Three-dimensional porous microcarrier bracket and method for preparing same by adopting ionic additive

Abstract

The invention discloses a three-dimensional porous microcarrier bracket and a method for preparing the three-dimensional porous microcarrier bracket by adopting an ionic additive. The method comprises the following steps: 1) mixing a biological material with a buffer solution, adding an ionic pore-forming additive, and mixing to obtain a completely co-dissolved premixed solution; 2) mixing an organic solvent with a nonionic surfactant to obtain an organic phase solution, and pre-cooling the organic phase solution; (3) preparing an emulsion: mixing the premixed solution with a curing agent under a refrigeration condition to obtain a water-phase solution; adding the water-phase solution into the organic-phase solution, emulsifying to obtain W / O type emulsion liquid drops, and then performing low-temperature light-shielding emulsion reaction to obtain emulsion; 4) sequentially removing liquid components from the emulsion, filtering out an organic phase by using a filter screen, and screening out liquid microspheres with particle sizes within a required particle size range; and 5) wetting the liquid microspheres with water, freezing, and drying to obtain the three-dimensional porous microcarrier. The preparation method can adjust the aperture of the three-dimensional porous microcarrier.

Description

[0001] This application is a divisional application of the application with the application date of August 3, 2020, the application number of 202010766076.0, and the title of 'a three-dimensional porous microcarrier scaffold and a method for preparing the same by using ionic additives'. TECHNICAL FIELD

[0002] The present application relates to a three-dimensional porous microcarrier scaffold and a method for preparing the same by using ionic additives, belonging to the field of microcarrier material preparation. BACKGROUND

[0003] The three-dimensional porous tissue engineering scaffold material prepared by freeze-drying method can well maintain the biological activity of biological macromolecules in recent years. The addition of additives during the preparation of ice glue can regulate the formation process of ice crystals, thereby changing the pore size, hardness and other properties of the scaffold material. Among the existing methods, the use of salt particles for porosity is one of the schemes for increasing the pore size. The main principle is to rely on the space occupation of salt particles existing in the saturated salt solution to affect the formation of ice crystals, thereby increasing the pore size. However, the most important problem of this method is that the density of salt particles is not consistent with the density of the solution, and it will be affected by gravity during the cross-linking reaction process and will be gathered at the bottom of the material, thereby causing uneven distribution of the pore size. Moreover, when the salt ion concentration is low, a large number of particles with a certain volume cannot exist, and a high salt ion concentration will inhibit the chemical cross-linking reaction. Therefore, it is urgent to develop a kind of porosity additive that can be uniformly distributed in the ice glue pre-mixing solution, so as to obtain a scaffold material with large pore size and uniform pore distribution, which is the top priority for the development of tissue engineering products. SUMMARY

[0004] The purpose of the present application is to provide a three-dimensional porous microcarrier scaffold and a method for preparing the same by using ionic additives.

[0005] The present application provides a method for preparing a three-dimensional porous microcarrier scaffold by using ionic additives, which comprises the following steps: 1) After mixing the biological material with the buffer solution, add the ionic porosity additive to obtain a completely miscible pre-mixing solution; Among them, the biological material includes artificially synthesized biological material and / or natural biological material; 2) Mix the organic solvent with the non-ionic surfactant to obtain the organic phase solution, and pre-cool it; 3) Emulsion configuration: mix the pre-mixing solution with the solidifying agent under refrigeration conditions to obtain the aqueous phase solution; add the aqueous phase solution to the organic phase solution for emulsification to obtain W / O type emulsion droplets, and then carry out low-temperature light-proof emulsion reaction to obtain the emulsion; 4) Remove the liquid components from the emulsion in sequence, filter out the organic phase by selecting a filter screen, and screen out the liquid microspheres with the particle size in the required particle size range; 5) wetting the liquid microspheres with water, then freezing, and drying after the freezing to obtain a three-dimensional porous microcarrier scaffold.

[0006] In the above preparation method, the artificial synthetic biomaterial is at least one selected from polyethylene glycol, polyethylene glycol derivative, polyethylene glycol diacrylate, polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid-alcohol copolymer, polydimethylsiloxane, polyanhydride, polyacid ester, polyamide, polylysine, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polymethacrylate, polyethylene, polycarbonate and polyethylene oxide.

[0007] In the above preparation process, the buffer solution can be at least one selected from deionized water, Tris buffer solution, acetic acid buffer solution, phosphate buffer solution, borate buffer solution, citrate buffer solution and carbonate buffer solution.

[0008] In the above preparation process, the natural biomaterial is at least one selected from collagen, proteoglycan, glycoprotein, gelatin, gelatin derivative, chitin, alginate, alginate derivative, agar, fibrinogen, matrigel, hyaluronic acid, laminin and fibronectin; and / or The ionic pore-forming additive is at least one selected from sodium chloride, potassium chloride, calcium chloride, magnesium chloride, barium chloride, sodium sulfate, sodium bisulfate, potassium sulfate, potassium bisulfate, sodium phosphate, monosodium phosphate, disodium phosphate, potassium phosphate, monopotassium phosphate, dipotassium phosphate, sodium nitrate and potassium nitrate.

[0009] In the above preparation method, the concentration of the buffer solution ranges from 0.005 to 1 M before mixing; In the premix solution, the mass-volume concentration of the biomaterial can be 0.1% to 30%, the mass-volume concentration of the ionic pore-forming additive can be 0.1% to 30%, and the balance is the buffer solution, based on a total amount of 100% and in g / mL; The stirring rate in step 1) can be 50 rpm to 300 rpm, specifically 300 rpm, 200 rpm to 300 rpm or 100 rpm to 300 rpm.

[0010] In the present application, the biomaterial can specifically be a mixture of hyaluronic acid and gelatin or a mixture of bovine bone collagen and chitin powder; and the ionic pore-forming additive can specifically be sodium bisulfate or potassium sulfate. The mass-volume concentration of the biomaterial in the premixed solution can be 3.01%, 2.0%, 2-3.01%, 0.1%-2%, 2%-15%, or 0.1%-20%, based on 100% of the total amount, in g / mL; the mass-volume concentration of the ionic pore-forming additive can be 0.5%, 0.1%-0.5%, 0.1%-10%, 0.5%-15%, or 0.1%-20%, and the balance is the buffer.

[0011] In the preparation method, the freezing temperature in step 5) can be 0- -196℃, specifically -80℃, -80- -196℃, 0- -80℃, -10- -196℃, or -10- -180℃. The drying is performed by freeze-drying, and the freeze-drying temperature can be 0- -50℃, and the time can be 10-96h, specifically 24h, 10-24h, 24-96h, or 20-80h.

[0012] In the preparation method, the volume ratio of the organic solvent to the non-ionic surfactant can be 5-100:1, specifically 100:5, 100:8, 12.5-20:1, 10-20:1, 10-50:1, or 5-75:1. In step 2), the stirring rate of the mixing can be 100-3000rpm, specifically 600rpm, 700rpm, 100-600rpm, 600-3000rpm, or 200-2000rpm, and the stirring time can be 10-120min, specifically 20min, 60min, 10-20min, 20-60min, 20-120min, or 10-100min. The precooling temperature can be 0- -80℃, specifically -30℃, -40℃, 0- -40℃, -30- -40℃, -40- -80℃, or -20- -70℃, and the time can be 2-24h, specifically 7h, 16h, or 7-16h. In step 2), the precooling step before mixing of the organic solution is also included.

[0013] In the preparation method, in step 3), the temperature of the refrigeration condition can be 0- -196℃; a low-temperature refrigerator is used to achieve the refrigeration condition, and the refrigerant used in the low-temperature refrigerator is at least one of liquid nitrogen, ethanol, trichloroethane, isopropyl alcohol, dichloromethane, ethyl acetate, ethylene glycol, propylene glycol, isobutane, n-hexane, chloroform, tetrahydrofuran, bromohexane, and acetonitrile. The volume ratio of the premixed solution to the curing agent can be 200-10000:1, specifically 200:1, 200-1000:1, 200-5000:1, or 200-7500:1. The volume ratio of the aqueous phase solution to the organic phase solution can be 1:5-40, specifically 1:15, 1:20, 1:15-20, 1:10-30, or 1:5-30. In step 3), the stirring rate of the emulsification can be 30-1000 rpm, specifically 1000 rpm, 750-1000 rpm, 500-1000 rpm, or 200-1000 rpm, the temperature can be 0-80℃, specifically room temperature (such as 25℃), and the time can be 10-60 min, specifically 20 min, 30 min, 20-30 min, 10-20 min, 10-30 min, 10-40 min, or 10-50 min. The rotation speed of the low-temperature light-avoiding emulsification reaction can be 10-2000 rpm, specifically 80 rpm, 10-80 rpm, 80-2000 rpm, 10-1000 rpm, or 10-1500 rpm, the temperature can be -80℃-0℃, specifically -30℃, -15℃, -30℃--15℃, -80℃--30℃, or -80℃--15℃, and the reaction time can be 2-48 h, specifically 16 h, 20 h, 24 h, 2-16 h, 16-20 h, 16-24 h, 10-30 h, or 5-40 h.

[0014] In the present application, the room temperature is a common sense known in the art, and can be specifically 10-30℃.

[0015] In the above preparation method, the organic solvent is at least one selected from the group consisting of hydrofluoroether, carbon tetrachloride, petroleum ether, cyclohexane, liquid paraffin, edible oil, soybean oil, olive oil, chloroform (also known as trichloromethane), dichloromethane, carbon tetrachloride, and tetrachloroethylene, and specifically trichloromethane and petroleum ether mixed at a volume ratio of 1:1. The non-ionic surfactant is at least one selected from the group consisting of sorbitan fatty acid ester, glycerol fatty acid ester, laurate, alkylphenol polyoxyethylene ether, high-carbon fatty alcohol polyoxyethylene ether, span, PO-500, monooleate, and Tween; and specifically Tween 20 is used in one embodiment. The curing agent is at least one selected from the group consisting of divinylbenzene, diisocyanate, N-hydroxysuccinimide N,N-methylenebisacrylamide, formaldehyde, glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, calcium chloride, tetramethyl ethylenediamine, ammonium sulfate, genipin, and transglutaminase.

[0016] In the preparation method, the liquid component is removed by using a filter screen cleaning device; The pore size of the filter screen can be 1-1000 microns, specifically 50 microns, 5-1000 microns, 1-5 microns, 50-250 microns, or 50-500 microns. The step 4) further includes a step of cleaning the liquid microspheres with a cleaning agent to remove the surface organic phase of the microcarrier material. The cleaning agent is selected from at least one of acetone, anhydrous copper sulfate, calcium chloride, sodium sulfate, anhydrous ethanol, medical alcohol, hydrofluoroether, sodium alkyl benzene sulfonate, fatty alcohol sodium sulfate, sodium tripolyphosphate, and deionized water.

[0017] The application also provides the porous microcarrier prepared by the above preparation method.

[0018] In the above three-dimensional porous microcarrier, the pore size of the porous microcarrier can be 20-500 microns, specifically 50-500 microns, and the porosity can be 85-95%.

[0019] The application has the following advantages: The ionic porogen used in the application is a soluble ionic compound, and the ionic porogen is used to adjust the pore size without producing incompatible phases, and after cross-linking, the pore size can be adjusted without removing the ionic porogen, so that the pore size is more uniform while the pore size is increased; the addition method is mutual solubility addition, and the solution is not saturated; the addition of soluble ionic compounds uses ice crystal to adjust the pore size at a reaction temperature of 0 to -196℃, the preparation method is simple, and the pore size of the three-dimensional porous microcarrier can be adjusted. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The electron microscope comparison chart of the porous microcarrier before and after the use of the porogen for the three-dimensional porous microcarrier prepared in Example 1 of the application. DETAILED DESCRIPTION

[0021] In the following examples, the experimental methods used are conventional methods unless otherwise specified.

[0022] In the following examples, the materials, reagents, etc. used are commercially available unless otherwise specified.

[0023] Example 1 (1) Preparation of aqueous solution: 1) The aqueous solution comprises synthetic biomaterials and / or natural biomaterials, ionic porogen additives, and curing agents Weigh a certain amount of bovine bone collagen, chitin powder, with a total amount of 100%, unit g / mL, 1% collagen powder + 1% chitin by mass fraction, dissolved with deionized water. Weigh the ionic pore-forming additive sodium bisulfate, add it to the premix and stir (speed 300 rpm), configure it to a final concentration of 0.5% w / v.

[0024] (2) Organic phase solution configuration: Measure chloroform and petroleum ether in a volume ratio of 1:1, mix well at a speed of 700 rpm for 1 hour, then add Tween 20 to a final concentration of 5% v / v (ml / ml), continue stirring at 700 rpm for 1 hour. Transfer to a -30℃ refrigerator for precooling for 10 hours.

[0025] (3) Emulsion preparation: 1) Add ethanol to the low-temperature refrigerator, cool to -40℃ using the low-temperature refrigerator; 2) Add 0.5% v / v (ml / ml) formaldehyde solution to the water phase, stir at a speed of 600 rpm on the stirrer for 20 minutes to obtain the water phase.

[0026] 3) The well-precooled organic phase solution is introduced into the low-temperature reaction stirrer by gear pump, and the stirrer is stirred at a speed of 500 rpm; 4) Add the water phase premix to the stirrer, the volume ratio of organic phase to water phase is 20:1, emulsification occurs (emulsification temperature room temperature, specifically 25℃), stirring speed 1000 rpm, fully stirred for 30 min to generate W / O type droplets, 5) After the emulsification process is completed, turn on the low-temperature emulsification reactor and adjust the speed to 100 rpm; 6) React at -15℃ for 16 hours in the dark; (4) Microcarrier collection and cleaning: 1) Collect the emulsion and use the filter screen cleaning device to remove the liquid components. A filter screen with a pore size of 50μm can be used to remove the organic phase. According to different purposes, three-dimensional porous microcarriers with the desired particle size range can be selected. In this way, the uniformity of the desired three-dimensional porous microcarriers can be further improved, and microcarriers with a particle size range of 50-500μm can be harvested, 2) Clean the emulsion: wash the collected microcarriers with a cleaning agent to remove the organic phase on the surface of the microcarrier material. Use well-precooled acetone mixed with deionized water at a ratio of 9:1 (temperature 0℃ precooling for 8h) to thoroughly rinse the collected material. Rinse a total of 5 times.

[0027] (5) Drying and screening: The uniform three-dimensional porous microcarriers after cleaning are wetted in a small amount of water, and are transferred to a freezing device or a freezing liquid at -80 degrees Celsius for freezing, and are transferred to a freeze dryer after freezing for freeze-drying at -40 degrees Celsius for 24 hours. The obtained three-dimensional porous microcarriers have a pore size of 50-500 microns, and the porosity can reach 85-95%.

[0028] By Figure 1 It can be known from the results that, by introducing an ionic additive, the pore size structure can be controlled to a large extent and uniformly in the process of preparing a scaffold material by a freeze-drying method. Compared with a traditional preparation method, the method is simple to operate, low in toxicity, and free of byproducts, and the obtained material has a large pore size, high porosity, and strong connectivity, and has a good biomedical prospect.

[0029] Example 2 (1) Preparation of a premix solution: A certain amount of hyaluronic acid and gelatin powder is weighed, with a total amount of 100% and a unit of g / mL, and 0.01% hyaluronic acid powder + 3% gelatin powder is used according to the mass fraction, and deionized water is fully dissolved. An ionic pore-forming additive, potassium sulfate, is weighed and added to the premix solution and stirred (at a rate of 300 rpm) to prepare a final concentration of 0.5%.

[0030] (2) Preparation of an organic phase solution: A certain amount of carbon tetrachloride and petroleum ether is measured and mixed uniformly at a volume ratio of 1:4, and is mixed at a speed of 1000 rpm for 1 hour, and then 8% v / v Tween 80 is added, and stirring is continued at 700 rpm for 1 hour. After being fully cooled, it is transferred to a -30℃ refrigerator for precooling for 16 hours.

[0031] (3) Preparation of an emulsion: 1) Ethanol is added to a low-temperature refrigerator, and the low-temperature refrigerator is cooled to -30 degrees Celsius; 2) 0.5% v / v (ml / ml) formaldehyde solution is added to the water phase, and stirring is performed on a stirrer at a speed of 600 rpm for 10 minutes to obtain the water phase.

[0032] 3) The fully pre-cooled organic phase solution is passed into the low-temperature reaction stirrer by a gear pump, and the stirrer is stirred at a speed of 500 rpm; 4) The water phase premix solution is added to the stirrer, and the volume ratio of the organic phase to the water phase is 15:1, emulsification occurs (emulsification temperature is room temperature, specifically 25℃), the stirring speed is 1000 rpm, and the emulsion droplets are fully stirred for 20 min to generate W / O type emulsion droplets, 5) After the emulsification process is completed, the low-temperature emulsification reactor is started, and the stirring speed is adjusted to 80 rpm; 6) Low-temperature reaction for 24 hours in the dark at -30℃; (4) Collection and cleaning of microcarriers: 1) Collect the emulsion, remove the liquid component using a suction filter screen washing device, and use a filter screen with a pore size of 50 um to filter out the organic phase. According to different uses, the three-dimensional porous microcarriers in the desired particle size range are screened out. Thus, the uniformity of the desired three-dimensional porous microcarriers can be further improved, and the three-dimensional porous microcarriers in the range of 50-500 um can be harvested, 2) Washing the emulsion: wash the collected microcarriers with a cleaning agent to remove the organic phase on the surface of the microcarrier material. Use a mixture of fully pre-cooled acetone and medical alcohol 3:1 (temperature 0°C pre-cooled for 8h), and fully rinse the collected material. Rinse a total of 5 times. Finally, rinse once with deionized water.

[0033] (5) Drying and screening: The uniform three-dimensional porous microcarriers after washing are wetted in a small amount of water and transferred to a -80°C freezing device or a freezing liquid for freezing. After freezing, they are transferred to a freeze dryer and freeze-dried at -40°C for 24 hours. The three-dimensional porous microcarriers obtained have micropores with a pore size of 50-500 microns, and the porosity can reach 85-95%.

[0034] Comparative Example 1 The method is the same as that in Example 1 of the present application, except that no porogen is used. The porous microcarriers before use of the porogen are obtained, and the results are as follows Figure 1 The electron microscope image of the porous microcarriers before use of the porogen in the three-dimensional porous microcarriers is shown in

[0035] The present application also relates to the following embodiments: Item 1. A method for preparing three-dimensional porous microcarrier scaffolds using ionic additives, comprising the following steps: 1) Mix the biological material with the buffer solution, then add the ionic porogenic additive to obtain a completely miscible premix; The biological material includes artificially synthesized biological material and / or natural biological material; 2) Mix the organic solvent with the non-ionic surfactant to obtain the organic phase solution, and pre-cool it; 3) Emulsion preparation: mix the premix with the solidifying agent under refrigeration conditions to obtain the aqueous phase solution; add the aqueous phase solution to the organic phase solution for emulsification to obtain W / O type emulsion droplets, then perform low-temperature light-protected emulsion reaction to obtain the emulsion; 4) Remove the liquid component from the emulsion, use a filter screen to filter out the organic phase, and screen out liquid microspheres in the desired particle size range; 5) Wet the liquid microspheres with water, then freeze them, and dry them after the freezing to obtain three-dimensional porous microcarriers.

[0036] Item 2. The method according to item 1, wherein the synthetic biomaterial is at least one selected from the group consisting of polyethylene glycol, polyethylene glycol derivative, polyethylene glycol diacrylate, polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid-alcohol copolymer, polydimethylsiloxane, polyanhydride, polyacid ester, polyamide, polylysine, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polymethacrylate, polyethylene, polycarbonate, and polyethylene oxide. The buffer solution can be at least one selected from the group consisting of deionized water, Tris buffer solution, acetic acid buffer solution, phosphate buffer solution, borate buffer solution, citrate buffer solution, and carbonate buffer solution. The natural biomaterial is at least one selected from the group consisting of collagen, proteoglycan, glycoprotein, gelatin, gelatin derivative, chitin, alginate, alginate derivative, agar, fibrinogen, matrigel, hyaluronic acid, laminin, and fibronectin; and / or The ionic pore-forming additive is at least one selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, barium chloride, sodium sulfate, sodium bisulfate, potassium sulfate, potassium bisulfate, sodium phosphate, monosodium phosphate, disodium phosphate, potassium phosphate, monopotassium phosphate, dipotassium phosphate, sodium nitrate, and potassium nitrate.

[0037] Item 3. The method according to item 1 or 2, wherein the concentration of the buffer solution is in the range of 0.005-1 M before mixing. In the premix solution, the mass / volume concentration of the biomaterial is 0.1%-30%, the mass / volume concentration of the ionic pore-forming additive is 0.1%-30%, and the balance is the buffer solution, based on a total amount of 100% g / mL. The stirring rate of the mixing in step 1) is in the range of 50-300 rpm.

[0038] Item 4. The method according to any one of items 1-3, wherein the freezing temperature in step 5) is in the range of 0--196 °C. The drying is performed by freeze-drying at a temperature in the range of 0--50 °C for a time in the range of 10-96 h.

[0039] Item 5. The method according to any one of items 1-4, wherein the volume ratio of the organic solvent to the non-ionic surfactant is in the range of 5-100:1. In step 2), the stirring rate of the mixing is in the range of 100-3000 rpm for a stirring time in the range of 10-120 min. The pre-cooling temperature is in the range of 0--80 °C for a time in the range of 2-24 h. The configuration device used in step 2) further comprises a pre-cooling step before the organic solution is mixed.

[0040] Item 6. The method according to any one of items 1-5, wherein in step 3), the temperature of the refrigeration condition is 0-196℃; and a low-temperature refrigerator is used to achieve the refrigeration condition, and the low-temperature refrigerator uses at least one of liquid nitrogen, ethanol, trichloroethane, isopropyl alcohol, dichloromethane, ethyl acetate, ethylene glycol, propylene glycol, isobutane, n-hexane, chloroform, tetrahydrofuran, bromohexane and acetonitrile as a refrigerant. The volume ratio of the premixed solution to the curing agent is 200-10000:1. The volume ratio of the aqueous solution to the organic phase solution is 1:5-40. In step 3), the stirring speed of the emulsification is 30-1000 rpm, the temperature is 0-80℃, and the time is 10-60 min. The stirring speed of the low-temperature light-proof emulsification reaction is 10-2000 rpm, the temperature is -80℃-0℃, and the reaction time is 2-48 h.

[0041] Item 7. The method according to any one of items 1-6, wherein the organic solvent is at least one of hydrofluoroether, carbon tetrachloride, petroleum ether, cyclohexane, liquid paraffin, edible oil, soybean oil, olive oil, chloroform, dichloromethane, carbon tetrachloride and tetrachloroethylene. The non-ionic surfactant is at least one of sorbitan fatty acid ester, glycerol fatty acid ester, laurate, alkylphenol polyoxyethylene ether, high-carbon fatty alcohol polyoxyethylene ether, span, PO-500, monooleate and Tween. The curing agent is at least one of divinylbenzene, diisocyanate, N-hydroxysuccinimide, N,N-methylenebisacrylamide, formaldehyde, glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, calcium chloride, tetramethyl ethylenediamine, ammonium sulfate, genipin and transglutaminase.

[0042] Item 8. The method according to any one of items 1-7, wherein the liquid components are removed by using a washing device with a filter screen. The pore size of the filter screen is 1-1000 μm. In step 4), the liquid microspheres are further washed with a cleaning agent to remove the surface organic phase of the microcarrier material. The cleaning agent is at least one of acetone, anhydrous copper sulfate, calcium chloride, sodium sulfate, anhydrous ethanol, medical alcohol, hydrofluoroether, sodium alkylbenzenesulfonate, fatty alcohol sodium sulfate, sodium tripolyphosphate and deionized water.

[0043] Item 9. The three-dimensional porous microcarriers prepared by the method of any one of items 1-7.

[0044] Item 10. The three-dimensional porous microcarriers according to item 9, characterized in that the three-dimensional porous microcarriers have a pore size of 20-500 microns and a porosity of 85-95%.

Claims

1. A method for preparing a three-dimensional porous microcarrier scaffold using an ionic additive, comprising the following steps: 1) mixing a biomaterial with a buffer solution, and then adding an ionic pore-forming additive to obtain a completely miscible premix; 2) mixing an organic solvent with a non-ionic surfactant to obtain an organic phase solution, and pre-cooling; 3) preparing an emulsion under refrigeration by mixing the premix with a solidifying agent to obtain an aqueous phase solution; 3-1) adding the aqueous phase solution to the organic phase solution to emulsify, obtaining W / O emulsion droplets, 3-2) then performing a low-temperature light-protected emulsion reaction to obtain an emulsion; 4) sequentially removing liquid components from the emulsion, filtering the organic phase using a filter screen, and screening microspheres of a desired particle size range; 5) wetting the microspheres with water, then freezing, and drying after the freezing to obtain a three-dimensional porous microcarrier, wherein the ionic pore-forming additive is adjusted to a pore size without generating incompatible phases by completely mixing with the solution.

2. The method of claim 1, wherein: the biomaterial comprises an artificially synthesized biomaterial and / or a natural biomaterial; the artificially synthesized biomaterial is selected from at least one of polyethylene glycol, polyethylene glycol derivatives, polyethylene glycol diacrylate, polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid-alcohol acid copolymer, polydimethylsiloxane, polyanhydride, polyacid ester, polyamide, polylysine, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polymethacrylate, polyethylene, polycarbonate, and polyethylene oxide; the buffer solution is selected from at least one of deionized water, Tris buffer solution, acetic acid buffer solution, phosphate buffer solution, borate buffer solution, citrate buffer solution, and carbonate buffer solution; the natural biomaterial is selected from at least one of collagen, proteoglycan, glycoprotein, gelatin, gelatin derivatives, chitin, alginate, alginate derivatives, agar, fibrinogen, Matrigel, hyaluronic acid, laminin, and fibronectin; and / or the ionic pore-forming additive is selected from at least one of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, barium chloride, sodium sulfate, sodium bisulfate, potassium sulfate, potassium bisulfate, sodium phosphate, monosodium phosphate, disodium phosphate, potassium phosphate, monopotassium phosphate, dipotassium phosphate, sodium nitrate, and potassium nitrate.

3. The method according to claim 1 or 2, characterized in that: the concentration of the buffer solution ranges from 0.005 to 1 M before mixing; in the premix, the mass-volume concentration of the biomaterial is 0.1% to 30%, the mass-volume concentration of the ionic pore-forming additive is 0.1% to 30%, and the balance is the buffer solution, based on a total of 100% in g / mL; the stirring rate for mixing in step 1) is 50 rpm to 300 rpm.

4. The method of claim 1 or 2, wherein: in step 5), the freezing temperature is 0 to -196°C; the drying is performed by freeze-drying at a temperature of 0 to -50°C for 10 to 96 hours.

5. The method of claim 1 or 2, wherein: the volume ratio of the organic solvent to the non-ionic surfactant is 5 to 100:

1. The stirring speed in step 2) is 100-3000 rpm, and the stirring time is 10-120 min; The pre-cooling temperature is -80-0℃, and the time is 2-24 h; Step 2) further comprises pre-cooling the configuration device used in step 2) before mixing the organic solvent and the non-ionic surfactant.

6. The method of claim 1 or 2, wherein: In step 3), the temperature of the refrigeration condition is -196-0℃; a low-temperature refrigerator is used to achieve the refrigeration condition, and the low-temperature refrigerator uses at least one of liquid nitrogen, ethanol, trichloroethane, isopropyl alcohol, dichloromethane, ethyl acetate, ethylene glycol, propylene glycol, isobutane, n-hexane, chloroform, tetrahydrofuran, bromohexane and acetonitrile as the refrigerant; The volume ratio of the pre-mixed solution to the solidifying agent is 200-10000:1; The volume ratio of the aqueous solution to the organic phase solution is 1:5-40; In step 3), the stirring speed of the emulsification is 30-1000 rpm, the temperature is 0-80℃, and the time is 10-60 min; The stirring speed of the low-temperature light-proof emulsification reaction is 10-2000 rpm, the temperature is -80-0℃, and the reaction time is 2-48 h.

7. The method of claim 1 or 2, wherein: The organic solvent is at least one of hydrofluoroether, carbon tetrachloride, petroleum ether, cyclohexane, liquid paraffin, edible oil, soybean oil, olive oil, chloroform, dichloromethane, carbon tetrachloride and tetrachloroethylene; The non-ionic surfactant is at least one of sorbitan fatty acid ester, glycerol fatty acid ester, laurate, alkylphenol polyoxyethylene ether, high-carbon fatty alcohol polyoxyethylene ether, span, PO-500, monooleate and Tween; The solidifying agent is at least one of divinylbenzene, diisocyanate, N-hydroxysuccinimide N,N-methylenebisacrylamide, formaldehyde, glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, calcium chloride, tetramethyl ethylenediamine, ammonium sulfate, genipin and transglutaminase.

8. The method of claim 1 or 2, wherein: The liquid components are removed by using a filter screen cleaning device; The pore size of the filter screen is 1-1000 μm; Step 4) further comprises cleaning the microspheres with a cleaning agent to remove the surface organic phase of the microcarrier material; The cleaning agent is at least one of acetone, anhydrous copper sulfate, calcium chloride, sodium sulfate, anhydrous ethanol, medical alcohol, hydrofluoroether, sodium alkylbenzenesulfonate, fatty alcohol sodium sulfate, sodium tripolyphosphate and deionized water.

9. The three-dimensional porous microcarrier prepared by the method of any one of claims 1-7.

10. The three-dimensional porous microcarrier of claim 9, wherein: The three-dimensional porous microcarrier has a pore size of 20-500 microns and a porosity of 85-95%. The three-dimensional porous microcarrier has a pore size of 20-500 microns and a porosity of 85-95%.

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