A method of enhancing the freeze-drying stability of porous microcarriers

Abstract

The present application relates to the engineering of biomaterials for cell culture, in particular to a method for enhancing the freeze-drying stability of porous microcarriers. The method comprises the following steps: (1) placing the porous microcarriers which have been sufficiently swollen in water, until the liquid surface is about to contact the porous microcarriers; (2) soaking the porous microcarriers obtained in step (1) in an organic solvent for dehydration treatment; (3) using a Pasteur dropper, a pipette or an electric pipette, transferring the dehydrated porous microcarriers in the container into a tray, sealing the top of the tray and placing it in liquid nitrogen or an ultra-low temperature freezer for pre-freezing; (4) placing the pre-frozen porous microcarriers in a freeze-drying box for freeze-drying. The method can effectively solve the problem of shrinkage, collapse and structural damage of the porous microcarrier scaffold during freeze-drying, and has the advantages of short drying period, low energy consumption and suitability for large-scale production of porous microcarriers.

Description

TECHNICAL FIELD

[0001] The present application relates to the engineering of biomaterials for cell culture, in particular to a method for enhancing the freeze-drying stability of porous microcarriers. BACKGROUND

[0002] Porous microcarriers are materials with highly porous structures, widely used in biomedical, drug delivery, tissue engineering, and chemical catalysis fields. In the biomedical field, porous microcarriers are often used as three-dimensional scaffolds for cell culture, providing space for cell attachment, growth, and differentiation. In drug delivery systems, they can serve as carriers for controlled release of drugs, achieving sustained release and targeted therapy.

[0003] Freeze-drying, also known as lyophilization or sublimation drying, is a drying technique that removes water from materials by freezing them and then removing the ice under vacuum. This technique can maximize the preservation of the original structure and biological activity of the materials, thus having irreplaceable advantages in the processing of heat-sensitive substances. In the preparation of porous microcarriers, freeze-drying is often used to fix cells or form porous structures to maintain the integrity and stability of their structures. Although freeze-drying technology plays an important role in the preparation of porous microcarriers, existing technologies still face many challenges in maintaining the structural stability of microcarriers. First, ice crystal growth during freezing can cause damage to the structure of the microcarriers, affecting their pore characteristics and mechanical strength. Second, shrinkage and collapse during the drying process can change the shape and size of the microcarriers, affecting their 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 influenced by many factors, including but not limited to freezing rate, drying temperature, vacuum degree, composition of materials, and pore structure. Too fast or too slow freezing rates can lead to uneven ice crystal formation, affecting the structural stability of the microcarriers. The control of drying temperature and vacuum degree is also crucial for water removal and preventing material collapse. In addition, the chemical composition and pore structure of the microcarriers determine their responsiveness and stability during freeze-drying.

[0004] To solve the above problems, researchers have tried various methods to improve freeze-drying technology, such as adjusting freezing parameters, using protective agents, developing new freeze-drying equipment, etc. For example, by controlling the freezing rate to obtain finer and more uniform ice crystals, using protective agents such as sucrose, trehalose, etc. to prevent damage to cells or proteins during freezing, and developing freeze-drying equipment that can achieve faster drying. However, these methods can only partially solve the problem of structural stability, and may bring new problems such as increased cost and complex operation. There is an urgent need for a new method to enhance the freeze-drying stability of porous microcarriers. SUMMARY

[0005] The technical problem solved by the present application is that improving the freeze-drying stability of microcarriers by improving the freeze-drying technology can only partially solve the problem of structural stability, and may bring new problems such as cost increase and complex operation. There is an urgent need for a new method to enhance the freeze-drying stability of porous microcarriers.

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

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

[0008] (1) The porous microcarriers (in the present application, the particles are particles of materials used in the field of biomedicine, which are commonly used materials known in the art) that have been fully swollen in water are placed for 5-60 min to remove excess water until the liquid surface just contacts the porous microcarriers.

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

[0010] (3) Use a Pasteur pipette, a pipettor or an electric pipettor to transfer the dehydrated porous microcarriers in the container into a tray (the selection of the tray is crucial to maintaining the structural stability of the microcarriers. The tray needs to ensure effective and uniform heat transfer while avoiding damage to the structure of the microcarriers during freeze-drying. The tray material can be polyethylene, polytetrafluoroethylene, stainless steel or polypropylene); after sealing the top of the tray, it is placed in liquid nitrogen or an ultra-low temperature freezer at-150℃ to-85℃ for pre-freezing for 6-48h. Pre-freezing at ultra-low temperature is an important step before freeze-drying, which aims to form small and uniform ice crystals at low temperature to reduce damage to the structure of the microcarriers.

[0011] (4) The pre-frozen porous microcarriers were placed in a freeze-drying oven for freeze-drying. The freeze-drying program was set to -60℃ to -20℃ for 6h to 12h, -20℃ to -0℃ for 6h to 12h, and 0℃ to 20℃ for 12h to 24h. At low temperatures, most of the organic solvents and ice crystals can be removed, while at relatively high temperatures, bound water and residual water can be removed.

[0012] The freeze-drying process, from -60℃ to -20℃ for 6 to 12 hours, aims to remove most of the bound water and ice crystal moisture. The low temperature at this stage prevents damage to the microcarrier structure while ensuring that ice crystals and most of the moisture are removed in a solid state.

[0013] Freeze-drying at -20℃ to -0℃ for 6 to 12 hours: The purpose of this stage is to further remove residual moisture from the ice crystals. Gradually increasing the temperature helps the ice crystals sublimate while avoiding the collapse of the microcarrier structure or the destruction of active ingredients caused by excessively high temperatures.

[0014] Freeze-drying at 0℃~20℃ for 12h~24h: Drying is carried out at a relatively high temperature, mainly to remove bound water and residual moisture from the microcarrier. Bound water is usually bonded to polar groups in the microcarrier through hydrogen bonds, requiring higher energy to remove. This stage helps to improve drying efficiency, ensure the degree of dryness of the microcarrier, and reduce the dissolution time after rehydration.

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

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

[0017] Furthermore, the porous microcarriers described in step (1) have a particle size of 50–999 μm after swelling in water. Porous microcarriers with a particle size of 50–999 μm can provide sufficient surface area for cell attachment and growth while maintaining the mechanical stability of the microcarriers. If the particle size is too high, it may lead to faster sedimentation of the microcarriers in water, which is not conducive to uniform cell distribution. Simultaneously, increased interference between microcarriers may affect the cell growth environment. If the particle size is too low, it may reduce the surface area of ​​the microcarriers, limiting cell attachment and growth. This increases the risk of microcarrier breakage during operation and affects the stability of cell culture.

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

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

[0020] Further, ethanol is selected for the dehydration treatment of the porous microcarriers in step (2); the specific dehydration steps are as follows:

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

[0022] (2-2) Add 20% ethanol solution to the swollen porous microcarriers, the ratio of the porous microcarriers to the ethanol solution is 1:10-1:50, continuously stir for 0.5-12 h, stand for 5-60 min, remove the excess ethanol solution after the porous microcarriers sink to the bottom until the liquid surface is about to contact the standing porous microcarriers; add 40% ethanol solution, the ratio of the porous microcarriers to the ethanol solution is 1:10-1:50, continuously stir for 0.5-12 h, stand for 5-60 min, remove the excess ethanol solution after the porous microcarriers sink to the bottom until the liquid surface is about to contact the standing porous microcarriers; add 60% ethanol solution, the ratio of the porous microcarriers to the ethanol solution is 1:10-1:50, continuously stir for 0.5-12 h, stand for 5-60 min, remove the excess ethanol solution after the porous microcarriers sink to the bottom until the liquid surface is about to contact the standing porous microcarriers; add 60% ethanol solution, the ratio of the porous microcarriers to the ethanol solution is 1:10-1:50, continuously stir for 0.5-12 h, stand for 5-60 min, remove the excess ethanol solution after the porous microcarriers sink to the bottom until the liquid surface is about to contact the standing porous microcarriers, and the obtained precipitate is standby.

[0023] Further, the tray top sealing method in step (3) is to seal the tray top with cling film, sealing film, or add a top cover with microporous structure to prevent material splashing.

[0024] Porous microcarriers have important applications in biomedical, drug delivery, tissue engineering, and chemical catalysis due to their highly porous structure. However, traditional freeze-drying techniques have many shortcomings in maintaining the structural stability of microcarriers, such as ice crystal growth, shrinkage, and collapse, which can severely affect the performance and application of microcarriers. In view of the limitations of existing technologies, the present application 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 the excess water is removed; second, the dehydrating ability of organic solvents is used to replace the water in the porous microcarriers to remove free water and bound water inside the microcarriers, reducing the possibility of ice crystal formation during freezing, thereby reducing damage to the structure of the microcarriers; third, pre-freezing treatment is performed by controlling the pre-freezing conditions such as temperature, time, and cooling rate to form fine and uniform ice crystals, reducing damage to the structure of the microcarriers; finally, through the gradient temperature drying step, the water in the microcarriers is effectively removed while the porous structure and biological activity of the microcarriers are maximally maintained.

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

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

[0027] Figure 1 The photo of the microcarriers after freezing in Example 1 using the method of the present application under an electron microscope.

[0028] Figure 2 The photo of the microcarriers after freezing in Example 2 using the method of the present application under an electron microscope.

[0029] Figure 3 The photo of the microcarriers after freezing in Example 3 using the method of the present application under an electron microscope.

[0030] Figure 4 The photo of the microcarriers after freezing in Example 4 using the method of the present application under an electron microscope.

[0031] Figure 5 The photo of the microcarriers after freezing using the existing common method under an electron microscope. DETAILED DESCRIPTION

[0032] The following description is merely exemplary of the present application and is not intended to limit the scope of the application, as described.

[0033] The following examples can be understood as part of the separate expression of the partial structure or method of the present application, or can be understood as the mutual combination of the examples to explain the larger range of structure or method of the present application. Unless otherwise specified, all raw materials of the present application are obtained from the market.

[0034] Example 1:

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

[0036] (1) Selecting gelatin (Henan Yaji Gelatin Co., Ltd., YEG796) as the material of the porous microcarriers. Remove the excess water in the swollen gelatin porous microcarriers.

[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 a ratio of 1:20 between the porous microcarriers and the ethanol solution, and continue to stir for 2 hours. Let it stand for 20 minutes, and then remove the excess ethanol solution until the liquid surface is about to contact the standing porous microcarriers. Add 40% ethanol solution, with a ratio of 1:20 between the porous microcarriers and the ethanol solution, and continue to stir for 2 hours. Let it stand for 20 minutes, and then remove the excess ethanol solution until the liquid surface is about to contact the standing porous microcarriers. Then, sequentially perform dehydration treatment with 60% ethanol solution and 80% ethanol solution.

[0038] (3) After dehydration, transfer the gelatin porous microcarriers to a stainless steel tray, and cover the top with a microporous top cover. Pre-freeze at -85°C for 12 hours.

[0039] (4) Perform program drying of the pre-frozen microcarriers in a freeze dryer, with a temperature setting of -50°C for 12 hours, -10°C for 12 hours, and 10°C for 12 hours.

[0040] The multiple microcarriers produced using the method of the present application are obviously spherical under electron microscopy as Figure 1 , effectively increasing its cell culture area, and the porosity of each batch is greater than 90%, thereby significantly improving the proliferation rate when used for cell culture, and can be well used for 3D biomimetic culture and large-scale culture expansion of cells.

[0041] Example 2:

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

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

[0044] (2) Ethanol was selected for the dehydration treatment of the porous microcarriers, 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 microcarriers, and the ratio of the porous microcarriers to the ethanol solution was 1:10, and the stirring was continued for 0.5 h, and the standing was continued for 30 min, and the excess ethanol solution was removed after the porous microcarriers settled at the bottom until the liquid surface was about to contact the standing porous microcarriers. Then, 40% ethanol solution was added, and the ratio of the porous microcarriers to the ethanol solution was 1:10, and the stirring was continued for 0.5 h, and the standing was continued for 30 min, and the excess ethanol solution was removed after the porous microcarriers settled at the bottom until the liquid surface was about to contact the standing porous microcarriers, and then the dehydration treatment with 60% ethanol solution and 80% ethanol solution was sequentially performed.

[0045] (3) The dehydrated porous microcarriers in the container were transferred into a tray using a Pasteur pipette, a pipettor, or an electric pipettor; the top of the tray was sealed with a plastic wrap, a parafilm, or a cap containing a microporous structure to prevent the material from splashing. The tray was placed in an ultra-low temperature freezer (-85℃) for pre-freezing for 24 h.

[0046] (4) The pre-frozen porous microcarriers were placed in a freeze-drying oven for freeze-drying, and the freeze-drying program was set as -20℃ for 12 h, -0℃ for 6 h, and 20℃ for 24 h.

[0047] The porous microcarriers produced by the method of the present application were obviously spherical under an electron microscope as Figure 2 , effectively increasing the cell culture area thereof, and the porosity of each batch thereof was greater than 90%, thereby significantly improving the proliferation speed thereof when used for cell culture, and the porous microcarriers could be well used for 3D biomimetic culture and large-scale culture expansion of cells.

[0048] Example 3:

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

[0050] (2) Select methanol as the porous microcarrier for dehydration treatment, and prepare 20%, 40%, 60%, and 80% methanol solutions in advance and stand at room temperature for 0.5 h. The specific dehydration steps are as follows: add 20% methanol solution to the swollen porous microcarrier, the ratio of porous microcarrier to methanol solution is 1:10, continuously stir for 0.5 h, stand for 30 min, remove the excess methanol solution after the porous microcarrier sinks to the bottom, until the liquid surface just contacts the standing porous microcarrier. Add 40% methanol solution, the ratio of porous microcarrier to methanol solution is 1:10, continuously stir for 0.5 h, stand for 30 min, remove the excess methanol solution after the porous microcarrier sinks to the bottom, until the liquid surface just contacts the standing porous microcarrier, then sequentially perform dehydration treatment with 60% methanol solution and 80% methanol solution.

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

[0052] (4) Place the pre-frozen porous microcarrier in a freeze dryer and perform freeze drying, with the freeze drying program set to -20°C for 12 h, -0°C for 12 h, and 20°C for 12 h.

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

[0054] Example 4:

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

[0056] (1) Stand the 3D-MIC microcarriers purchased from Qingdao Marine Food Nutrition and Health Innovation Institute for 5-60 min to remove excess water until the liquid surface just contacts the standing porous microcarrier.

[0057] (2) Select isopropanol as a porous microcarrier for dehydration treatment, 20%, 40%, 60%, 80% isopropanol solution is prepared in advance and room temperature is placed for 0.5h. The specific dehydration steps are as follows: add 20% isopropanol solution to the swollen porous microcarrier, the ratio of porous microcarrier to isopropanol solution is 1:10, continue to stir for 0.5h, stand for 30min, remove the excess isopropanol solution after the porous microcarrier sinks to the bottom, until the liquid surface just contacts the static porous microcarrier. Add 40% isopropanol solution, the ratio of porous microcarrier to isopropanol solution is 1:10, continue to stir for 0.5h, stand for 30min, remove the excess isopropanol solution after the porous microcarrier sinks to the bottom, until the liquid surface just contacts the static porous microcarrier, then sequentially carry out 60% isopropanol solution and 80% isopropanol solution dehydration treatment.

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

[0059] (4) Place the pre-frozen porous microcarrier in the freeze dryer for freeze drying, and the freeze drying program is set to -20℃ for 6h, -0℃ for 12h, and 20℃ for 24h.

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

[0061] Comparative example:

[0062] Select a porous microcarrier with gelatin as the material. Remove the excess water in the swollen gelatin porous microcarrier. Transfer the gelatin porous microcarrier into a stainless steel tray, and add a micro-porous top cover to the open top. Pre-freeze at -85℃ for 12h. Perform program drying on the pre-frozen microcarrier in the freeze dryer, and continuously freeze dry at room temperature for 60h.

[0063] As can be seen from Figure 5 , the microcarrier produced by the general method is obviously irregular spherical under electron microscope Figure 2 , greatly reducing its cell culture area, and the porosity is less than 75%, resulting in a significant reduction in its proliferation rate when used for cell culture, and it cannot be well used for 3D biomimetic culture and large-scale culture expansion of cells.

Claims

1. A method for enhancing the freeze-drying stability of porous microcarriers, characterized in that... Includes the following steps: (1) Let the porous microcarrier that has been fully swollen in water stand for 5 to 60 minutes until the liquid surface is about to contact the standing porous microcarrier. (2) The porous microcarrier obtained in step (1) is immersed in an organic solvent for dehydration; ethanol is selected as the porous microcarrier for dehydration in step (2); the specific dehydration steps are as follows: (2-1) Prepare 20%, 40%, 60%, and 80% ethanol solutions in advance and let them stand at room temperature for 0.5 to 6 hours; (2-2) Add 20% ethanol solution to the swollen porous microcarrier, with a ratio of porous microcarrier to ethanol solution of 1:10 to 1:

50. Stir continuously for 0.5 to 12 hours, let stand for 5 to 60 minutes, and remove excess ethanol solution after the porous microcarrier has settled to the bottom, until the liquid surface is about to contact the settled porous microcarrier; add 40% ethanol solution, with a ratio of porous microcarrier to ethanol solution of 1:10 to 1:50, stir continuously for 0.5 to 12 hours, let stand for 5 to 60 minutes, and remove excess ethanol solution after the porous microcarrier has settled to the bottom, until the liquid surface is about to contact the settled porous microcarrier; add 60% ethanol solution, with a ratio of porous microcarrier to ethanol solution of 1:10 to 1:50, stir continuously for 0.5 to 12 hours, and let stand for 5 to 60 minutes. After the porous microcarrier settles to the bottom, remove excess ethanol solution until the liquid surface is about to contact the stationary porous microcarrier; add 60% ethanol solution, the ratio of porous microcarrier to ethanol solution is 1:10~1:50, stir continuously for 0.5~12h, let stand for 5~60 min, after the porous microcarrier settles to the bottom, remove excess ethanol solution until the liquid surface is about to contact the stationary porous microcarrier, and the obtained precipitate is ready for use. (3) Use a Pasteur pipette or electric pipette to transfer the dehydrated porous microcarrier in the container into a tray, seal the top of the tray and place it in liquid nitrogen or an ultra-low temperature freezer at -150℃ to -85℃ for pre-freezing for 6 to 48 hours; (4) The pre-frozen porous microcarrier was placed in a freeze dryer for freeze drying. The freeze drying program was set to freeze-dry at -60℃ to -20℃ for 6h to 12h, at -20℃ to -0℃ for 6h to 12h, and at 0℃ to 20℃ for 12h to 24h.

2. The method as described in claim 1, characterized in that: The porous microcarrier mentioned in step (1) is a commercially available cell culture microcarrier.

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

4. The method as described in claim 1, characterized in that: The porous microcarrier described in step (1) has a particle size of 50~999μm when it swells in water.

5. The method as described in claim 1, characterized in that: Step (3) The method for sealing the top of the tray is to seal the top of the tray with plastic wrap, sealing film or a top cover with a microporous structure.

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