Method for enhancing freeze drying stability of porous microcarrier

Abstract

The invention relates to biological material engineering for cell culture, in particular to a method for enhancing freeze-drying stability of a porous microcarrier. Comprising the following steps: (1) standing a porous microcarrier which is fully swelled in water until the liquid level is about to be in contact with the porous microcarrier which is stood; (2) soaking the porous microcarrier obtained in the step (1) in an organic solvent for dehydration treatment; (3) transferring the dehydrated porous microcarrier in the container into a tray by using a pasteurization dropper, a pipettor or an electric pipettor, sealing the top of the tray, and pre-freezing in liquid nitrogen or an ultralow-temperature refrigerator; and (4) freeze-drying the pre-frozen porous microcarrier in a freeze drying box. The method provided by the invention can effectively solve the problems of shrinkage, collapse and structural damage of the porous microcarrier scaffold in the freeze drying process, and is short in drying period, low in energy consumption and suitable for large-scale production of porous microcarriers.

Description

Technical Field

[0001] The invention relates to biomaterial engineering for cell culture, and in particular to a method for enhancing the freeze-drying stability of porous microcarriers. Background Art

[0002] Porous microcarriers are highly porous materials that are widely used in biomedicine, drug delivery, tissue engineering, chemical catalysis and other fields. In the biomedical field, porous microcarriers are often used as three-dimensional scaffolds for cell culture, providing space for cells to attach, grow and differentiate. In drug delivery systems, they can be used as carriers for controlled release of drugs to achieve sustained release and targeted therapy of drugs.

[0003] Freeze drying, also known as freeze drying or sublimation drying, is a drying technology that removes moisture under vacuum conditions after freezing the material. This technology can maximize the preservation of the original structure and biological activity of the material, so it has irreplaceable advantages in the treatment of heat-sensitive substances. In the preparation process of porous microcarriers, freeze drying is often used to fix cells or form porous structures to maintain their structural integrity and stability. Although freeze drying technology plays an important role in the preparation of porous microcarriers, the existing technology still faces many challenges in maintaining the structural stability of microcarriers. First, the growth of ice crystals during the freezing process may lead to the destruction of the microcarrier structure, thereby affecting its pore characteristics and mechanical strength. Secondly, the shrinkage and collapse during the drying process will change the shape and size of the microcarrier, thereby affecting its performance in practical applications. In addition, traditional freeze drying technology has a long cycle and high energy consumption, which is not conducive to large-scale production and application. The structural stability of porous microcarriers during freeze drying is affected by many factors, including but not limited to freezing rate, drying temperature, vacuum degree, material composition and pore structure. Too fast or too slow freezing rate may lead to uneven formation of ice crystals, thereby affecting the structural stability of microcarriers. Control of drying temperature and vacuum is also critical to remove moisture and prevent material collapse. In addition, the chemical composition and pore structure of the microcarriers also determine their responsiveness and stability during freeze-drying.

[0004] In order to solve the above problems, researchers have tried many methods to improve freeze-drying technology, such as adjusting freezing parameters, using protective agents, and developing new freeze-drying equipment. For example, by controlling the freezing rate to obtain finer and more uniform ice crystals, using protective agents such as sucrose and trehalose to prevent cells or proteins from being damaged during the freezing process, and developing freeze-drying equipment that can achieve faster drying. However, these methods can often only partially solve the problem of structural stability and may bring new problems such as increased costs and complex operations. A new method to enhance the freeze-drying stability of porous microcarriers is urgently needed. Summary of the invention

[0005] The technical problem to be solved by the present invention is that currently improving the freeze-drying stability of microcarriers by improving freeze-drying technology can only partially solve the structural stability problem, and may bring new problems such as increased costs and complicated operations. A new method for enhancing the freeze-drying stability of porous microcarriers is urgently needed.

[0006] In view of the problems of the prior art, the present invention provides a method for maintaining structural stability of porous microcarriers during freeze-drying. By optimizing and introducing new protective agents, freeze-drying parameters and improving freeze-drying processes, the original pore structure and biological activity of the microcarriers can be maintained while significantly improving their structural stability, reducing shrinkage and collapse during the drying process, shortening the drying cycle, and reducing energy consumption, thereby providing technical support for the large-scale production and application of porous microcarriers. The method of the present invention can effectively solve the problem of shrinkage, collapse and structural damage of porous microcarrier scaffolds during freeze-drying, and has a short drying cycle and low energy consumption, and is suitable for large-scale production of porous microcarriers.

[0007] To achieve the above object, the technical solution of the present invention is: a method for enhancing the freeze-drying stability of porous microcarriers, comprising the following steps:

[0008] (1) The porous microcarriers (in the present invention, the particles are particles of materials used in the biomedical field, all of which are commonly known materials in the art) that have been fully swollen in water are allowed to stand for 5 to 60 minutes to remove excess water until the liquid surface is about to contact the stationary porous microcarriers.

[0009] (2) Soaking the porous microcarrier obtained in step (1) in an organic solvent for dehydration treatment to remove free water and bound water inside the microcarrier.

[0010] (3) Use a Pasteur pipette, pipette or electric pipette to transfer the dehydrated porous microcarriers in the container into a tray (the selection of the tray is crucial to maintaining the stability of the microcarrier structure. The tray needs to ensure effective and uniform heat transfer while avoiding damage to the microcarrier structure during the freeze-drying process. The tray material can be: polyethylene, polytetrafluoroethylene, stainless steel, polypropylene); seal the top of the tray and place it in liquid nitrogen or an ultra-low temperature refrigerator at -150℃ to -85℃ for pre-freezing for 6 to 48 hours. Pre-freezing at ultra-low temperature is an important step before freeze-drying. Its purpose is to form tiny and uniform ice crystals at low temperatures to reduce damage to the microcarrier structure.

[0011] (4) The pre-frozen porous microcarriers are placed in a freeze drying oven for freeze drying, and the freeze drying program is set to freeze drying at -60°C to -20°C for 6h to 12h, freeze drying at -20°C to -0°C for 6h to 12h, and freeze drying at 0°C to 20°C for 12h to 24h. Most of the organic solvents and ice crystal water can be removed under low temperature conditions, and bound water and residual water can be removed at relatively high temperatures.

[0012] Among them, freeze drying at -60℃~-20℃ for 6h~12h: The low-temperature freeze drying at this stage is to remove most of the bound water and ice crystal water. At this stage, the temperature is low, which can avoid the destruction of the microcarrier structure and ensure that the ice crystals and most of the water are removed in the solid state.

[0013] -20℃~-0℃ freeze drying for 6h~12h: The purpose of this stage is to further remove the residual ice crystal moisture. The gradual increase in temperature is conducive to the sublimation of ice crystals, while avoiding the collapse of the microcarrier structure or the destruction of active ingredients caused by excessive temperature.

[0014] 0℃~20℃ lyophilization for 12h~24h: Drying at relatively high temperature is mainly to remove bound water and residual water in the microcarriers. Bound water is usually hydrogen bonded to polar groups in the microcarriers and requires higher energy to remove. This stage helps to improve the drying efficiency, ensure the dryness of the microcarriers, and reduce the dissolution time after rehydration.

[0015] Furthermore, the porous microcarrier in step (1) is a commercially available microcarrier for cell culture, such as 3D-MIC microcarrier, Cytopore, Cytodex, Cytoline, 3D At least one of .

[0016] Furthermore, the porous microcarrier in step (1) is a self-prepared microcarrier such as a porous gelatin microcarrier, such as at least one of a porous chitosan microcarrier and a porous sodium alginate microcarrier. The preparation method can refer to patents CN201710184720.1 and CN202410625621.2.

[0017] Furthermore, the particle size of the porous microcarrier swollen in water in step (1) is 50 to 999 μm. Porous microcarriers with a particle size of 50 to 999 μm can provide sufficient surface area for cell attachment and growth while maintaining the mechanical stability of the microcarrier. If the particle size is too high, the sedimentation rate of the microcarrier in water may be accelerated, which is not conducive to the uniform distribution of cells. At the same time, the mutual interference between the microcarriers increases, which may affect the growth environment of the cells. If the particle size is too low, the surface area of ​​the microcarrier may be reduced, limiting the attachment and growth of cells. The risk of microcarrier breakage during operation is increased, affecting the stability of cell culture.

[0018] Furthermore, the organic solvent in step (2) can be selected from ethanol, methanol, acetone, butanone, dichloromethane, ethyl acetate, isopropanol, dimethylformamide, chloroform, and 2-methyl-2,4-pentanediol.

[0019] Furthermore, the dehydration method of step (2) is gradient dehydration: prepare 20%, 40%, 60%, and 80% organic solvent solutions in advance; add the porous microcarrier to the 20% organic solvent solution, stir and let stand, and take the precipitate for use; then add the porous microcarrier to the 40% organic solvent solution, stir and let stand, and take the precipitate for use; then add the porous microcarrier to the 60% organic solvent solution, stir and let stand, and take the precipitate for use; then add the porous microcarrier to the 80% organic solvent solution, stir and let stand, take the precipitate, and complete the dehydration.

[0020] Furthermore, in step (2), ethanol is selected as the porous microcarrier for dehydration treatment; the specific dehydration steps are:

[0021] (2-1) Prepare 20%, 40%, 60%, or 80% ethanol solutions in advance and let stand at room temperature for 0.5 to 6 hours;

[0022] (2-2) adding 20% ​​ethanol solution to the swollen porous microcarriers, the ratio of the porous microcarriers to the ethanol solution being 1:10 to 1:50, stirring continuously for 0.5 to 12 hours, standing for 5 to 60 minutes, removing excess ethanol solution after the porous microcarriers sink to the bottom, until the liquid surface is about to contact the stationary porous microcarriers; adding 40% ethanol solution, the ratio of the porous microcarriers to the ethanol solution being 1:10 to 1:50, stirring continuously for 0.5 to 12 hours, standing for 5 to 60 minutes, removing excess ethanol solution after the porous microcarriers sink to the bottom, until the liquid surface is about to contact the stationary porous microcarriers. microcarrier; adding 60% ethanol solution, the ratio of porous microcarrier to ethanol solution is 1:10 to 1:50, stirring continuously for 0.5 to 12 hours, standing for 5 to 60 minutes, removing excess ethanol solution after the porous microcarrier sinks to the bottom, until the liquid surface is about to contact the stationary porous microcarrier; adding 60% ethanol solution, the ratio of porous microcarrier to ethanol solution is 1:10 to 1:50, stirring continuously for 0.5 to 12 hours, standing for 5 to 60 minutes, removing excess ethanol solution after the porous microcarrier sinks to the bottom, until the liquid surface is about to contact the stationary porous microcarrier, and the obtained precipitate is ready for use.

[0023] Furthermore, in step (3), the method for sealing the top of the tray is to seal the top of the tray with a plastic wrap, or to add a top cover containing a microporous structure to prevent the material from splashing.

[0024] Porous microcarriers have important applications in biomedicine, drug delivery, tissue engineering and chemical catalysis due to their highly porous structure. However, traditional freeze-drying technology has many shortcomings in maintaining the structural stability of microcarriers, such as ice crystal growth, shrinkage and collapse, which will seriously affect the performance and application of microcarriers. In view of the limitations of the prior art, the present invention significantly improves the structural stability of microcarriers during freeze-drying by optimizing freeze-drying parameters and process flow. The method includes several key steps: first, the porous microcarriers are fully swollen in water and excess water is removed; second, the dehydration capacity of the organic solvent is used to replace the water in the porous microcarriers to remove the free water and bound water inside the microcarriers, reduce the possibility of ice crystal formation during the freezing process, and thus reduce the damage to the microcarrier structure; then, a pre-freezing treatment is performed, and by controlling the pre-freezing conditions, such as temperature, time and cooling rate, small and uniform ice crystals are formed to reduce damage to the microcarrier structure; finally, through the gradient temperature control drying step, the water in the microcarrier is effectively removed while maintaining its porous structure and biological activity to the greatest extent.

[0025] The beneficial effects of the present invention are:

[0026] The present invention introduces organic solvent dehydration and shaping treatment during the freeze-drying process of porous microcarriers and optimizes the pre-freezing and freeze-drying steps, which not only significantly improves the structural stability and integrity of the microcarriers and reduces the damage caused by ice crystal growth, but also improves production efficiency and product quality, while reducing energy consumption and production costs, providing an efficient and environmentally friendly innovative method for achieving large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a photo of the microcarrier after freezing in Example 1 of the method of the present invention under an electron microscope.

[0028] Figure 2 This is a photograph of the microcarrier after freezing in Example 2 of the method of the present invention under an electron microscope.

[0029] Figure 3 This is a photo of the microcarrier after freezing in Example 3 of the method of the present invention under an electron microscope.

[0030] Figure 4 This is a photograph of the microcarrier after freezing in Example 4 of the method of the present invention under an electron microscope.

[0031] Figure 5 The electron microscope photograph of the microcarrier after freezing using the commonly used method. DETAILED DESCRIPTION

[0032] The following description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0033] The following examples can be understood as a part of the local structure or method of the present invention alone, and can also be understood as the combination of the examples to explain the connotation of the larger structure or method of the present invention. Unless otherwise specified, all raw materials of the present invention are obtained from commercial sales.

[0034] Embodiment 1:

[0035] A method for enhancing the freeze-drying stability of porous microcarriers, comprising the following steps:

[0036] (1) Select a porous microcarrier made of gelatin (Henan Yaqi Gelatin Co., Ltd., YEG796) and remove excess water from the swollen porous microcarrier.

[0037] (2) Prepare 20%, 40%, 60%, and 80% ethanol solutions in advance and let them stand at room temperature for 2 hours. Add 20% ethanol solution to the swollen porous microcarriers, with the ratio of porous microcarriers to ethanol solution being 1:20, continue stirring for 2 hours, let it stand for 20 minutes, remove excess ethanol solution after the porous microcarriers sink to the bottom, until the liquid surface is about to contact the stationary porous microcarriers. Add 40% ethanol solution, with the ratio of porous microcarriers to ethanol solution being 1:20, continue stirring for 2 hours, let it stand for 20 minutes, remove excess ethanol solution after the porous microcarriers sink to the bottom, until the liquid surface is about to contact the stationary porous microcarriers, and then perform dehydration treatments with 60% ethanol solution and 80% ethanol solution in turn.

[0038] (3) After dehydration, transfer the gelatin porous microcarriers into a stainless steel tray, cover the upper opening with a microporous cover, and prefreeze at -85°C for 12 hours.

[0039] (4) The pre-frozen microcarriers were dried in a freeze dryer at a temperature of -50°C for 12 hours, -10°C for 12 hours, and 10°C for 12 hours.

[0040] The microcarriers produced by the method of the present invention are obviously spherical under electron microscope. Figure 1 , effectively increasing its cell culture area, and the porosity of each batch is greater than 90%, which significantly improves its proliferation rate when used for cell culture, and can be well used for 3D bionic culture and large-scale culture expansion of cells.

[0041] Embodiment 2:

[0042] A method for enhancing the freeze-drying stability of porous microcarriers, comprising the following steps:

[0043] (1) The 3D-MIC microcarrier aqueous solution purchased from Qingdao Marine Food Nutrition and Health Innovation Institute was allowed to stand for 60 min, and excess water was removed until the liquid surface was about to contact the stationary porous microcarrier.

[0044] (2) Ethanol was selected as the porous microcarrier for dehydration treatment, and 20%, 40%, 60%, and 80% ethanol solutions were prepared in advance and allowed to stand at room temperature for 0.5 h. The specific dehydration steps were as follows: 20% ethanol solution was added to the swollen porous microcarrier, the ratio of the porous microcarrier to the ethanol solution was 1:10, stirring was continued for 0.5 h, and the solution was allowed to stand for 30 min. After the porous microcarrier sank to the bottom, the excess ethanol solution was removed until the liquid surface was about to contact the stationary porous microcarrier. 40% ethanol solution was added, the ratio of the porous microcarrier to the ethanol solution was 1:10, stirring was continued for 0.5 h, and the solution was allowed to stand for 30 min. After the porous microcarrier sank to the bottom, the excess ethanol solution was removed until the liquid surface was about to contact the stationary porous microcarrier, and then the dehydration treatment with 60% ethanol solution and 80% ethanol solution was performed in sequence.

[0045] (3) Use a Pasteur pipette, pipette or electric pipette to transfer the dehydrated porous microcarriers in the container into a tray; cover the top of the tray with plastic wrap, sealing film or add a top cover with a microporous structure to prevent the material from splashing. Place the tray in an ultra-low temperature refrigerator (-85°C) for pre-freezing for 24 hours.

[0046] (4) The pre-frozen porous microcarriers were placed in a freeze drying chamber for freeze drying. The freeze drying program was set to -20°C for 12 h, -0°C for 6 h, and 20°C for 24 h.

[0047] The microcarriers produced by the method of the present invention are obviously spherical under electron microscope. Figure 2 , effectively increasing its cell culture area, and the porosity of each batch is greater than 90%, which significantly improves its proliferation rate when used for cell culture, and can be well used for 3D bionic culture and large-scale culture expansion of cells.

[0048] Embodiment 3:

[0049] (1) The 3D-MIC microcarriers purchased from Qingdao Marine Food Nutrition and Health Innovation Institute were allowed to stand for 60 min to remove excess water until the liquid surface was about to contact the stationary porous microcarriers.

[0050] (2) Methanol was selected as the porous microcarrier for dehydration treatment, and 20%, 40%, 60%, and 80% methanol solutions were prepared in advance and allowed to stand at room temperature for 0.5 h. The specific dehydration steps were as follows: 20% methanol solution was added to the swollen porous microcarrier, the ratio of the porous microcarrier to the methanol solution was 1:10, stirring was continued for 0.5 h, and the solution was allowed to stand for 30 min. After the porous microcarrier sank to the bottom, the excess methanol solution was removed until the liquid surface was about to contact the stationary porous microcarrier. 40% methanol solution was added, the ratio of the porous microcarrier to the methanol solution was 1:10, stirring was continued for 0.5 h, and the solution was allowed to stand for 30 min. After the porous microcarrier sank to the bottom, the excess methanol solution was removed until the liquid surface was about to contact the stationary porous microcarrier, and then the dehydration treatment with 60% methanol solution and 80% methanol solution was performed in sequence.

[0051] (3) Use a Pasteur pipette, pipette or electric pipette to transfer the dehydrated porous microcarriers in the container into the tray; cover the top of the tray with plastic wrap, sealing film or add a top cover with a microporous structure to prevent the material from splashing. Place the tray in an ultra-low temperature refrigerator (-85°C) for pre-freezing for 12 hours.

[0052] (4) The pre-frozen porous microcarriers were placed in a freeze drying chamber for freeze drying. The freeze drying program was set to -20°C freeze drying for 12 h, -0°C freeze drying for 12 h, and 20°C freeze drying for 12 h.

[0053] The microcarriers produced by the method of the present invention are obviously spherical under electron microscope. Figure 3 , effectively increasing its cell culture area, and the porosity of each batch is greater than 90%, which significantly improves its proliferation rate when used for cell culture, and can be well used for 3D bionic culture and large-scale culture expansion of cells.

[0054] Embodiment 4:

[0055] A method for enhancing the freeze-drying stability of porous microcarriers, comprising the following steps:

[0056] (1) The 3D-MIC microcarriers purchased from Qingdao Marine Food Nutrition and Health Innovation Institute were allowed to stand for 5 to 60 min to remove excess water until the liquid surface was about to contact the stationary porous microcarriers.

[0057] (2) Isopropanol was selected as the porous microcarrier for dehydration treatment, and 20%, 40%, 60%, and 80% isopropanol solutions were prepared in advance and allowed to stand at room temperature for 0.5 h. The specific dehydration steps are as follows: 20% isopropanol solution was added to the swollen porous microcarrier, the ratio of the porous microcarrier to the isopropanol solution was 1:10, stirring was continued for 0.5 h, and the solution was allowed to stand for 30 min. After the porous microcarrier sank to the bottom, the excess isopropanol solution was removed until the liquid surface was about to contact the stationary porous microcarrier. 40% isopropanol solution was added, the ratio of the porous microcarrier to the isopropanol solution was 1:10, stirring was continued for 0.5 h, and the solution was allowed to stand for 30 min. After the porous microcarrier sank to the bottom, the excess isopropanol solution was removed until the liquid surface was about to contact the stationary porous microcarrier, and then the dehydration treatment of 60% isopropanol solution and 80% isopropanol solution was performed in sequence.

[0058] (3) Use a Pasteur pipette, pipette or electric pipette to transfer the dehydrated porous microcarriers in the container into a tray; cover the top of the tray with plastic wrap, sealing film or add a top cover with a microporous structure to prevent the material from splashing. Place the tray in liquid nitrogen for pre-freezing for 6 hours.

[0059] (4) The pre-frozen porous microcarriers were placed in a freeze drying chamber for freeze drying. The freeze drying program was set to -20°C for 6 h, -0°C for 12 h, and 20°C for 24 h.

[0060] The microcarriers produced by the method of the present invention are obviously spherical under electron microscope. Figure 4 , effectively increasing its cell culture area, and the porosity of each batch is greater than 90%, which significantly improves its proliferation rate when used for cell culture, and can be well used for 3D bionic culture and large-scale culture expansion of cells.

[0061] Comparative Example:

[0062] Select porous microcarriers made of gelatin. Remove excess water from the swollen porous gelatin microcarriers. Transfer the porous gelatin microcarriers to a stainless steel tray and cover the upper opening with a microporous top cover. Prefreeze at -85°C for 12 hours. Program-dry the prefrozen microcarriers in a freeze dryer and continue freeze-drying at room temperature for 60 hours.

[0063] from Figure 5 It can be seen that the microcarriers produced by the general method are obviously irregular spherical under the electron microscope. Figure 2 , greatly reducing its cell culture area, and its porosity is less than 75%, which leads to a significant decrease in its proliferation rate when used for cell culture, and it cannot be well used for 3D bionic culture and large-scale culture expansion of cells.

Claims

1. A method for enhancing the freeze-drying stability of porous microcarriers, characterized in that The following steps are involved: (1) The porous microcarriers that have been fully swollen in water are allowed to stand for 5 to 60 minutes until the liquid surface is about to contact the stationary porous microcarriers; (2) immersing the porous microcarrier obtained in step (1) in an organic solvent for dehydration treatment; (3) Use a Pasteur pipette, pipette or electric pipette to transfer the dehydrated porous microcarriers in the container into a tray, seal the top of the tray and place it in liquid nitrogen or an ultra-low temperature refrigerator at -150°C to -85°C for pre-freezing for 6 to 48 hours; (4) The pre-frozen porous microcarriers are placed in a freeze drying oven for freeze drying. The freeze drying program is set to freeze drying at -60°C to -20°C for 6 h to 12 h, freeze drying at -20°C to -0°C for 6 h to 12 h, and freeze drying at 0°C to 20°C for 12 h to 24 h.

2. The method according to claim 1, characterized in that: The porous microcarrier in step (1) is a commercially available microcarrier for cell culture.

3. The method according to claim 1, characterized in that: The porous microcarrier in step (1) is a self-prepared microcarrier such as a porous gelatin microcarrier.

4. The method according to claim 1, characterized in that: The particle size of the porous microcarrier swollen in water in step (1) is 50 to 999 μm.

5. The method according to claim 1, characterized in that: The organic solvent in step (2) can be selected from ethanol, methanol, acetone, butanone, dichloromethane, ethyl acetate, isopropanol, dimethylformamide, chloroform, and 2-methyl-2,4-pentanediol.

6. The method according to claim 1, characterized in that: The dehydration method of step (2) is gradient dehydration: prepare 20%, 40%, 60%, and 80% organic solvent solutions in advance; add the porous microcarrier to the 20% organic solvent solution, stir and let stand, and take the precipitate for use; then add the porous microcarrier to the 40% organic solvent solution, stir and let stand, and take the precipitate for use; then add the porous microcarrier to the 60% organic solvent solution, stir and let stand, and take the precipitate for use; then add the porous microcarrier to the 80% organic solvent solution, stir and let stand, and take the precipitate to complete the dehydration.

7. The method according to claim 1, characterized in that: Step (2) uses ethanol as the porous microcarrier for dehydration treatment; the specific dehydration steps are: (2-1) Prepare 20%, 40%, 60%, or 80% ethanol solutions in advance and let stand at room temperature for 0.5 to 6 hours; (2-2) adding 20% ​​ethanol solution to the swollen porous microcarriers, the ratio of the porous microcarriers to the ethanol solution being 1:10 to 1:50, stirring continuously for 0.5 to 12 hours, standing for 5 to 60 minutes, removing excess ethanol solution after the porous microcarriers sink to the bottom, until the liquid surface is about to contact the stationary porous microcarriers; adding 40% ethanol solution, the ratio of the porous microcarriers to the ethanol solution being 1:10 to 1:50, stirring continuously for 0.5 to 12 hours, standing for 5 to 60 minutes, removing excess ethanol solution after the porous microcarriers sink to the bottom, until the liquid surface is about to contact the stationary porous microcarriers. microcarrier; adding 60% ethanol solution, the ratio of porous microcarrier to ethanol solution is 1:10 to 1:50, stirring continuously for 0.5 to 12 hours, standing for 5 to 60 minutes, removing excess ethanol solution after the porous microcarrier sinks to the bottom, until the liquid surface is about to contact the stationary porous microcarrier; adding 60% ethanol solution, the ratio of porous microcarrier to ethanol solution is 1:10 to 1:50, stirring continuously for 0.5 to 12 hours, standing for 5 to 60 minutes, removing excess ethanol solution after the porous microcarrier sinks to the bottom, until the liquid surface is about to contact the stationary porous microcarrier, and the obtained precipitate is ready for use.

8. The method according to claim 1, characterized in that: The method for sealing the top of the tray in step (3) is to seal the top of the tray with a plastic wrap, a sealing film or a top cover containing a microporous structure.

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