and the liquid emulsifier are processed to form a water-in-oil emulsion.

17. The microcarrier according to claim 16, wherein, The concentration of the polysaccharide in the polysaccharide solution is 10%-50 % w / w.

18. The microcarrier according to claim 16, wherein, The steps further include, after or simultaneously with the addition of the cross-linking agent, adding a positively charged substance or a substance that promotes cell adhesion, to cause the polysaccharide microspheres to have a positive charge or cell adhesion molecules on the surface by physical adsorption or chemical reaction.

19. The microcarrier according to claim 16, wherein, The water-immiscible organic solvent is a vegetable oil.

20. The microcarrier of claim 16, wherein, The emulsifier includes lecithin, carrageenan, guar gum, xanthan gum, polysorbate, cellulose, fatty acid monoglyceride and diglyceride, sucrose ester and sucroglyceride, fatty acid polyglycerol ester, polyglycerol polyricinoleate, stearoyl lactylate, sorbitan ester.

21. The microcarrier according to claim 20, wherein, The volume fraction of the polyglycerol polyricinoleate in the organic solvent is 1-10 %.

22. The microcarrier of claim 16, wherein, The cross-linking agent is epichlorohydrin.

23. The microcarrier according to claim 22, wherein, The molar ratio of the epichlorohydrin to the monosaccharide residues in the polysaccharide is 0.23-0.47:

1.

24. The microcarrier of claim 16, wherein, The time of the cross-linking reaction is greater than 20 hours.

25. Use of the microcarriers of any one of claims 1-24 as a material for culturing adherent cells.

26. A method for adherent cell culture and cell recovery using the microcarriers of any one of claims 1-24, wherein, The method includes the following steps: mixing the microcarriers, the culture medium, and the adherent cells, culturing the cells, then washing the microcarriers, simultaneously or stepwise adding polysaccharidases and proteolytic or polypeptidolytic enzymes to dissolve the microcarriers, confirming that the microcarriers are completely dissolved, and recovering the adherent cells.

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