Cell culture microcarrier as well as preparation method and application thereof

Abstract

The invention relates to a cell culture microcarrier as well as a preparation method and application thereof. The cell culture microcarrier comprises a polymer porous microsphere with an inside-outside through pore channel structure. The cell culture micro-carrier comprises a biodegradable micro-carrier and also comprises a non-degradable micro-carrier, so that the growth of cells in the carrier is facilitated, and the mass transfer of nutrient substances and metabolites is also facilitated, thereby promoting the growth of the cells, enabling the cultured cells to keep good activity and function, and being high in proliferation efficiency; the method is suitable for three-dimensional culture of somatic cells, stem cells, tumor cells and the like.

Description

[0001] This application claims the priority of a Chinese patent application filed on July 10, 2023, with application number 2023108392984, and invention name “A cell culture microcarrier, preparation method and application thereof”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention belongs to the technical field of biomaterials, and relates to a cell culture microcarrier and a preparation method and application thereof, and in particular to a microcarrier that can be used for culturing somatic cells, stem cells and tumor cells and a preparation method and application thereof. Background Art

[0003] Stem cells have been widely used in clinical research on a variety of diseases and have shown good results in the treatment of some diseases. There are still some common problems in the application of stem cells. First, the clinical application of stem cells requires a large amount, but the number of stem cells is far from meeting the wide clinical needs; on the other hand, after the stem cells are injected into the body, they will be quickly cleared, and the implantation rate is low, which affects the therapeutic effect of the cells.

[0004] Porous microcarriers can be a good solution to the above two problems. First, porous microcarriers can provide a large specific surface area, which is suitable for large-scale cell culture. On the other hand, compared with 2D culture, 3D culture provides cells with a 3D growth environment that is closer to the body, which is more advantageous for maintaining cell activity and function. In addition, the space provided by porous microcarriers allows cells to enter the microcarriers for culture, protecting the cells from mechanical damage during injection and attacks by immune cells in the body, thereby increasing the cell implantation rate and enhancing the therapeutic effect of the cells. Similarly, culturing tumor cells in 3D porous microcarriers can greatly improve the cell harvest rate on the one hand, and on the other hand, the 3D growth environment is more conducive to simulating the real in vivo tumor microenvironment than the 2D environment, which is used for tumor pathology research and the screening of therapeutic drugs.

[0005] At present, porous microcarriers at home and abroad are mainly based on natural polymer materials. Natural polymer materials such as gelatin, chitosan, cellulose, etc. have good biocompatibility and have sites that are conducive to cell adhesion and growth. However, natural polymer materials have poor batch reproducibility and unclear composition, making it difficult to control their culture effects, mechanical properties, degradation properties and other characteristics.

[0006] In addition, from the perspective of the structure of porous microspheres, there are still some shortcomings due to the limitation of the pore-forming method. The pore-forming method of porous microcarriers is usually formed by freeze drying, gas foaming, solution casting / particle dissolution, etc. The freeze drying method uses ice crystal growth and nucleation as pore-forming agents, and removes the pore-forming agents by freeze drying technology to finally form pores. The macropores formed by this method are amorphous structures, lamellar, and the outer pores are trumpet-shaped, but the pore permeability is relatively poor, and some inner pores are closed. The gas foaming method is to dissolve the foaming agent in the inner water phase, form a W / O / W type emulsion through two-step emulsification, and then induce bubble generation to form porous microcarriers. The pores formed by this method are interconnected, but the pores are randomly distributed and of different sizes, which easily lead to uneven cell growth microenvironment. Solution casting or particle dissolution is to dissolve the solidified salt particles to form macroporous microcarriers. This method has poor pore connectivity.

[0007] Most of the porous microcarriers prepared so far have uneven internal pores, poor connectivity, and lack of wall pores (secondary pores), which affects the mass transfer of nutrients and metabolites, resulting in deterioration or even death of cells growing inside the carriers. Summary of the invention

[0008] In view of the deficiencies in the prior art, the object of the present invention is to provide a cell culture microcarrier and a preparation method and application thereof, and in particular to provide a microcarrier that can be used for culturing somatic cells, stem cells and tumor cells and a preparation method and application thereof.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a cell culture microcarrier, wherein the cell culture microcarrier comprises a polymer porous microsphere having an inner and outer through-hole structure.

[0011] Preferably, the cell culture microcarrier has a primary pore structure and a secondary pore structure; the pore size of the primary pore is 5-200μm, for example, 10μm, 30μm, 40μm, 50μm, 60μm, 70μm, 80μm, 90μm, 100μm, 110μm, 120μm, 150μm, 200μm, etc.; the pore size of the secondary pore is 0.1-50μm, for example, 0.5μm, 1μm, 1.5μm, 2μm, 2.5μm, 3μm, 4μm, 5μm, 8μm, 10μm, 15μm, 20μm, 25μm, 30μm, 35μm, 40μm, 50μm, etc.

[0012] Preferably, the primary pores have a diameter of 10-150 μm, more preferably 30-100 μm.

[0013] Preferably, the secondary pores have a diameter of 0.1-30 μm, more preferably 1-10 μm.

[0014] The cell culture microcarrier involved in the present invention has a porous through structure, with primary holes and secondary holes (holes on the wall) in a specific pore size range, the secondary holes are relatively small, and are present on the hole wall of the primary holes, which is convenient for the transportation of nutrients; the primary holes are relatively large, forming large cavities and windows, which are convenient for cells to migrate and grow inside the microspheres. This double-porous microcarrier structure combining primary holes and secondary holes is conducive to the growth of cells inside the carrier, and is also conducive to the mass transfer of nutrients and metabolites, thereby promoting the growth of cells, so that the cultured cells maintain good activity and function, and high proliferation efficiency. And the cell culture microcarrier includes both biodegradable microcarriers and non-degradable microcarriers.

[0015] Preferably, the particle size of the cell culture microcarrier is 50-1000 μm, for example, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, etc., preferably 100-500 μm, and more preferably 150-300 μm.

[0016] The particle size of the cell culture microcarrier involved in the present invention is controlled within the above-mentioned specific range, which is beneficial to provide sufficient growth space for cells, but not too large to cause problems such as difficulty in internal nutrient delivery and difficulty in absorption operation; on the other hand, the particle size of the microcarrier is not too small to limit the cell growth space, and to avoid problems such as excessive aggregation of microspheres in the later stage of cell culture, thereby affecting the space utilization and application of the microcarrier.

[0017] Preferably, the raw materials for preparing the matrix of the porous microspheres include modified or unmodified polymer materials and polymer monomers.

[0018] Preferably, the polymer material comprises any one of polycaprolactone, polylactic acid, polylactic glycolic acid, polyglycolic acid, polyhydroxyalkanoate, poly-L-glutamic acid, polymethacrylic acid, polystyrene, polyethylene oxide, polypropylene oxide copolymer, polymethyl methacrylate, polyethylene glycol, monomethoxypolyethylene glycol polylactic acid copolymer, polystyrene and polypropylene oxide copolymer, polystyrene and polyethylene glycol copolymer, polystyrene and polyacrylic acid copolymer, polystyrene and polyhydroxyethyl acrylate copolymer, polymethacrylate and polyacrylic acid copolymer, polymethyl methacrylate and polyhydroxyethyl acrylate copolymer, polymethacrylate and methacrylic acid copolymer, methyl methacrylate and β-hydroxyethyl methacrylate copolymer, polychloromethylstyrene or polyglycidyl methacrylate, or a combination of at least two thereof.

[0019] Preferably, the polymer monomer includes any one of glycidyl methacrylate, methyl methacrylate, butyl methacrylate, octadecyl methacrylate, hydroxyethyl methacrylate or styrene, or a combination of at least two thereof.

[0020] The present invention selects synthetic polymer materials or polymer monomers as the matrix materials of the porous microspheres because the synthetic polymer materials or polymer monomers have stable properties and can achieve controllable properties of the microcarriers such as degradation rate and mechanical properties.

[0021] Preferably, the cell culture microcarrier further comprises any one or a combination of at least two of proteins, glycoproteins, polysaccharides, polypeptides or adhesion materials modified on the surface of the polymer porous microspheres.

[0022] In order to enable cells to adhere and grow well, especially stem cells are sensitive to the chemical properties of the microenvironment, the surface of the microcarrier can be modified, such as any one or a combination of at least two of proteins, glycoproteins, polysaccharides, polypeptides or adhesion materials.

[0023] Preferably, the protein comprises any one of gelatin, collagen, laminin, elastin, fibronectin, fibroblast growth factor, bone morphogenetic protein, nerve growth factor, epidermal growth factor, interleukin transforming growth factor or cadherin, or a combination of at least two thereof.

[0024] Preferably, the polypeptide comprises any one or a combination of at least two of RGD, YIGSR, IKVAV, PHSRN, LDV, REDV, KRSR, DGEA, GFOGER, FHRRIKA, TYRSRKY, RRETAWA, poly-lysine, poly-ornithine or E7 polypeptide.

[0025] The above-mentioned polypeptides are derived from the amino acid sequences of the adhesion sites on the extracellular matrix, which are beneficial to the adhesion and proliferation of cells. For example, the amino acid sequences from fibronectin include RGD, PHSRN, LDV, REDV, and KRSR, the amino acid sequences from collagen include DGEA and GFOGER, the amino acid sequences of bone sialoprotein include FHRRIKA and RGD, the amino acid sequences of fibroblast growth factor-2 include TYRSRKY, the amino acid sequences of laminin include YIGSR and IKVAV, poly-lysine and poly-ornithine are polypeptides with positive charges, which can promote the adhesion of cells, and the E7 polypeptide has affinity with cells and can promote the adhesion of cells.

[0026] Preferably, the adhesive material includes any one or a combination of at least two of polydopamine or its derivatives.

[0027] In a second aspect, the present invention provides a method for preparing the cell culture microcarrier according to the first aspect, the preparation method comprising the following steps:

[0028] (1) mixing a polymer material, a surfactant and an oil phase solvent to obtain an oil phase; or mixing a polymer monomer, a surfactant, an initiator, a crosslinking agent and an optional oil phase solvent to obtain an oil phase; using an aqueous solution containing an osmotic pressure regulator as an inner aqueous phase; using an aqueous solution containing a surfactant as an outer aqueous phase;

[0029] (2) mixing and emulsifying the oil phase and the internal water phase to obtain oil-in-water primary emulsions of two particle size ranges;

[0030] (3) mixing and emulsifying the oil-in-water primary emulsions of two particle size ranges with an external water phase to obtain a water-in-oil-in-water secondary emulsion;

[0031] (4) solidifying the multiple emulsion to obtain the cell culture microcarrier.

[0032] In step (1), when the oil phase is prepared by mixing polymer monomers, surfactants, initiators and crosslinking agents, the reaction system may or may not contain an oil phase solvent.

[0033] Preferably, the polymer material comprises any one of polycaprolactone, polylactic acid, polylactic glycolic acid, polyglycolic acid, polyhydroxyalkanoate, poly-L-glutamic acid, polymethacrylic acid, polystyrene, polyethylene oxide, polypropylene oxide copolymer, polymethyl methacrylate, polyethylene glycol, monomethoxypolyethylene glycol polylactic acid copolymer, polystyrene and polypropylene oxide copolymer, polystyrene and polyethylene glycol copolymer, polystyrene and polyacrylic acid copolymer, polystyrene and polyhydroxyethyl acrylate copolymer, polymethacrylate and polyacrylic acid copolymer, polymethyl methacrylate and polyhydroxyethyl acrylate copolymer, polymethacrylate and methacrylic acid copolymer, methyl methacrylate and β-hydroxyethyl methacrylate copolymer, polychloromethylstyrene or polyglycidyl methacrylate, or a combination of at least two thereof.

[0034] Preferably, the polymer monomer includes any one of glycidyl methacrylate, methyl methacrylate, butyl methacrylate, octadecyl methacrylate, hydroxyethyl methacrylate or styrene, or a combination of at least two thereof.

[0035] Preferably, the surfactant in the oil phase is selected from oil-soluble small molecule surfactants and / or block copolymers;

[0036] Preferably, the oil-soluble small molecule surfactant is selected from any one of sorbitan monostearate, polyglycerol fatty acid ester, polyoxyethylene fatty acid ester (polyoxyethylene sorbitan 4.5 oleate), sorbitan monooleate, and glycidyl methacrylate, or a combination of at least two thereof;

[0037] Preferably, the block copolymer includes a diblock polymer and a multiblock copolymer;

[0038] Preferably, the diblock polymer is selected from any one or a combination of at least two of polyoxyethylene polyoxypropylene ether block copolymers, polyethylene glycol polylactic acid copolymers, polystyrene polyethylene glycol copolymers, polystyrene polyacrylic acid copolymers, polystyrene polyhydroxyethyl acrylate copolymers, polymethyl methacrylate polyacrylic acid copolymers or polymethyl methacrylate polyhydroxyethyl acrylate copolymers;

[0039] Preferably, the crosslinking agent is selected from divinyl aromatic compounds and / or acrylate compounds;

[0040] Preferably, the crosslinking agent is selected from divinylbenzene, ethylene glycol dimethacrylate or trimethoxypropane trimethyl acrylate;

[0041] Preferably, the initiator is selected from azo di-initiators and / or peroxide initiators;

[0042] Preferably, the initiator is selected from benzoyl peroxide or azobisisobutyronitrile;

[0043] Preferably, the oil phase solvent comprises any one or a combination of at least two of chloroform, acetone, tetrahydrofuran, toluene, dichloromethane, chloroform, dichloroethane or ethyl acetate;

[0044] According to the solubility characteristics of different polymer materials, suitable oil phase solvents are selected. These oil phase solvents can be removed by volatilization or solvent extraction to solidify the polymer microspheres.

[0045] Preferably, the osmotic pressure regulator is a metal ion salt or a sugar, the metal ion salt is selected from any one or a combination of at least two of sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium sulfate, potassium sulfate, magnesium sulfate or calcium sulfate, and the sugar is selected from any one or a combination of at least two of glucose, sucrose, agarose or trehalose.

[0046] Preferably, the concentration of the osmotic pressure regulator in the inner aqueous phase is 0.001-20%, for example 0.005%, 0.05%, 0.1%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 5%, 7%, 10%, 12%, 13%, 14%, 15%, 17%, 18%, 20%, etc., and more preferably 0.005-5%.

[0047] Preferably, the surfactant in the external aqueous phase includes a non-ionic water-soluble surfactant.

[0048] Preferably, the nonionic water-soluble surfactant comprises polyvinyl alcohol.

[0049] Preferably, the concentration of the nonionic water-soluble surfactant in the external aqueous phase is 0.05-10%, for example 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 8%, 10%, etc., more preferably 1-5%.

[0050] Preferably, the mixed emulsification in step (2) and step (3) is carried out by any one of vortex oscillation, mechanical stirring emulsification, ultrasonic emulsification, homogenization emulsification, membrane emulsification or microfluidic emulsification, or a combination of at least two of them, preferably homogenization emulsification and membrane emulsification.

[0051] Preferably, in the present invention, the primary pores and secondary pores on the cell culture microcarrier can be obtained by the following method: in step (2), two oil-in-water primary emulsions with particle size ranges are prepared, which are 0.1-50 μm and 5-150 μm respectively, and then in step (3), the two primary emulsions are co-encapsulated in the external aqueous phase to form a double emulsion, and finally primary pores and secondary pores with two specific pore size ranges are obtained.

[0052] Preferably, the two primary emulsions are co-encapsulated in the external aqueous phase in a volume ratio range of 1:1000-1000:1.

[0053] Preferably, the curing treatment method in step (4) includes solvent volatilization curing method or polymerization curing method;

[0054] When a polymer material is selected as the preparation material of the matrix, a solvent volatilization solidification method is adopted, and the solidification liquid is an aqueous solution, and more preferably deionized water.

[0055] When a polymer monomer is selected as the preparation material of the matrix, a polymerization curing method is adopted, preferably a temperature-raising polymerization curing method, the reaction temperature is 10-90° C., and the reaction time is 2-24 hours.

[0056] When polymer monomers are selected as the material for preparing the matrix, step (4) also requires the addition of a cross-linking agent for cross-linking.

[0057] Preferably, when the polymer molecular chain contains hydroxyl groups, the crosslinking agent is a bicyclic epoxy crosslinking agent selected from any one of 1,4-butanediol diglycidyl ether and 1,2-ethylene glycol diglycidyl ether or a combination of at least two thereof;

[0058] Preferably, when the polymer molecular chain contains carboxyl groups, the crosslinking agent is a polyamino macromolecule selected from any one or a combination of at least two of chitosan, polylysine or protein;

[0059] Preferably, when the polymer molecular chain contains carboxyl groups, the crosslinking agent further comprises a carboxyl activator selected from diimide activators; further preferably, any one or a combination of at least two of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N,N'-dicyclohexylcarbodiimide, and N-hydroxysuccinimide;

[0060] Preferably, when the polymer molecular chain contains epoxy groups, the crosslinking agent is a polycarboxyl compound or a polysaccharide; selected from any one or a combination of at least two of ethylene glycol, propylene glycol, butylene glycol, glycerol, glucose, sucrose or raffinose;

[0061] Preferably, the polysaccharide is selected from any one of dextran, agarose or chitosan, or a combination of at least two thereof.

[0062] Preferably, the surface of the product is modified after cross-linking after the curing treatment in step (4).

[0063] The modification method is selected according to the properties of different modified active substances, such as coating with polydopamine as the intermediate layer, layer-by-layer assembly through electrostatic adsorption, polymer surface active groups and physical adsorption, etc. It is further preferred to use polydopamine as the intermediate layer or directly use polydopamine for coating.

[0064] Using polydopamine as the middle layer is suitable for the modification of polymer materials and active substances of all surface properties. The concentration of polydopamine is preferably 0.001-20 mg / mL, and more preferably 0.01-0.5 mg / mL; the dopamine incubation time is 1-48 h.

[0065] In a third aspect, the present invention provides a use of the cell culture microcarrier according to the first aspect in cell culture.

[0066] Preferably, the cell comprises a somatic cell, a stem cell or a tumor cell.

[0067] Preferably, the somatic cells include VERO cells, CHO cells, WISH cells, L929 cells, MDCK cells, fibroblasts, endothelial cells, myoblasts, osteoblasts, chondrocytes, hepatocytes, kidney cells or epidermal cells.

[0068] Preferably, the stem cells include bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells, amniotic mesenchymal stem cells, peripheral blood mesenchymal stem cells, dental pulp mesenchymal stem cells, liver stem cells, liver precursor cells, myocardial stem cells, skin epidermal stem cells, intestinal epithelial stem cells, retinal stem cells, pancreatic stem cells, neuronal stem cells, muscle stem cells, amniotic epithelial stem cells, limbal stem cells, corneal epithelial stem cells, embryonic stem cells or induced pluripotent stem cells.

[0069] Preferably, the tumor cells include bladder cancer cells, prostate cancer cells, thyroid cancer cells, glioblastoma cells, malignant melanoma cells, lung cancer cells, rhabdomyosarcoma cells, renal cell adenocarcinoma cells, gastric adenocarcinoma cells, endometrial cancer cells, liver cancer cells, cervical cancer cells, colon cancer cells, brain glioma cells, neuroblastoma cells, breast cancer cells, osteosarcoma cells, oral epidermoid cancer cells, ovarian cancer cells or esophageal cancer cells.

[0070] Compared with the prior art, the present invention has the following beneficial effects:

[0071] The cell culture microcarrier involved in the present invention has a porous through structure, with primary holes and secondary holes (holes on the wall) in a specific pore size range, the secondary holes are relatively small, and are present on the hole wall of the primary holes, which is convenient for the transportation of nutrients; the primary holes are relatively large, forming large cavities and windows, which are convenient for cells to migrate and grow inside the microspheres. This double-porous microcarrier structure combining primary holes and secondary holes is not only conducive to the growth of cells inside the carrier, but also conducive to the mass transfer of nutrients and metabolites, thereby promoting the growth of cells, so that the cultured cells maintain good activity and function, and high proliferation efficiency. And the cell culture microcarrier includes both biodegradable microcarriers and non-degradable microcarriers. And the preparation method of the cell culture microcarrier involved in the present invention is simple, highly operable, and suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 is an electron microscope image of the cell culture microcarrier prepared in Example 1;

[0073] Figure 2 This is a laser confocal image of cells cultured on the cell culture microcarriers prepared in Example 1;

[0074] Figure 3 is an electron microscope image of the cell culture microcarrier prepared in Example 2;

[0075] Figure 4 This is a laser confocal image of cells cultured on the cell culture microcarriers prepared in Example 2;

[0076] Figure 5is the cell proliferation multiple of the cells cultured on the cell culture microcarriers prepared in Example 2 for 96 hours;

[0077] Figure 6 This is a confocal image of cells cultured on the cell culture microcarriers prepared in Example 2 for 96 hours;

[0078] Figure 7 This is a confocal image of cells cultured on smooth-surface microcarriers for 96 h;

[0079] Figure 8 is an electron microscopic image of the cell culture microcarrier prepared in Example 3;

[0080] Fig. 9 This is a laser confocal image of cells cultured on the cell culture microcarriers prepared in Example 3;

[0081] Fig.10 is an electron microscopic image of the cell culture microcarrier prepared in Example 4;

[0082] Fig.11 This is a laser confocal image of cells cultured on the cell culture microcarriers prepared in Example 4;

[0083] Fig.12 is an electron microscopic image of the cell culture microcarrier prepared in Example 5;

[0084] Fig.13 This is a laser confocal image of cells cultured on the cell culture microcarriers prepared in Example 5;

[0085] Fig.14 is an electron microscopic image of the cell culture microcarrier prepared in Example 6;

[0086] Fig.15 This is a laser confocal image of cells cultured on the cell culture microcarriers prepared in Example 6;

[0087] Fig.16 is an electron microscopic image of the cell culture microcarrier prepared in Example 7;

[0088] Fig.17 This is a laser confocal image of cells cultured on the cell culture microcarriers prepared in Example 7;

[0089] Fig.18 is an electron microscope image of the cell culture microcarrier prepared in Example 8;

[0090] Fig.19 This is a laser confocal image of cells cultured on the cell culture microcarriers prepared in Example 8;

[0091] Fig. 20 is an electron microscopic image of the cell culture microcarrier prepared in Example 9;

[0092] Fig.21 This is a laser confocal image of cells cultured on the cell culture microcarriers prepared in Example 9;

[0093] Fig. 22 This is a laser confocal image of cells cultured on the cell culture microcarriers prepared in Example 9. DETAILED DESCRIPTION

[0094] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0095] Example 1

[0096] This embodiment provides a cell culture microcarrier, and the preparation method thereof is as follows:

[0097] (1) Preparation:

[0098] 4 mL of 0.15% NaCl aqueous solution was used as the inner aqueous phase W1, 2.376 g of 50 / 50 PLGA (Shandong Academy of Biomedical Sciences Co., Ltd.) and 0.024 g of surfactant span80 (Sinopharm Group) were dissolved in 16 mL of ethyl acetate as the oil phase O; 1.5% PVA aqueous solution was prepared as the outer aqueous phase W2.

[0099] (2) Preparation of primary emulsion:

[0100] 2 mL of the internal aqueous phase W1 was added to 8 mL of the oil phase O and emulsified with a homogenizer at 3000 rpm for 1.5 min to obtain a primary emulsion A with a particle size distribution range of 10-100 μm;

[0101] 2 mL of the internal aqueous phase W1 was added to 8 mL of the oil phase O and emulsified with a homogenizer at 8000 rpm for 5 min to obtain a primary emulsion B with a particle size distribution range of 5-20 μm.

[0102] (3) Preparation of double emulsion:

[0103] The primary emulsions of the two particle size ranges were added to 80 mL of the external aqueous phase W2 under the action of a magnetic stirrer and magnetically stirred at 560 rpm for 3 min to obtain a double emulsion;

[0104] (4) Solidification of emulsion droplets:

[0105] The multiple emulsion was added to 500 mL of deionized water, stirred with a stirring paddle for 15 min, replaced with 1000 mL of deionized water and stirred overnight to solidify the microcarriers into spheres, and microspheres with a particle size of 150-200 μm were sieved out with a sieve.

[0106] The sieved microspheres were observed by scanning electron microscope. Figure 1 As shown in the figure, it can be seen that the microsphere has an internal and external through-hole channel structure, and has a primary pore structure and a secondary pore (wall pore) structure, wherein the pore size range of the primary pore is 30-70 μm, and the pore size range of the secondary pore is 5-10 μm.

[0107] (5) Surface modification of microcarriers:

[0108] Prepare 10 mM Tris buffer with pH = 8.5, dissolve dopamine in the buffer (0.1 mg / mL), mix with 4 mL dopamine solution / mL microspheres, and mix on a vertical mixer for 24 hours; then wash with deionized water until the supernatant is clear to obtain dopamine-modified microcarriers.

[0109] Application Example 1

[0110] The cell culture microcarrier prepared in Example 1 was used for cell culture, specifically as follows:

[0111] (1) sterilizing the dopamine-modified microcarriers by irradiation at 15 kGy for 1 h;

[0112] (2) PBS and rat bone marrow mesenchymal stem cell culture medium were replaced three times;

[0113] (3) According to the microcarrier 3×10 per 0.12 mL sedimentation volume 5 The cells were inoculated with SD rat bone marrow mesenchymal stem cells and cultured at 37°C for 6 h. Then, the cells were observed using a laser confocal microscope. Figure 2 As shown, the results show that cells can enter the interior of the microcarriers and grow (the arrows indicate that the cells enter the pores of the microspheres).

[0114] Example 2

[0115] This embodiment provides a cell culture microcarrier, and the preparation method thereof is as follows:

[0116] (1) Preparation:

[0117] 4 mL of 0.15% NaCl aqueous solution was used as the inner aqueous phase W1, 1.6 g of 50 / 50 PLGA (Shandong Academy of Biomedical Sciences Co., Ltd.), 1.44 g of polymethyl methacrylate (PMMA) (9011-14-7, Sigma-Aldrich (Shanghai) Trading Co., Ltd.) and 0.16 g of surfactant span80 (Sinopharm Group) were dissolved in 16 mL of ethyl acetate as the oil phase O; 1.5% PVA aqueous solution was prepared as the outer aqueous phase W2.

[0118] (2) Preparation of primary emulsion:

[0119] 2 mL of the internal aqueous phase W1 was added to 8 mL of the oil phase O and emulsified with a homogenizer at 3000 rpm for 5 min to obtain a primary emulsion A with a particle size distribution range of 10-80 μm;

[0120] 2 mL of the internal aqueous phase W1 was added to 8 mL of the oil phase O, and firstly emulsified by homogenizer at 8000 rpm for 5 min, and then the primary emulsion B with a particle size distribution range of 1-5 μm was obtained by rapid membrane emulsification.

[0121] (3) Preparation of double emulsion:

[0122] Primary emulsion A and primary emulsion B were added to 120 mL of external aqueous phase W2 under the action of a magnetic stirrer, and magnetically stirred at 400 rpm for 3 min to obtain a double emulsion;

[0123] (4) Solidification of emulsion droplets:

[0124] The multiple emulsion was added to 170 mL of deionized water, stirred with a stirring paddle for 15 min, replaced with 700 mL of deionized water and stirred overnight to solidify the microcarriers into spheres, and microspheres with a particle size of 150-200 μm were sieved out with a sieve.

[0125] The sieved microspheres were observed by scanning electron microscope. Figure 3 As shown in the figure, it can be seen that the microsphere has an internal and external through-hole channel structure, and has a primary pore structure and a secondary pore (wall pore) structure, wherein the pore size range of the primary pore is 30-80 μm, and the pore size range of the secondary pore is 1-10 μm.

[0126] (5) Surface modification of microcarriers:

[0127] Prepare 10 mM Tris buffer with pH = 8.5, dissolve dopamine in the buffer (0.1 mg / mL), mix with 4 mL dopamine solution / mL microspheres, and mix on a vertical mixer for 24 hours; then wash with deionized water until the supernatant is clear to obtain dopamine-modified microcarriers.

[0128] Application Example 2

[0129] The cell culture microcarrier prepared in Example 2 was used for cell culture, specifically as follows:

[0130] (1) sterilizing the dopamine-modified microcarriers by irradiation at 15 kGy for 1 h;

[0131] (2) PBS and rat bone marrow mesenchymal stem cell culture medium were replaced three times;

[0132] (3) According to the microcarrier 3×10 per 0.12 mL sedimentation volume 5The cells were inoculated with SD rat bone marrow mesenchymal stem cells and cultured at 37°C for 24 h. Then, the cells were observed using a laser confocal microscope. Figure 4 As shown, the results show that cells can enter the interior of the microcarriers and grow (the marked area represents cells growing inside the microspheres).

[0133] (4) The cell culture microcarriers prepared in Example 2 and the dopamine-modified microcarriers with smooth surfaces (control samples) were used for cell culture (the same method as above). After 96 hours of cell culture, the statistical results were as follows: Figure 5 As shown in the figure, it can be seen that the proliferation rate of cells on macroporous microcarriers is 10 times, which is twice that of smooth surface microcarriers, indicating that macroporous microcarriers have an advantage over smooth surface microcarriers in terms of cell proliferation. Figure 6 and Figure 7 shown.

[0134] The preparation method of the dopamine-modified microcarrier with smooth surface is as follows:

[0135] (1') 1.6 g 50 / 50 PLGA (Shandong Academy of Biomedical Sciences Co., Ltd.), 1.44 g polymethyl methacrylate (PMMA) (9011-14-7, Sigma-Aldrich (Shanghai) Trading Co., Ltd.) and 0.16 g surfactant span80 (Sinopharm Group) were dissolved in 16 mL ethyl acetate as the oil phase O; 1.5% PVA aqueous solution was prepared as the water phase W.

[0136] (2') 15 mL of the oil phase was added to 120 mL of the water phase and magnetically stirred at 400 rpm for 3 min to obtain an emulsion.

[0137] (3') The emulsion was added to 170 mL of deionized water and stirred with a stirring paddle for 15 min. Then, 700 mL of deionized water was added and stirred overnight to solidify the microcarriers into spheres. Microspheres with a particle size of 150-200 μm were sieved with a sieve.

[0138] (4') Prepare 10 mM Tris buffer at pH = 8.5, dissolve dopamine in the buffer (0.1 mg / mL), mix at a rate of 4 mL dopamine solution / mL microspheres, and mix on a vertical mixer for 24 h; then wash with deionized water until the supernatant is clear.

[0139] Example 3

[0140] This embodiment provides a cell culture microcarrier, and the preparation method thereof is as follows:

[0141] (1) Preparation:

[0142] 4 mL of 0.15% NaCl aqueous solution was used as the inner aqueous phase W1, 2.34 g of polymethyl methacrylate (PMMA) (9011-14-7, Sigma-Aldrich (Shanghai) Trading Co., Ltd.) and 0.06 g of polystyrene polyhydroxyethyl acrylate (Shandong Academy of Biomedical Sciences Co., Ltd.) were dissolved in 16 mL of ethyl acetate as the oil phase O; 1.5% PVA aqueous solution was prepared as the outer aqueous phase W2.

[0143] (2) Preparation of primary emulsion:

[0144] 2 mL of the internal aqueous phase W1 was added to 8 mL of the oil phase O and emulsified by vortex oscillation for 5 min to obtain a primary emulsion A with a particle size distribution range of 50-200 μm;

[0145] 2 mL of the internal aqueous phase W1 was added to 8 mL of the oil phase O, and firstly emulsified by homogenizer at 8000 rpm for 5 min, and then the primary emulsion B with a particle size distribution range of 1-10 μm was obtained by rapid membrane emulsification.

[0146] (3) Preparation of double emulsion:

[0147] Primary emulsion A and primary emulsion B were added to 120 mL of external aqueous phase W2 under the action of a magnetic stirrer, and magnetically stirred at 300 rpm for 5 min to obtain a double emulsion;

[0148] (4) Solidification of emulsion droplets:

[0149] The double emulsion was added into 300 mL of deionized water, stirred with a stirring paddle for 15 min, and then replaced with 1400 mL of deionized water and stirred overnight to solidify the microcarriers into spheres.

[0150] The obtained microspheres were observed by scanning electron microscopy. Figure 8 As shown, the particle size of the microsphere is about 300 μm, and the microsphere has internal and external through-hole channels as well as a primary pore structure and a secondary pore (wall pore) structure, wherein the pore size range of the primary pore is 80-100 μm, presenting an open pore structure, and the pore size range of the secondary pore is 1-5 μm.

[0151] (5) Surface modification of microcarriers:

[0152] Prepare 10mM Tris buffer at pH=8.5, dissolve dopamine in the buffer (0.1mg / mL), mix with 4mL dopamine solution / mL microspheres, mix on a vertical mixer for 24h; then wash with deionized water until the supernatant is clear. Then disperse 0.2g microspheres in 10mg / mL RGD PBS solution, mix on a vertical mixer for 24h, and remove unreacted RGD with deionized water.

[0153] Application Example 3

[0154] The cell culture microcarrier prepared in Example 3 was used for cell culture, specifically as follows:

[0155] (1) sterilizing the RGD-modified microcarriers by irradiation at 15 kGy for 1 h;

[0156] (2) replacing PBS and human amniotic membrane mesenchymal stem cell culture medium three times;

[0157] (3) According to the microcarrier 3×10 per 0.12 mL sedimentation volume 5 Human amniotic mesenchymal stem cells were inoculated into each cell and cultured at 37°C for 24 h. The cells were then observed using a laser confocal microscope. Fig. 9 As shown, the results showed that cells could attach and grow on the microspheres.

[0158] (4) The cell culture microcarriers prepared in Example 3 and the RGD-modified microcarriers with smooth surface (control sample) were used for cell culture (same method as above). After 7 days of cell culture, the cell proliferation multiple of the cell culture microcarriers prepared in Example 3 was 6 times, which was 4.3 times that of the smooth surface microcarriers and 2 times that of the 2D culture plate, indicating the advantage of macroporous microcarriers over smooth surface microcarriers in cell proliferation.

[0159] The preparation method of the RGD-modified microcarrier with a smooth surface is as follows:

[0160] (1′) 2.34 g of polymethyl methacrylate (PMMA) (9011-14-7, Sigma-Aldrich (Shanghai) Trading Co., Ltd.) and 0.06 g of polystyrene polyhydroxyethyl acrylate (Shandong Academy of Biomedical Sciences Co., Ltd.) were dissolved in 16 mL of ethyl acetate as the oil phase O; a 1.5% PVA aqueous solution was prepared as the water phase W.

[0161] (2') 15 mL of the oil phase was added to 120 mL of the water phase under the action of a magnetic stirrer and magnetically stirred at 300 rpm for 5 min to obtain an emulsion.

[0162] (3') The emulsion was added to 300 mL of deionized water and stirred with a stirring paddle for 15 min. Then, 1400 mL of deionized water was added and stirred overnight to allow the microcarriers to solidify into spheres.

[0163] (4') Prepare 10 mM Tris buffer at pH = 8.5, dissolve dopamine in the buffer (0.1 mg / mL), mix with 4 mL dopamine solution / mL microspheres, mix on a vertical mixer for 24 h; then wash with deionized water until the supernatant is clear. Then disperse 0.2 g of microspheres in 10 mg / mL RGD PBS solution, mix on a vertical mixer for 24 h, and remove unreacted RGD with deionized water to obtain.

[0164] Example 4

[0165] This embodiment provides a cell culture microcarrier, and the preparation method thereof is as follows:

[0166] (1) Preparation:

[0167] 4 mL of 0.15% KCl aqueous solution was used as the inner aqueous phase W1, 15 g of styrene monomer, 5 g of crosslinking agent divinylbenzene, 0.6 g of initiator benzoyl peroxide, and 6 g of surfactant polymethyl methacrylate and polyhydroxyethyl acrylate were stirred as the oil phase O; 1.5% PVA aqueous solution was prepared as the outer aqueous phase W2.

[0168] (2) Preparation of primary emulsion:

[0169] 2 mL of the internal aqueous phase W1 was added to 8 mL of the oil phase O and emulsified with a homogenizer at 3000 rpm for 5 min to obtain a primary emulsion A with a particle size distribution range of 10-80 μm;

[0170] 2 mL of the internal aqueous phase W1 was added to 8 mL of the oil phase O, and firstly emulsified by homogenizer at 8000 rpm for 5 min, and then the primary emulsion B with a particle size distribution range of 1-10 μm was obtained by rapid membrane emulsification.

[0171] (3) Preparation of double emulsion:

[0172] Primary emulsion A and primary emulsion B were added to 120 mL of external aqueous phase W2 under the action of a magnetic stirrer, and magnetically stirred at 300 rpm for 3 min to obtain a double emulsion;

[0173] (4) Solidification of emulsion droplets:

[0174] The double emulsion was placed in a reactor, the polymerization temperature was 75°C, the time was 20 hours, after the reaction, it was washed several times with distilled water and ethanol to wash away components such as surfactants, extracted, and dried to obtain the product.

[0175] The obtained microspheres were observed by scanning electron microscopy. Fig.10As shown in the figure, it can be seen that the microsphere particle size is 200-300μm, has an internal and external through-channel structure, and has a primary pore structure and a secondary pore (wall pore) structure, wherein the pore size range of the primary pore is 10-40μm, and the pore size range of the secondary pore is 1-5μm.

[0176] (5) Surface modification of microcarriers:

[0177] 0.5 g of microcarriers were dispersed in 0.5 mg / mL type I collagen acetic acid solution and incubated on a shaker at 4°C for 24 h; then washed with deionized water to obtain type I collagen modified microcarriers.

[0178] Application Example 4

[0179] The cell culture microcarrier prepared in Example 4 was used for cell culture, specifically as follows:

[0180] (1) sterilizing the type I collagen modified microcarriers by irradiation at 15 kGy for 1 h;

[0181] (2) replacing PBS and human adipose-derived mesenchymal stem cell culture medium three times respectively;

[0182] (3) According to the microcarrier 3×10 per 0.12 mL sedimentation volume 5 Human adipose-derived mesenchymal stem cells were inoculated into 100 cells and cultured at 37°C for 24 h. The cells were then observed using a laser confocal microscope. Fig.11 As shown, the results showed that cells could attach and grow on the microspheres.

[0183] (4) The cell culture microcarrier prepared in Example 4 and the smooth surface type I collagen modified microcarrier (control sample) were used for cell culture (the method is the same as above). After 7 days of cell culture, the cell proliferation multiple of the cell culture microcarrier prepared in Example 4 was 22 times, which was twice that of the smooth surface microcarrier and 2.4 times that of the 2D culture plate, indicating that the macroporous microcarrier has an advantage over the smooth surface microcarrier in terms of cell proliferation.

[0184] The preparation method of the smooth-surfaced type I collagen-modified microcarrier is as follows:

[0185] (1') 15 g of styrene monomer, 5 g of crosslinking agent divinylbenzene, 0.6 g of initiator benzoyl peroxide, and 6 g of surfactant polymethyl methacrylate and polyhydroxyethyl acrylate were stirred to form the oil phase O; and a 1.5% PVA aqueous solution was prepared to form the water phase W.

[0186] (2') 15 mL of the oil phase was added to 120 mL of the water phase under the action of a magnetic stirrer and magnetically stirred at 300 rpm for 5 min to obtain an emulsion.

[0187] (3') The emulsion was placed in a reaction kettle, and the polymerization temperature was 75°C for 20 h. After the reaction, it was washed several times with distilled water and ethanol to remove components such as surfactants, extracted, and dried to obtain microcarriers.

[0188] (4') 0.5 g of microcarriers were dispersed in 0.5 mg / mL type I collagen acetic acid solution and incubated at 4° C. on a shaker for 24 h; then washed with deionized water to obtain type I collagen modified microcarriers.

[0189] Example 5

[0190] This embodiment provides a cell culture microcarrier, and the preparation method thereof is as follows:

[0191] (1) Preparation:

[0192] 4 mL of 0.15% glucose aqueous solution was used as the inner aqueous phase W1, 2.4 g of PLA (Shandong Academy of Biomedical Sciences Co., Ltd.) and 0.06 g of surfactant span80 (Sinopharm Group) were dissolved in 16 mL of dichloroethane as the oil phase O; 1.5% PVA aqueous solution was prepared as the outer aqueous phase W2.

[0193] (2) Preparation of primary emulsion:

[0194] 3 mL of the internal aqueous phase W1 was added to 12 mL of the oil phase O and emulsified at 1000 rpm for 10 min using magnetic stirring to obtain a primary emulsion A with a particle size distribution range of 10-100 μm;

[0195] 1 mL of the internal aqueous phase W1 was added to 4 mL of the oil phase O, and firstly emulsified by homogenizer at 7000 rpm for 5 min, and then the primary emulsion B with a particle size distribution range of 1-10 μm was obtained by rapid membrane emulsification.

[0196] (3) Preparation of double emulsion:

[0197] Primary emulsion A and primary emulsion B were added to 120 mL of external aqueous phase W2 under the action of a magnetic stirrer, and magnetically stirred at 400 rpm for 5 min to obtain a double emulsion;

[0198] (4) Solidification of emulsion droplets:

[0199] The double emulsion was added to 1400 mL of deionized water and stirred overnight to solidify the microcarriers into spheres, and microspheres with a particle size of 150-200 μm were sieved out using a sieve.

[0200] The sieved microspheres were observed by scanning electron microscope. Fig.12As shown in the figure, it can be seen that the microsphere has an internal and external through-hole channel structure, and has a primary pore structure and a secondary pore (wall pore) structure, wherein the pore size range of the primary pore is 30-80 μm, and the pore size range of the secondary pore is 1-10 μm.

[0201] (5) Surface modification of microcarriers:

[0202] 0.5 g of microcarriers were dispersed in PBS, 0.07 g of EDC and 0.03 g of NHS were added, the mixture was reacted at 25°C for 24 h, and the mixture was washed with deionized water. The obtained microcarriers were dispersed in 1 mL of PBS, 10 mg of RGD was added, the mixture was reacted at 25°C for 24 h, and the mixture was washed with deionized water to obtain RGD-modified microcarriers.

[0203] Application Example 5

[0204] The cell culture microcarrier prepared in Example 5 was used for cell culture, specifically as follows:

[0205] (1) sterilizing the RGD-modified microcarriers by irradiation at 15 kGy for 1 h;

[0206] (2) Replace with PBS three times; then replace with human bone marrow mesenchymal stem cell culture medium three times;

[0207] (3) According to the microcarrier 3×10 per 0.12 mL sedimentation volume 5 Human bone marrow mesenchymal stem cells were inoculated and cultured at 37°C for 24 h. Then, the cells were observed using a laser confocal microscope. Fig.13 As shown, the results showed that cells could attach and grow on the microspheres.

[0208] (4) The cell culture microcarriers prepared in Example 5 and the RGD-modified microcarriers with smooth surface (control sample) were used for cell culture (same method as above). After 7 days of cell culture, the cell proliferation multiple of the cell culture microcarriers prepared in Example 5 was 20 times, which was 1.6 times that of the smooth surface microcarriers and 2.3 times that of the 2D culture plate, indicating that the macroporous microcarriers have an advantage over the smooth surface microcarriers in terms of cell proliferation.

[0209] The preparation method of the RGD-modified microcarrier with a smooth surface is as follows:

[0210] (1') 2.4 g PLA (Shandong Academy of Biomedical Sciences Co., Ltd.) and 0.06 g surfactant span80 (Sinopharm Group) were dissolved in 16 mL dichloroethane as the oil phase O; 1.5% PVA aqueous solution was prepared as the water phase W.

[0211] (2') 15 mL of the oil phase was added to 120 mL of the water phase under the action of a magnetic stirrer and magnetically stirred at 400 rpm for 3 min to obtain an emulsion.

[0212] (3') The emulsion was added to 300 mL of deionized water and stirred with a stirring paddle for 15 min. Then, 1400 mL of deionized water was added and stirred overnight to allow the microcarriers to solidify into spheres.

[0213] (4') 0.5 g of microcarriers were dispersed in PBS, 0.07 g of EDC and 0.03 g of NHS were added, the mixture was reacted at 25°C for 24 h, and the mixture was washed with deionized water. The obtained microcarriers were dispersed in 1 mL of PBS, 10 mg of RGD was added, the mixture was reacted at 25°C for 24 h, and the mixture was washed with deionized water to obtain RGD-modified microcarriers.

[0214] Example 6

[0215] This embodiment provides a cell culture microcarrier, and the preparation method thereof is as follows:

[0216] (1) Preparation:

[0217] 4 mL of 0.15% NaCl aqueous solution was used as the inner aqueous phase W1, 3 g of methyl methacrylate and methacrylic acid copolymer P (MMA-MAA) (Shandong Academy of Biomedical Sciences Co., Ltd.) and 0.03 g of polystyrene polyhydroxyethyl acrylate (Shandong Academy of Biomedical Sciences Co., Ltd.) were dissolved in 24 mL of ethyl acetate as the oil phase O; 1.5% PVA aqueous solution was prepared as the outer aqueous phase W2.

[0218] (2) Preparation of primary emulsion:

[0219] 3 mL of the internal aqueous phase W1 was added to 12 mL of the oil phase O and emulsified with a homogenizer at 3000 rpm for 10 min to obtain a primary emulsion A with a particle size distribution range of 5-50 μm;

[0220] 1 mL of the internal aqueous phase W1 was added to 4 mL of the oil phase O, and firstly emulsified by homogenizer at 10000 rpm for 5 min, and then the primary emulsion B with a particle size distribution range of 1-3 μm was obtained by rapid membrane emulsification.

[0221] (3) Preparation of double emulsion:

[0222] Primary emulsion A and primary emulsion B were added to 120 mL of external aqueous phase W2 under the action of a magnetic stirrer, and magnetically stirred at 500 rpm for 5 min to obtain a double emulsion;

[0223] (4) Solidification of emulsion droplets:

[0224] The double emulsion was added to 1400 mL of deionized water and stirred overnight to solidify the microcarriers into spheres, and microspheres with a particle size of 100-150 μm were sieved out using a sieve.

[0225] The sieved microspheres were observed by scanning electron microscope. Fig.14 As shown in the figure, it can be seen that the microsphere has an internal and external through-hole channel structure, and has a primary pore structure and a secondary pore (wall pore) structure, wherein the pore size range of the primary pore is 10-20 μm, and the pore size range of the secondary pore is 1-2 μm.

[0226] (5) Surface modification of microcarriers:

[0227] 0.5 g of microcarriers were dispersed in 0.5 mg / mL type I collagen acetic acid solution and incubated on a shaker at 4°C for 24 h; then washed with deionized water to obtain type I collagen modified microcarriers.

[0228] Application Example 6

[0229] The cell culture microcarrier prepared in Example 6 was used for cell culture, specifically as follows:

[0230] (1) sterilizing the type I collagen modified microcarriers by irradiation at 15 kGy for 1 h;

[0231] (2) PBS and rat bone marrow mesenchymal stem cell culture medium were replaced three times;

[0232] (3) According to the microcarrier 3×10 per 0.12 mL sedimentation volume 5 Human bone marrow mesenchymal stem cells were inoculated and cultured at 37°C for 24 h. Then, the cells were observed using a laser confocal microscope. Fig.15 As shown, the results showed that cells could attach and grow on the microspheres.

[0233] (4) The cell culture microcarrier prepared in Example 6 and the smooth surface type I collagen modified microcarrier (control sample) were used for cell culture (the method is the same as above). After 7 days of cell culture, the cell proliferation multiple of the cell culture microcarrier prepared in Example 6 was 29 times, which was 2.1 times that of the smooth surface microcarrier and 3.2 times that of the 2D culture plate, indicating that the macroporous microcarrier has an advantage over the smooth surface microcarrier in terms of cell proliferation.

[0234] The preparation method of the smooth-surfaced type I collagen-modified microcarrier is as follows:

[0235] (1′) 3 g of methyl methacrylate and methacrylic acid copolymer P (MMA-MAA) (Shandong Academy of Biomedical Sciences Co., Ltd.) and 0.03 g of polystyrene polyhydroxyethyl acrylate (Shandong Academy of Biomedical Sciences Co., Ltd.) were dissolved in 24 mL of ethyl acetate as the oil phase O; a 1.5% PVA aqueous solution was prepared as the water phase W.

[0236] (2') 15 mL of the oil phase was added to 120 mL of the water phase under the action of a magnetic stirrer and magnetically stirred at 400 rpm for 3 min to obtain an emulsion.

[0237] (3') The emulsion was added to 300 mL of deionized water and stirred with a stirring paddle for 15 min. Then, 1400 mL of deionized water was added and stirred overnight to allow the microcarriers to solidify into spheres.

[0238] (4') 0.5 g of microcarriers were dispersed in 0.5 mg / mL type I collagen acetic acid solution and incubated at 4° C. on a shaker for 24 h; then washed with deionized water to obtain type I collagen modified microcarriers.

[0239] Example 7

[0240] This embodiment provides a cell culture microcarrier, and the preparation method thereof is as follows:

[0241] (1) Preparation:

[0242] 4 mL of 0.15% sucrose aqueous solution was used as the inner aqueous phase W1, 2.4 g of poly(glycidyl methacrylate) (PGMA) (Shandong Academy of Biomedical Sciences Co., Ltd.) and 0.06 g of glycidyl methacrylate (Shandong Academy of Biomedical Sciences Co., Ltd.) were dissolved in 16 mL of ethyl acetate as the oil phase O; 1.5% PVA aqueous solution was prepared as the outer aqueous phase W2.

[0243] (2) Preparation of primary emulsion:

[0244] 3 mL of the internal aqueous phase W1 was added to 12 mL of the oil phase O and emulsified with magnetic stirring at 400 rpm for 2 min to obtain a primary emulsion A with a particle size distribution range of 20-100 μm;

[0245] 1 mL of the internal aqueous phase W1 was added to 4 mL of the oil phase O, and firstly emulsified by homogenizer at 10000 rpm for 2 min, and then the primary emulsion B with a particle size distribution range of 1-3 μm was obtained by rapid membrane emulsification.

[0246] (3) Preparation of double emulsion:

[0247] Primary emulsion A and primary emulsion B were added to 120 mL of external aqueous phase W2 under the action of a magnetic stirrer, and magnetically stirred at 400 rpm for 5 min to obtain a double emulsion;

[0248] (4) Solidification of emulsion droplets:

[0249] The double emulsion was added into 1400 mL of deionized water and stirred overnight to solidify the microcarriers into spheres.

[0250] The obtained microspheres were observed by scanning electron microscopy. Fig.16 As shown in the figure, it can be seen that the particle size of the microspheres is 200-300μm, the surface is relatively smooth, has an internal and external through-channel structure, and has a primary pore structure and a secondary pore (wall pore) structure, wherein the pore size range of the primary pore is 30-100μm, and the pore size range of the secondary pore is 1-5μm.

[0251] (5) Surface modification of microcarriers:

[0252] 0.5 g of microcarriers were dispersed in 1 mL of PBS, 0.07 g of EDC and 0.03 g of NHS were added, reacted at 25°C for 24 h, washed with PBS, the obtained microcarriers were dispersed in 1 mL of PBS, 10 mg of GFOGER was added, incubated at 25°C for 24 h, and washed with deionized water to obtain GFOGER-modified microcarriers.

[0253] Application Example 7

[0254] The cell culture microcarrier prepared in Example 7 was used for cell culture, specifically as follows:

[0255] (1) The GFOGER-modified microcarriers were sterilized by irradiation at 15 kGy for 1 h;

[0256] (2) PBS and L929 cell culture medium were replaced three times respectively;

[0257] (3) According to the microcarrier 3×10 per 0.12 mL sedimentation volume 5 L929 cells were inoculated and cultured at 37°C for 24 h, and then observed using a laser confocal microscope. Fig.17 As shown, the results showed that cells could attach and grow on the microspheres.

[0258] (4) The cell culture microcarriers prepared in Example 7 and the microcarriers modified with GFOGER having a smooth surface (control sample) were used for cell culture (the method is the same as above). After 7 days of cell culture, the proliferation multiple of the cell culture microcarriers prepared in Example 7 was 20 times, which was 1.5 times that of the microcarriers having a smooth surface and 2 times that of the 2D culture plate, indicating that the macroporous microcarriers have an advantage in cell proliferation over the microcarriers having a smooth surface.

[0259] The preparation method of the GFOGER-modified microcarrier with smooth surface is as follows:

[0260] (1′) 2.4 g of poly(glycidyl methacrylate) (PGMA) (Shandong Academy of Biomedical Sciences Co., Ltd.) and 0.06 g of glycidyl methacrylate (Shandong Academy of Biomedical Sciences Co., Ltd.) were dissolved in 16 mL of ethyl acetate as the oil phase O; a 1.5% PVA aqueous solution was prepared as the water phase W.

[0261] (2') 15 mL of the oil phase was added to 120 mL of the water phase under the action of a magnetic stirrer and magnetically stirred at 400 rpm for 3 min to obtain an emulsion.

[0262] (3') The emulsion was added to 300 mL of deionized water and stirred with a stirring paddle for 15 min. Then, 1400 mL of deionized water was added and stirred overnight to allow the microcarriers to solidify into spheres.

[0263] (4′) 0.5 g of microcarriers were dispersed in 1 mL of PBS, 0.07 g of EDC and 0.03 g of NHS were added, the mixture was reacted at 25° C. for 24 h, and the mixture was washed with PBS. The obtained microcarriers were dispersed in 1 mL of PBS, 10 mg of GFOGER was added, the mixture was incubated at 25° C. for 24 h, and the mixture was washed with deionized water to obtain GFOGER-modified microcarriers.

[0264] Example 8

[0265] This embodiment provides a cell culture microcarrier, and the preparation method thereof is as follows:

[0266] (1) Preparation:

[0267] 4 mL of 0.15% NaCl aqueous solution was used as the inner aqueous phase W1, 15 g of styrene monomer (Sinopharm Group), 5 g of crosslinking agent divinylbenzene (Sinopharm Group), 0.6 g of initiator benzoyl peroxide (Sinopharm Group), and 8 g of surfactant span80 (Sinopharm Group) were stirred and mixed; 1.5% PVA aqueous solution was prepared as the outer aqueous phase W2.

[0268] (2) Preparation of primary emulsion:

[0269] 1 mL of the internal aqueous phase W1 was added to 4 mL of the oil phase O and emulsified with a homogenizer at 4000 rpm for 2 min to obtain a primary emulsion with a particle size distribution range of 5-30 μm;

[0270] 3 mL of the internal aqueous phase W1 was added to 12 mL of the oil phase O, and firstly emulsified by homogenizer at 8000 rpm for 2 min, and then the primary emulsion B with a particle size distribution range of 1-5 μm was obtained by rapid membrane emulsification.

[0271] (3) Preparation of double emulsion:

[0272] Primary emulsion A and primary emulsion B were added to 120 mL of external aqueous phase W2 under the action of a magnetic stirrer, and magnetically stirred at 400 rpm for 5 min to obtain a double emulsion;

[0273] (4) Solidification of emulsion droplets:

[0274] The double emulsion was placed in a reactor, the polymerization temperature was 75°C, the time was 20 hours, after the reaction, it was washed several times with distilled water and ethanol to wash away components such as surfactants, extracted, and dried to obtain the product.

[0275] The obtained microspheres were observed by scanning electron microscopy. Fig.18 As shown in the figure, it can be seen that it has an internal and external through-channel structure, a large porosity, a small pore spacing, and a primary pore structure and a secondary pore (wall pore) structure, wherein the pore size range of the primary pore is 5-20 μm, and the pore size range of the secondary pore is 1-5 μm.

[0276] (5) Surface modification of microcarriers:

[0277] 0.5 g of microspheres were dispersed in 1 mL of 0.05 mg / mL laminin Tri-HCl solution, incubated at 4°C for 24 h, and washed with deionized water to obtain laminin-modified microcarriers.

[0278] Application Example 8

[0279] The cell culture microcarrier prepared in Example 8 was used for cell culture, specifically as follows:

[0280] (1) sterilizing the laminin-modified microcarriers by irradiation at 15 kGy for 1 h;

[0281] (2) replacing PBS and human umbilical cord mesenchymal stem cell culture medium three times;

[0282] (3) According to the microcarrier 3×10 per 0.12 mL sedimentation volume 5 Human umbilical cord mesenchymal stem cells were inoculated and cultured at 37°C for 24 h, and then observed using a laser confocal microscope. Fig.19 As shown, the results showed that cells could attach and grow on the microspheres.

[0283] (4) The cell culture microcarrier prepared in Example 8 and the laminin-modified microcarrier with a smooth surface (control sample) were used for cell culture (same method as above). After 7 days of cell culture, the cell proliferation multiple of the cell culture microcarrier prepared in Example 8 was 27 times, which was 2.5 times that of the smooth surface microcarrier and 3 times that of the 2D culture plate, indicating the advantage of macroporous microcarrier over smooth surface microcarrier in cell proliferation.

[0284] The preparation method of the laminin-modified microcarrier with smooth surface is as follows:

[0285] (1') 15 g of styrene monomer (Sinopharm Group), 5 g of crosslinking agent divinylbenzene (Sinopharm Group), 0.6 g of initiator benzoyl peroxide (Sinopharm Group), and 8 g of surfactant span80 (Sinopharm Group) were stirred and mixed; 1.5% PVA aqueous solution was prepared as the aqueous phase W.

[0286] (2') 15 mL of the oil phase was added to 120 mL of the water phase under the action of a magnetic stirrer and magnetically stirred at 400 rpm for 3 min to obtain an emulsion.

[0287] (3') The emulsion was placed in a reaction kettle, and the polymerization temperature was 75°C for 20 h. After the reaction, it was washed several times with distilled water and ethanol to remove components such as surfactants, extracted, and dried to obtain microcarriers.

[0288] (4') 0.5 g of microspheres were dispersed in 1 mL of 0.05 mg / mL laminin Tri-HCl solution, incubated at 4° C. for 24 h, and washed with deionized water to obtain laminin-modified microcarriers.

[0289] Example 9

[0290] This embodiment provides a cell culture microcarrier, and the preparation method thereof is as follows:

[0291] (1) Preparation:

[0292] 4 mL of 0.15% NaCl aqueous solution was used as the inner aqueous phase W1, 2.4 g of polycaprolactone (Shandong Academy of Biomedical Sciences Co., Ltd.) and 0.06 g of polymethyl methacrylate polyhydroxyethyl acrylate (Shandong Academy of Biomedical Sciences Co., Ltd.) were dissolved in 20 mL of chloroform as the oil phase O; 1.5% PVA aqueous solution was prepared as the outer aqueous phase W2.

[0293] (2) Preparation of primary emulsion:

[0294] 1 mL of the internal aqueous phase W1 was added to 4 mL of the oil phase O and emulsified with a homogenizer at 5000 rpm for 2 min to obtain a primary emulsion with a particle size distribution range of 5-30 μm;

[0295] 3 mL of the internal aqueous phase W1 was added to 12 mL of the oil phase O, and firstly emulsified by homogenizer at 8000 rpm for 2 min, and then the primary emulsion B with a particle size distribution range of 1-5 μm was obtained by rapid membrane emulsification.

[0296] (3) Preparation of double emulsion:

[0297] Primary emulsion A and primary emulsion B were added to 120 mL of external aqueous phase W2 under the action of a magnetic stirrer, and magnetically stirred at 400 rpm for 5 min to obtain a double emulsion;

[0298] (4) Solidification of emulsion droplets:

[0299] The double emulsion was added to 1400 mL of deionized water and stirred overnight to solidify the microcarriers into spheres, and the screening particle size range was 150-200 μm.

[0300] The obtained microspheres were observed by scanning electron microscopy. Fig. 20 As shown in the figure, it can be seen that it has an internal and external through-channel structure, a large porosity, a small pore spacing, and a primary pore structure and a secondary pore (wall pore) structure, wherein the pore size range of the primary pore is 5-20 μm, and the pore size range of the secondary pore is 1-5 μm.

[0301] (5) Surface modification of microcarriers:

[0302] 0.5 g of microcarriers were dispersed in 1 mL of PBS, 0.09 g of EDC and 0.06 g of NHS were added, reacted at 25°C for 24 h, washed with PBS, the obtained microspheres were dispersed in 1 mL of PBS, 10 mg of gelatin was added, incubated at 40°C for 24 h, and washed with PBS to obtain gelatin-modified microcarriers.

[0303] Application Example 9

[0304] The cell culture microcarrier prepared in Example 9 was used for cell culture, specifically as follows:

[0305] (1) sterilizing the gelatin-modified microcarriers by irradiation at 15 kGy for 1 h;

[0306] (2) replacing PBS and human umbilical cord mesenchymal stem cell culture medium three times;

[0307] (3) According to the microcarrier 3×10 per 0.12 mL sedimentation volume 5 Human umbilical cord mesenchymal stem cells were inoculated and cultured at 37°C for 24 h, and then observed using a laser confocal microscope. Fig.21 As shown, the results showed that cells could attach and grow on the microspheres.

[0308] (4) The cell culture microcarrier prepared in Example 9 and the smooth-surfaced gelatin-modified microcarrier (control sample) were used for cell culture (same method as above). After 7 days of cell culture, the cell proliferation multiple of the cell culture microcarrier prepared in Example 9 was 28 times, which was 1.9 times that of the smooth-surfaced microcarrier and 3.1 times that of the 2D culture plate, indicating the advantage of macroporous microcarrier over smooth-surfaced microcarrier in cell proliferation.

[0309] The preparation method of the smooth-surfaced gelatin-modified microcarrier is as follows:

[0310] (1′) 2.4 g polycaprolactone (Shandong Academy of Biomedical Sciences Co., Ltd.) and 0.06 g polymethyl methacrylate polyhydroxyethyl acrylate (Shandong Academy of Biomedical Sciences Co., Ltd.) were dissolved in 20 mL chloroform as the oil phase O; 1.5% PVA aqueous solution was prepared as the water phase W.

[0311] (2') 15 mL of the oil phase was added to 120 mL of the water phase under the action of a magnetic stirrer and magnetically stirred at 400 rpm for 5 min to obtain an emulsion.

[0312] (3') The emulsion was added to 300 mL of deionized water and stirred with a stirring paddle for 15 min. Then, 1400 mL of deionized water was added and stirred overnight to allow the microcarriers to solidify into spheres.

[0313] (4') 0.5 g of microcarriers were dispersed in 1 mL of PBS, 0.09 g of EDC and 0.06 g of NHS were added, the mixture was reacted at 25 °C for 24 h, and the mixture was washed with PBS. The obtained microspheres were dispersed in 1 mL of PBS, 10 mg of gelatin was added, the mixture was incubated at 40 °C for 24 h, and the mixture was washed with PBS to obtain gelatin-modified microcarriers.

[0314] Application Example 10

[0315] The cell culture microcarrier prepared in Example 9 was used to culture breast cancer cells MCF7, as follows:

[0316] (1) sterilizing the gelatin-modified microcarriers by irradiation at 15 kGy for 1 h;

[0317] (2) Replace PBS and culture medium three times respectively;

[0318] (3) According to the microcarrier 3×10 per 0.12 mL sedimentation volume 5 The cells were inoculated into MCF7 and cultured at 37°C for 24 h. Then, the cells were observed using a laser confocal microscope. Fig. 22 As shown, the results showed that cells could attach and grow on the microspheres.

[0319] (4) The cell culture microcarriers prepared in Example 9 and the smooth-surfaced gelatin-modified microcarriers (control samples) were used to culture breast cancer MCF7 cells (same method as above). After 7 days of cell culture, the cell proliferation multiple of the cell culture microcarriers prepared in Example 9 was 9 times, which was 2.1 times that of the smooth-surfaced microcarriers and 3 times that of the 2D culture plates, indicating the advantages of macroporous microcarriers over smooth-surfaced microcarriers in terms of cell proliferation.

[0320] The preparation method of the gelatin-modified microcarrier with a smooth surface is the same as that of Application Example 9.

[0321] In summary, by regulating various parameters in the preparation process, cell culture microcarriers with different particle sizes, different pore size distributions, different surface roughnesses, and different surface coatings can be prepared, and cells can be loaded thereon and grow normally.

[0322] The applicant declares that the present invention illustrates a cell culture microcarrier and its preparation method and application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0323] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0324] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A cell culture microcarrier, characterized in that: The cell culture microcarrier comprises a polymer porous microsphere with an inner and outer through-hole structure.

2. The cell culture microcarrier according to claim 1, characterized in that: The cell culture microcarrier has a primary pore structure and a secondary pore structure; the pore size of the primary pore is 5-200 μm, and the pore size of the secondary pore is 0.1-50 μm; Preferably, the primary pores have a pore size of 10-150 μm, more preferably 30-100 μm; Preferably, the secondary pores have a diameter of 0.1-30 μm, more preferably 1-10 μm.

3. The cell culture microcarrier according to claim 1 or 2, characterized in that: The particle size of the cell culture microcarrier is 50-1000 μm, preferably 100-500 μm, and more preferably 150-300 μm.

4. The cell culture microcarrier according to any one of claims 1 to 3, characterized in that: The raw materials for preparing the matrix of the porous microspheres include modified or unmodified polymer materials and polymer monomers; Preferably, the polymer material comprises any one of polycaprolactone, polylactic acid, polylactic glycolic acid, polyglycolic acid, polyhydroxyalkanoate, poly-L-glutamic acid, polymethacrylic acid, polystyrene, polyethylene oxide, polyoxypropylene copolymer, polymethyl methacrylate, polyethylene glycol, monomethoxy polyethylene glycol polylactic acid copolymer, polystyrene and polyoxypropylene copolymer, polystyrene and polyethylene glycol copolymer, polystyrene and polyacrylic acid copolymer, polystyrene and polyhydroxyethyl acrylate copolymer, polymethacrylate and polyacrylic acid copolymer, polymethyl methacrylate and polyhydroxyethyl acrylate copolymer, polymethacrylate and methacrylic acid copolymer, methyl methacrylate and β-hydroxyethyl methacrylate copolymer, polychloromethylstyrene or polyglycidyl methacrylate, or a combination of at least two thereof; Preferably, the polymer monomer includes any one of glycidyl methacrylate, methyl methacrylate, butyl methacrylate, octadecyl methacrylate, hydroxyethyl methacrylate or styrene, or a combination of at least two thereof.

5. The cell culture microcarrier according to any one of claims 1 to 4, characterized in that: The cell culture microcarrier further comprises any one or a combination of at least two of proteins, glycoproteins, polysaccharides, polypeptides or adhesion materials modified on the surface of the polymer porous microsphere; Preferably, the protein comprises any one or a combination of at least two of gelatin, collagen, laminin, elastin, fibronectin, fibroblast growth factor, bone morphogenetic protein, nerve growth factor, epidermal growth factor, interleukin transforming growth factor or cadherin; Preferably, the polypeptide comprises any one or a combination of at least two of RGD, YIGSR, IKVAV, PHSRN, LDV, REDV, KRSR, DGEA, GFOGER, FHRRIKA, TYRSRKY, RRETAWA, poly-lysine, poly-ornithine or E7 polypeptide; Preferably, the adhesive material includes any one or a combination of at least two of polydopamine or its derivatives.

6. The method for preparing a cell culture microcarrier according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: (1) mixing a polymer material, a surfactant, and an oil phase solvent to obtain an oil phase; or mixing a polymer monomer, a surfactant, an initiator, a crosslinking agent, and an optional oil phase solvent to obtain an oil phase; using an aqueous solution containing an osmotic pressure regulator as an inner aqueous phase; using an aqueous solution containing a surfactant as an outer aqueous phase; (2) mixing and emulsifying the oil phase and the internal water phase to obtain oil-in-water primary emulsions of two particle size ranges; (3) mixing and emulsifying the oil-in-water primary emulsions of two particle size ranges with an external water phase to obtain a water-in-oil-in-water secondary emulsion; (4) solidifying the multiple emulsion to obtain the cell culture microcarrier.

7. The method for preparing a cell culture microcarrier according to claim 6, characterized in that: The surfactant in the oil phase is selected from oil-soluble small molecule surfactants and / or block copolymers; Preferably, the oil-soluble small molecule surfactant is selected from any one of sorbitan monostearate, polyglycerol fatty acid ester, polyoxyethylene fatty acid ester, glycidyl methacrylate, and sorbitan monooleate, or a combination of at least two thereof; Preferably, the block copolymer includes a diblock polymer and a multiblock copolymer; Preferably, the diblock polymer is selected from any one or a combination of at least two of polyoxyethylene polyoxypropylene ether block copolymers, polyethylene glycol polylactic acid copolymers, polystyrene polyethylene glycol copolymers, polystyrene polyacrylic acid copolymers, polystyrene polyhydroxyethyl acrylate copolymers, polymethyl methacrylate polyacrylic acid copolymers or polymethyl methacrylate polyhydroxyethyl acrylate copolymers; Preferably, the crosslinking agent is selected from divinyl aromatic compounds and / or acrylate compounds; Preferably, the crosslinking agent is selected from divinylbenzene, ethylene glycol dimethacrylate or trimethoxypropane trimethyl acrylate; Preferably, the initiator is selected from azo di-initiators and / or peroxide initiators; Preferably, the initiator is selected from benzoyl peroxide or azobisisobutyronitrile; Preferably, the oil phase solvent comprises any one of acetone, tetrahydrofuran, toluene, dichloromethane, chloroform, dichloroethane or ethyl acetate, or a combination of at least two thereof; Preferably, the osmotic pressure regulator is a metal ion salt or a sugar, the metal ion salt is selected from any one or a combination of at least two of sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium sulfate, potassium sulfate, magnesium sulfate or calcium sulfate, and the sugar is selected from any one or a combination of at least two of glucose, sucrose, agarose or trehalose; Preferably, the concentration of the osmotic pressure regulator in the inner water phase is 0.001-20%, more preferably 0.005-5%.

8. The method for preparing a cell culture microcarrier according to claim 6 or 7, characterized in that: The surfactant in the external water phase includes a nonionic water-soluble surfactant; Preferably, the nonionic water-soluble surfactant comprises polyvinyl alcohol; Preferably, the concentration of the nonionic water-soluble surfactant in the external aqueous phase is 0.05-10%, more preferably 1-5%; Preferably, the mixed emulsification in step (2) and step (3) is carried out by any one of vortex oscillation, mechanical stirring emulsification, ultrasonic emulsification, homogenization emulsification, membrane emulsification or microfluidic emulsification, or a combination of at least two of them, preferably homogenization emulsification and membrane emulsification; Preferably, the curing treatment method in step (4) includes solvent volatilization curing method or polymerization curing method; Preferably, the surface of the product is modified after the curing treatment in step (4).

9. Use of the cell culture microcarrier according to any one of claims 1 to 5 in cell culture.

10. The use according to claim 9, characterized in that: The cells include somatic cells, stem cells or tumor cells; Preferably, the somatic cells include VERO cells, CHO cells, WISH cells, L929 cells, MDCK cells, fibroblasts, endothelial cells, myoblasts, osteoblasts, chondrocytes, hepatocytes, kidney cells or epidermal cells; Preferably, the stem cells include bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells, amniotic mesenchymal stem cells, peripheral blood mesenchymal stem cells, dental pulp mesenchymal stem cells, liver stem cells, liver precursor cells, myocardial stem cells, skin epidermal stem cells, intestinal epithelial stem cells, retinal stem cells, pancreatic stem cells, neuronal stem cells, muscle stem cells, amniotic epithelial stem cells, limbal stem cells, corneal epithelial stem cells, embryonic stem cells or induced pluripotent stem cells; Preferably, the tumor cells include bladder cancer cells, prostate cancer cells, thyroid cancer cells, glioblastoma cells, malignant melanoma cells, lung cancer cells, rhabdomyosarcoma cells, renal cell adenocarcinoma cells, gastric adenocarcinoma cells, endometrial cancer cells, liver cancer cells, cervical cancer cells, colon cancer cells, brain glioma cells, neuroblastoma cells, breast cancer cells, osteosarcoma cells, oral epidermoid cancer cells, ovarian cancer cells or esophageal cancer cells.

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