Preparation method of controllable dissolution surface modification microcarrier and cell culture method

Abstract

The invention relates to the technical field of microcarriers, and provides a preparation method of a controllable dissolution surface modification microcarrier and a cell culture method.The preparation method comprises the steps that a gelatin solution is prepared based on the preset solution concentration; obtaining a target microcarrier, and soaking the target microcarrier in the gelatin solution to obtain a first mixed solution; performing first constant-temperature shaking treatment on the first mixed solution; carrying out sedimentation treatment on the first mixed solution subjected to the first constant-temperature shaking treatment, and removing supernate to obtain a gelatin-modified target microcarrier; obtaining a cross-linking agent solution, and soaking the gelatin-modified target microcarrier in the cross-linking agent solution to obtain a second mixed solution; performing second constant-temperature shaking treatment on the second mixed solution; carrying out sedimentation treatment on the second mixed solution subjected to the second constant-temperature shaking treatment, and removing supernate to obtain a gelatin-crosslinked target microcarrier. According to the microcarrier provided by the invention, the adherent growth advantage of cells can be maintained, and meanwhile, large-scale suspension culture of adherent cells and controllable separation between the cells and the microcarrier can be realized.

Description

Technical Field

[0001] The invention relates to a preparation method of a controllably dissolvable surface-modified microcarrier and a cell culture method, belonging to the technical field of microcarriers. Background Art

[0002] Biopharmaceuticals usually use cells as production plants for large-scale cell culture, and the cultured cells produce target products such as monoclonal antibodies, viruses, and exosomes. Cell culture microsphere carriers (microcarriers for short) are micron-sized particles with a three-dimensional structure that can provide support for adherent cells to attach and grow. Compared with traditional two-dimensional culture, cell culture microcarriers combined with bioreactors can achieve large-scale cell proliferation in a short period of time while maintaining their specific morphology and phenotype.

[0003] In the related art, after the cells are cultured to the surface of the microcarrier, the existing microcarriers often lack an effective cell separation mechanism. This results in the adhesion between the cells and the microcarriers being too tight when harvesting the cells, making it difficult to achieve gentle and efficient separation. As a result, the cell loss rate is high and the harvesting efficiency is low, which greatly affects the production efficiency. Therefore, it is necessary to develop a new type of microcarrier that can achieve controllable separation between cells and microcarriers while maintaining the advantages of cell adhesion growth. Summary of the invention

[0004] The present invention provides a method for preparing a controllably dissolvable surface-modified microcarrier and a cell culture method, the main purpose of which is to achieve controllable separation between cells and microcarriers while maintaining the cell wall growth advantage.

[0005] To achieve the above object, the first aspect of the present invention provides a method for preparing a controllably dissolvable surface-modified microcarrier, comprising:

[0006] Based on a predetermined solution concentration, a gelatin solution is prepared;

[0007] Obtaining a target microcarrier, and immersing the target microcarrier in the gelatin solution to obtain a first mixed solution; wherein the matrix of the target microcarrier is pectin and sodium alginate;

[0008] Performing a first constant temperature shaking treatment on the first mixed solution; wherein the time of the first constant temperature shaking treatment is 1 to 5 hours;

[0009] performing sedimentation treatment on the first mixed solution after the first constant temperature shaking treatment, and removing the supernatant to obtain the target microcarrier modified with gelatin;

[0010] Obtaining a crosslinking agent solution, and immersing the target microcarrier modified with gelatin in the crosslinking agent solution to obtain a second mixed solution; wherein the crosslinking agent is a genipin solution or a glutaraldehyde solution;

[0011] Performing a second constant temperature shaking treatment on the second mixed solution; wherein the time of the first constant temperature shaking treatment is 1 to 7 hours;

[0012] The second mixed solution after the second constant temperature shaking treatment is subjected to sedimentation treatment, and the supernatant is removed to obtain the target microcarrier after gelatin cross-linking.

[0013] Optionally, the step of preparing a gelatin solution based on a preset solution concentration comprises:

[0014] Weighing gelatin according to a preset solution concentration; wherein the preset solution concentration ranges from 1% to 16%;

[0015] Gelatin is added into deionized water, and stirred in a 60° C. water bath until completely dissolved, to obtain the gelatin solution.

[0016] Optionally, the method further comprises:

[0017] Using ultrapure water to wash the gelatin-modified target microcarrier once or multiple times to remove unreacted gelatin;

[0018] or,

[0019] Using ultrapure water to wash the target microcarrier after gelatin cross-linking once or multiple times to remove unreacted cross-linking agent;

[0020] The target microcarrier has a blue or transparent spherical or spherical appearance;

[0021] The diameter of the target microcarrier is 10-1000 μm;

[0022] The density of the target microcarrier is 1.02-1.05 g / cm3.

[0023] The second aspect of the present invention provides a cell culture method of a controllably dissolvable surface-modified microcarrier, wherein the controllably dissolvable surface-modified microcarrier used in the cell culture method is prepared using the above-mentioned preparation method, and the cell culture method comprises:

[0024] Obtaining target microcarriers and target cultured cells, and pretreating the target microcarriers;

[0025] Inoculating the target cultured cells into the pretreated target microcarriers for proliferation, to obtain a target cultured cell culture medium containing cells and microcarriers; wherein the target cultured cells are anchorage-dependent cells;

[0026] The target culture cell culture medium is allowed to settle for a certain period of time, and after removing the supernatant waste culture medium, a target microcarrier dissolving agent corresponding to the target microcarrier is added; wherein the mixing volume ratio of the target microcarrier to the target microcarrier dissolving agent is 1:5-10, and the dissolution time is 30s to 5min;

[0027] After complete dissolution, complete culture medium is added to terminate the trypsin reaction and the culture medium of the target cultured cells after the target microcarriers are dissolved is centrifuged to separate the proliferated target cultured cells.

[0028] Optionally, the target microcarrier is based on pectin and sodium alginate, and the surface is modified with gelatin. The target microcarrier dissolving agent is a mixed solution of trypsin solution and chelating agent, and the chelating agent is EDTA solution or sodium citrate solution, and the concentration of the chelating agent is 4mM / L to 20mM / L. M / L, the concentration of the trypsin solution is 0.001% to 0.5%, trypsin is used to hydrolyze gelatin, and the chelating agent is used to chelate calcium ions to dissolve the target microcarrier and completely separate it from the proliferated target cultured cells.

[0029] Optionally, the pre-treating the target microcarrier comprises:

[0030] Sterilize the target microcarrier using 75% alcohol;

[0031] Wash the sterilized target microcarrier once or multiple times using sterile water;

[0032] The washed target microcarriers are added to complete culture medium for incubation and re-suspending.

[0033] Optionally, before inoculating the target cultured cells into the pretreated target microcarriers for proliferation, the method further comprises:

[0034] Counting the target cultured cells and the target microcarriers to obtain the number of the target cells and the target microcarriers;

[0035] The target culture cell number of the target culture cells corresponding to the target microcarrier number is determined, and the ratio of the number of the target microcarriers to the number of the target culture cells is 1:10-20.

[0036] Optionally, after the target culture cell culture medium is allowed to settle for a certain period of time and the supernatant waste culture medium is removed, before the target microcarrier dissolving agent corresponding to the target microcarrier is added, the method further comprises:

[0037] Using an inverted optical microscope to obtain the confluence of the target cultured cells proliferating on the surface of the target microcarrier;

[0038] When the confluence exceeds a preset confluence threshold, it is determined that the proliferation of the target cultured cells is complete.

[0039] Optionally, the cell culture method further comprises:

[0040] The target cultured cells are stained with calcein and propidium iodide; wherein the target cultured cells include living cells and dead cells, the living cells are stained with calcein and exhibit green fluorescence under microscopic observation, and the dead cells are stained with propidium iodide and exhibit red fluorescence under microscopic observation.

[0041] Optionally, after separating the proliferated target cultured cells, the method further includes counting the proliferated target cultured cells to obtain the number of the proliferated target cultured cells and harvesting the cells, specifically including:

[0042] After removing the supernatant and discarded culture medium from the target culture medium containing cells and microcarriers after proliferation, the target microcarriers are washed once or multiple times using a phosphate buffer;

[0043] Adding a dissolving agent to decompose the matrix between cells of the target microcarrier and completely dissolve the target microcarrier;

[0044] Complete medium was added to terminate the reaction, and the cells were resuspended after centrifugation;

[0045] The proliferated target cultured cells are counted by a blood cell counting plate to obtain the number of the proliferated target cultured cells, the proliferation multiples, and the harvested cells.

[0046] Compared with the problems described in the background technology, the present invention first prepares a gelatin solution based on a preset solution concentration, then obtains a target microcarrier, and immerses the target microcarrier in the gelatin solution to obtain a first mixed solution; wherein, the matrix of the target microcarrier is pectin and sodium alginate, and the present invention uses a gelatin solution to modify the surface of the microcarrier, thereby increasing the protein content on the surface of the microcarrier, thereby improving the adhesion of the cells, and gelatin, as a polypeptide mixture, contains a variety of biologically active groups, which can promote the interaction between cells and microcarriers; then, the first mixed solution is subjected to a first constant temperature shaking treatment; wherein, the time for the first constant temperature shaking treatment is 1 to 5 hours; constant temperature shaking helps to uniformly distribute and preliminarily fix the gelatin molecules on the surface of the microcarrier, 1 to 5 hours The treatment time ensures that gelatin is in full contact with the surface of the microcarrier, laying the foundation for subsequent cross-linking; next, the first mixed solution after the first constant temperature shaking treatment is subjected to sedimentation treatment, and the supernatant is removed to obtain the target microcarrier modified with gelatin; the sedimentation process separates the microcarrier modified with gelatin from the solution, and the removal of the supernatant removes the unbound gelatin and other impurities, ensuring the purity and uniformity of the surface modification of the microcarrier; then, a cross-linking agent solution is obtained, and the target microcarrier modified with gelatin is immersed in the cross-linking agent solution to obtain a second mixed solution; the use of the cross-linking agent further strengthens the binding force between the gelatin layer and the microcarrier, improves the mechanical strength of the microcarrier, makes it more stable in the subsequent cell culture and operation process, and reduces cell loss. The second mixed solution is then subjected to a second constant temperature shaking treatment; wherein, the time of the first constant temperature shaking treatment is 1 to 7 hours; the 1 to 7 hours of the second constant temperature shaking treatment provides sufficient time for the cross-linking reaction, ensures the chemical bonding between the gelatin layer and the microcarrier, and further enhances the stability and durability of the microcarrier. Finally, the second mixed solution after the second constant temperature shaking treatment is subjected to sedimentation treatment, and the supernatant is removed to obtain the target microcarrier after gelatin cross-linking, and the target microcarrier is blue or transparent spherical or quasi-spherical, with a diameter of 10 μm to 1000 μm, and can be maintained without cracks or breakage for 30 days under the condition of continuous high-speed stirring of 40-80 rpm in the culture medium. Therefore, the preparation method of the present invention not only improves the cell adhesion of the microcarrier, but also enhances its stability and durability during the cell culture process, realizes the controllable separation between cells and microcarriers, reduces the loss during cell harvesting, and improves the overall efficiency and quality of cell culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 A schematic flow chart of a method for preparing a controllably dissolvable surface-modified microcarrier provided in one embodiment of the present invention;

[0048] Figure 2 A schematic flow chart of a method for preparing a controllably dissolvable surface-modified microcarrier provided in another embodiment of the present invention;

[0049] Figure 3 A schematic diagram of a process for culturing cells of a controllably dissolvable surface-modified microcarrier provided in one embodiment of the present invention;

[0050] Figure 4 A schematic diagram of a cell culture method for a controllably dissolvable surface-modified microcarrier provided in another embodiment of the present invention;

[0051] Figure 5 A schematic diagram of a cell culture method for a controllably dissolvable surface-modified microcarrier provided in another embodiment of the present invention;

[0052] Figure 6 A schematic diagram of a cell culture method for a controllably dissolvable surface-modified microcarrier provided in another embodiment of the present invention;

[0053] Figure 7 An experimental diagram of dissolving a controllably dissolvable surface-modified microcarrier using a dissolving agent provided in one embodiment of the present invention;

[0054] Figure 8 This is a staining experiment diagram of live cells and dead cells of Vero cells provided in one embodiment of the present invention;

[0055] Fig. 9 This is a staining experiment diagram of live cells and dead cells of MSCs cells provided in one embodiment of the present invention;

[0056] Fig.10 A schematic diagram of the structure of an electronic device for implementing a method for preparing a surface-modified microcarrier with controllable dissolution or a method for culturing cells of a surface-modified microcarrier with controllable dissolution provided by an embodiment of the present invention;

[0057] Fig.11 A schematic diagram of the stability and mechanical strength test of the controllable dissolution surface-modified microcarrier provided in one embodiment of the present invention;

[0058] Fig.12 This is a schematic diagram comparing the surface-modified microsphere carrier provided by one embodiment of the present invention before and after being suspended and stirred in a culture medium for 30 days.

[0059] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings in conjunction with the embodiments. DETAILED DESCRIPTION

[0060] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0061] It should be noted that although the functional modules are divided in the device schematic and the logical order is shown in the flowchart, in some cases, the steps shown or described may be performed in a different order than the module division in the device or the order in the flowchart.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein are only for the purpose of describing the embodiments of this application and are not intended to limit this application.

[0063] Biopharmaceuticals are a technology that uses organisms (such as cells or bacteria) to produce drugs. In this field, cell culture technology plays a vital role. Target microcarriers (microcarriers for short) are three-dimensional micron-sized particles used to support the growth of adherent cells. Compared with traditional two-dimensional cell culture, the combination of microcarriers and bioreactors can achieve a large number of cell proliferation in a shorter time while maintaining the specific morphology and phenotype of the cells. This is of great significance for the production of monoclonal antibodies, viral vectors and other biological products.

[0064] There are still some significant deficiencies in the relevant technology. The main problems include:

[0065] First, it is difficult to separate cells from microcarriers. After the cells grow and cover the surface of the microcarriers, it is difficult to achieve efficient separation of cells from microcarriers using existing microcarriers, resulting in high cell loss when harvesting cells.

[0066] Second, the cost is high. Some existing soluble microcarriers rely on expensive raw materials, such as RGD synthetic peptide, which increases the overall cost.

[0067] Third, the dissolution time is too long. The dissolution process of some microcarriers requires a long time (e.g., 15 minutes or even longer), which may affect the activity and harvesting efficiency of the cells. In addition, the dissolution process may damage the cells. A long dissolution process can easily damage the cells and reduce the cell harvesting rate.

[0068] Based on this, the embodiment of the present application provides a method for preparing a controllably dissolvable surface-modified microcarrier and a cell culture method, and the execution subject of the method for preparing a controllably dissolvable surface-modified microcarrier and the cell culture method includes but is not limited to at least one of the electronic devices such as a server and a terminal that can be configured to execute the method provided by the embodiment of the present application. In other words, the method for preparing a controllably dissolvable surface-modified microcarrier and the cell culture method can be executed by software or hardware installed in a terminal device or a server device. The server includes but is not limited to: a single server, a server cluster, a cloud server or a cloud server cluster, etc.

[0069] Embodiment 1:

[0070] Reference Figure 1 FIG. 1 is a flow chart of a method for preparing a controlled dissolution surface-modified microcarrier according to an embodiment of the present invention. In this embodiment, the method for preparing a controlled dissolution surface-modified microcarrier includes but is not limited to S101 to S107:

[0071] S101. Prepare a gelatin solution based on a preset solution concentration.

[0072] It is understandable that the main purpose of step S101 is to provide a protein-modified coating for the microcarrier to enhance cell adhesion and promote cell growth. Gelatin, as a polypeptide mixture, contains a variety of bioactive groups that can bind to corresponding receptors on the cell surface and provide a three-dimensional network structure required for cell growth.

[0073] The concentration of gelatin solution is preset according to the specific needs of cell culture and the characteristics of microcarriers. Usually, the concentration ranges from 1% to 16% to ensure that it can provide sufficient protein matrix without being too viscous to affect the pore structure of microcarriers.

[0074] Further, refer to Figure 2 FIG. 1 is a schematic flow chart of a method for preparing a controllably dissolvable surface-modified microcarrier according to another embodiment of the present invention, wherein S101 may also include but is not limited to S201 to S202.

[0075] S201. Weighing a corresponding weight of gelatin according to a preset solution concentration; wherein the preset solution concentration ranges from 1% to 16%.

[0076] It is understandable that when preparing gelatin solution, the amount of gelatin needs to be accurately controlled according to the preset solution concentration and the final solution volume. Therefore, it is necessary to calculate and weigh the corresponding weight of gelatin according to the required final volume and the preset solution concentration (1%-16%). This step is the key to ensure the accurate concentration of the solution, which is crucial for the success of subsequent cell culture.

[0077] For example, in some embodiments, 100 milliliters (mL) of gelatin solution is required to be prepared, and the desired solution concentration is 8% (weight / volume). The required gelatin weight = solution volume × target concentration. The required gelatin weight is calculated as follows:

[0078] 0.1L×8%=0.1L×8g / L=0.8g

[0079] Therefore, according to the calculation results, 0.8 grams of gelatin needs to be weighed.

[0080] S202, adding gelatin into deionized water, and stirring in a 60° C. water bath until completely dissolved, to obtain a gelatin solution.

[0081] It is understandable that the purpose of S202 is to completely dissolve the gelatin to form a uniform solution and provide uniform surface modification for the microcarrier. The weighed gelatin is added to the pre-prepared deionized water, wherein the use of deionized water is to ensure the purity of the solution and avoid the introduction of impurities that may affect cell culture. The mixture of gelatin and deionized water is heated in a 60°C water bath. This temperature can promote the movement of gelatin molecules and accelerate the dissolution process, while also avoiding the damage to the molecular structure of gelatin caused by excessively high temperatures. During the heating process, the mixture is continuously stirred to ensure that the gelatin is evenly dispersed in the water. Stirring helps prevent gelatin from agglomerating and ensures the uniformity of the solution. Continue heating and stirring until the gelatin is completely dissolved. The criterion for complete dissolution is that the solution presents a uniform and consistent transparency without visible particles or lumps.

[0082] S102, obtaining a target microcarrier, and immersing the target microcarrier in the gelatin solution to obtain a first mixed solution; wherein the matrix of the target microcarrier is pectin and sodium alginate.

[0083] S103, performing a first constant temperature shaking treatment on the first mixed solution; wherein the time of the first constant temperature shaking treatment is 1 to 5 hours.

[0084] It can be understood that the purpose of S102 to S103 is to ensure that the surface of the microcarrier can evenly adsorb gelatin to form a protein modification layer that is beneficial to cell adhesion and growth.

[0085] In some embodiments, microcarriers with pectin and sodium alginate as matrices can be selected. These natural polysaccharides provide good biocompatibility and three-dimensional structure for microcarriers. The microcarriers are added to the previously prepared gelatin solution in a certain proportion (for example, volume ratio or mass ratio) to form a first mixed solution. This step is to allow the microcarriers to fully contact the gelatin so that gelatin molecules are adsorbed on their surface. The container containing the microcarriers and the gelatin solution can be placed in a constant temperature shaker. Set the temperature of the shaker to the ideal temperature for cell culture, usually around 37°C, to simulate the environment in the body, turn on the shaking function, and keep the solution in the container in a uniform shaking motion. The shaking rate and mode should be adjusted according to the size and density of the microcarriers to ensure that the microcarriers are fully suspended in the solution to avoid sedimentation. And the shaking treatment time can be set to 1 to 5 hours. This time period allows the gelatin molecules to be fully adsorbed on the surface of the microcarriers while maintaining the dynamic state of the solution to promote uniform distribution.

[0086] S104, performing sedimentation treatment on the first mixed solution after the first constant temperature shaking treatment, and removing the supernatant to obtain the target microcarrier modified with gelatin.

[0087] It is understood that after the constant temperature shaking treatment is completed, the shaker is turned off and the container is left to stand, allowing the microcarriers to naturally settle to the bottom of the container under the action of gravity. Since the density of the microcarriers is greater than the density of the solution, they will gradually settle. After the microcarriers settle, the gelatin solution on the upper layer is removed while avoiding disturbing the settled microcarriers. This step is called "removing the supernatant" and the purpose is to remove the unadsorbed gelatin and other soluble impurities, thereby obtaining the target microcarriers modified with gelatin.

[0088] In some embodiments, after removing the supernatant to obtain the target microcarrier modified with gelatin, an appropriate amount of ultrapure water or sterile water may be added to the settled microcarrier. The addition of an appropriate amount of cleaning solution is to suspend the microcarrier for further cleaning. The container is gently shaken or stirred to resuspend the microcarrier in the cleaning solution and then allowed to settle again. The steps of settling and removing the supernatant are repeated at least twice to ensure thorough cleaning.

[0089] S105, obtaining a cross-linking agent solution, and immersing the target microcarrier modified with gelatin in the cross-linking agent solution to obtain a second mixed solution.

[0090] S106, performing a second constant temperature shaking treatment on the second mixed solution; wherein the time of the first constant temperature shaking treatment is 1 to 7 hours.

[0091] It is understandable that steps S105 and S106 are intended to enhance the bonding force between the gelatin layer and the microcarriers through chemical cross-linking, improve the stability and mechanical strength of the microcarriers, and ensure the durability and ability of the microcarriers to resist physical damage during cell culture.

[0092] Among them, suitable cross-linking agents can be selected according to the required chemical and biological properties. Commonly used cross-linking agents include multivalent aldehyde compounds, such as glutaraldehyde. Genipin can also be used as a cross-linking agent. Genipin is the product of gardenia glycoside hydrolyzed by β-glucosidase. It is an excellent natural biological cross-linking agent that can be cross-linked with proteins, collagen, gelatin and chitosan to make biomaterials. Its toxicity is much lower than glutaraldehyde and other commonly used chemical cross-linking agents. After completing the sedimentation and cleaning steps (S104), the surface of the microcarrier has been evenly covered with gelatin. These microcarriers are resuspended in an appropriate amount of solution, ready for cross-linking treatment, and the gelatin-modified microcarriers are transferred to the cross-linking agent solution to form a second mixed solution. The container containing the microcarrier and the cross-linking agent solution is placed back in a constant temperature shaker, and a suitable temperature (usually 25°C to 37°C) and shaking speed are set to perform constant temperature shaking treatment. The shaking time is determined according to the type of cross-linking agent and the required degree of cross-linking, and is usually 1 to 7 hours.

[0093] S107, performing sedimentation treatment on the second mixed solution after the second constant temperature shaking treatment, and removing the supernatant to obtain the target microcarrier after gelatin cross-linking.

[0094] It can be understood that the purpose of step S107 is to remove unreacted cross-linking agent and other soluble impurities that may be produced during the cross-linking process, ensure the cleanliness of the microcarrier surface, and prepare for subsequent application or storage. The target microcarrier obtained in step 107 is blue or transparent, spherical or quasi-spherical, with a diameter of 10 μm to 1000 μm and a density of 1.02-1.05 g / cm3. It can remain without cracks or breakage for 30 days under continuous high-speed stirring of 40-80 rpm in the culture medium, and has good stability.

[0095] In some embodiments, after the constant temperature shaking treatment (step S106) is completed, the constant temperature shaker is turned off, and the solution containing the cross-linked gelatin microcarriers is allowed to stand, so that the microcarriers settle to the bottom of the container under the action of gravity. After the microcarriers are fully settled, a sterile pipette or straw is used to gently suck out and remove the transparent supernatant on the upper layer of the solution to avoid disturbing the settled microcarriers; an appropriate amount of ultrapure water or sterile water can also be added to the settled microcarriers, and the microcarriers can be gently shaken or stirred to resuspend the microcarriers to remove the unreacted crosslinker attached to the microcarriers, and the microcarriers are allowed to settle again, and the new supernatant is removed. This cleaning process can be repeated 2 to 3 times to ensure that all unreacted crosslinkers and impurities are completely removed.

[0096] In summary, compared with the background technology, the embodiment of the present application provides a method for preparing a controllable dissolution surface-modified microcarrier, by preparing a gelatin solution based on a preset solution concentration, then obtaining a target microcarrier, and immersing the target microcarrier in the gelatin solution to obtain a first mixed solution; wherein, the matrix of the target microcarrier is pectin and sodium alginate, and the present invention uses a gelatin solution to modify the surface of the microcarrier, thereby increasing the protein content on the surface of the microcarrier, thereby improving the adhesion of the cells, and gelatin, as a polypeptide mixture, contains a variety of biologically active groups, which can promote the interaction between cells and microcarriers; then, the first mixed solution is subjected to a first constant temperature shaking treatment; wherein, the time of the first constant temperature shaking treatment is 1 to 5 hours; constant temperature shaking helps to evenly distribute the gelatin molecules on the surface of the microcarrier Distribution and preliminary fixation, the treatment time of 1 to 5 hours ensures sufficient contact between gelatin and the surface of the microcarrier, laying the foundation for subsequent cross-linking; next, the first mixed solution after the first constant temperature shaking treatment is subjected to sedimentation treatment, and the supernatant is removed to obtain the target microcarrier modified with gelatin; the sedimentation process separates the microcarrier modified with gelatin from the solution, and the removal of the supernatant removes the unbound gelatin and other impurities, ensuring the purity and uniformity of the surface modification of the microcarrier; then, a cross-linking agent solution is obtained, and the target microcarrier modified with gelatin is immersed in the cross-linking agent solution to obtain a second mixed solution; the use of the cross-linking agent further strengthens the binding force between the gelatin layer and the microcarrier, improves the mechanical strength of the microcarrier, makes it more stable in the subsequent cell culture and operation process, and reduces cell loss. The second mixed solution is then subjected to a second constant temperature shaking treatment; wherein, the time of the first constant temperature shaking treatment is 1 to 7 hours; the 1 to 7 hours of the second constant temperature shaking treatment provides sufficient time for the cross-linking reaction, ensures the chemical bonding between the gelatin layer and the microcarrier, and further enhances the stability and durability of the microcarrier. Finally, the second mixed solution after the second constant temperature shaking treatment is subjected to sedimentation treatment, and the supernatant is removed to obtain the target microcarrier after gelatin cross-linking. Therefore, the preparation method of the present invention not only improves the cell adhesion of the microcarrier, but also enhances its stability and durability during cell culture, realizes controllable separation between cells and microcarriers, reduces the loss during cell harvesting, and improves the overall efficiency and quality of cell culture.

[0097] like Fig.11 As shown in the figure, compared with the unmodified microspheres, the average diameter of the surface-modified microspheres increased by about 20 μm. From day 0 to day 1, the surface-modified microspheres immersed in the culture medium showed obvious swelling phenomenon, and the diameter increased by about 20 μm. During the subsequent suspension and stirring process, the average diameter of the microspheres remained stable at about 190 μm from day 2 to day 30, indicating that the mechanical strength of the surface-modified microspheres was improved.

[0098] like Fig.12As shown, no microsphere breakage was found during the optical microscope observation after 30 days of suspension and stirring, and the culture medium was not contaminated, indicating that the surface-modified microspheres have good stability and high mechanical strength and can be used for large-scale dynamic culture of adherent cells.

[0099] Embodiment 2:

[0100] Reference Figure 3 As shown, it is a schematic flow diagram of a cell culture method of a controllably dissolvable surface-modified microcarrier provided in one embodiment of the present invention. The controllably dissolvable surface-modified microcarrier used in this embodiment is prepared by the preparation method of the controllably dissolvable surface-modified microcarrier described in any of the above embodiments. The cell culture method of the controllably dissolvable surface-modified microcarrier includes but is not limited to S301 to S304:

[0101] S301, obtaining target microcarriers and target cultured cells, and pretreating the target microcarriers.

[0102] It is understood that obtaining the target microcarrier refers to obtaining the desired controllably soluble surface-modified microcarrier from the above preparation method, such as gelatin cross-linked pectin / sodium alginate microspheres, etc. Pretreatment of the target microcarrier may include sterilization treatment, and activation treatment of the microcarrier, etc., to make it suitable for the adherent growth of the target cultured cells.

[0103] Further, refer to Figure 4 FIG. 1 is a schematic flow chart of a method for culturing cells using a controllably dissolvable surface-modified microcarrier according to another embodiment of the present invention, wherein S301 may also include but is not limited to S401 to S403.

[0104] S401, sterilize the target microcarrier using 75% alcohol.

[0105] It is understandable that 75% alcohol solution has a good disinfection and sterilization effect, and can effectively kill bacteria, fungi and other microorganisms that may exist on the surface and inside of the microcarrier. Soaking the prepared microcarrier in 75% alcohol solution for a certain period of time can achieve the disinfection and sterilization of the microcarrier, and prepare for the subsequent cell culture aseptically.

[0106] S402, using sterile water to wash the sterilized target microcarriers once or multiple times.

[0107] It is understandable that although alcohol has a disinfecting effect, the residual alcohol has a certain toxic effect on cells. The microcarriers disinfected with alcohol need to be washed several times with sterile pure water to completely remove the alcohol residue and ensure the sterility and non-toxicity of the microcarriers.

[0108] S403, adding the washed target microcarriers to complete culture medium for incubation.

[0109] It can be understood that complete culture medium refers to a culture medium containing various nutrients, growth factors, hormones, etc. required for cell growth. Adding the disinfected and cleaned microcarriers to the complete culture medium for incubation can allow a layer of nutrients to be adsorbed on the surface of the microcarriers, which is conducive to the subsequent cell adhesion growth. At the same time, the sterile environment of the complete culture medium can also ensure the sterile state of the microcarriers, creating suitable conditions for cell culture.

[0110] Through the above-mentioned S401-S403 pretreatment steps, the prepared microcarriers can be thoroughly disinfected, pollutants can be removed, and the microcarriers can be suspended in a nutrient environment suitable for cell growth for incubation, so as to fully prepare for the subsequent S302 cell inoculation and culture, thereby improving the efficiency of cell culture.

[0111] S302, inoculating the target cultured cells into the pretreated target microcarriers for proliferation, and obtaining a target cultured cell culture medium containing the target cultured cells and the target microcarriers.

[0112] It can be understood that the target cultured cells obtained in step S301 are inoculated into the pretreated target microcarriers, and the cells are cultured under conditions of cell culture medium, certain temperature, humidity and carbon dioxide concentration, so that the cells grow and proliferate on the surface of the microcarriers to form a cell culture medium of a cell / microcarrier complex.

[0113] Further, refer to Figure 5 FIG. 1 is a schematic flow chart of a method for culturing cells using a controllably dissolvable surface-modified microcarrier according to another embodiment of the present invention. Before S302 , the method may also include but is not limited to S501 to S502 .

[0114] S501. Count the target cells and microcarriers to obtain the number of target cultured cells and target microcarriers.

[0115] It is understandable that before inoculating cells onto microcarriers for culture, the microcarriers need to be counted first to understand the actual number of microcarriers. The microcarrier suspension can be sampled and counted using a hemacytometer or an automatic particle counter. The number of microcarriers can provide a basis for determining a reasonable cell inoculation amount.

[0116] Further, refer to Figure 6 FIG. 4 is a schematic flow chart of a method for culturing cells using a controllably dissolvable surface-modified microcarrier according to another embodiment of the present invention, wherein S501 may also include but is not limited to S601 to S603.

[0117] S601. Wash the target microcarrier once or multiple times using phosphate buffer.

[0118] Among them, phosphate buffered saline (PBS) is a commonly used physiological saline solution used in the cell culture process. The prepared microcarriers are washed with PBS once or multiple times to remove residual substances and impurities on the surface and inside of the microcarriers, preparing for subsequent counting. PBS can also maintain the isotonic environment of the microcarriers to avoid damage to the microcarriers.

[0119] S602, adding trypsin to decompose the matrix between the target microcarriers.

[0120] It is understandable that microcarriers may form some aggregates due to cross-linking and other reasons during the preparation process. Adding a protease enzyme such as trypsin can hydrolyze and decompose the matrix structure between these aggregated microcarriers, so that the microcarriers are completely dispersed into single particles, avoiding counting errors and ensuring counting accuracy.

[0121] S603, counting the target microcarriers using a blood cell counting plate to obtain the number of target microcarriers.

[0122] Among them, the blood cell counting plate is a standard cell / particle counting tool. The dispersed microcarrier suspension processed in the first two steps is added to the counting area of ​​the counting plate, and the clearly visible single microcarrier particles are counted under a microscope to calculate the actual number of microcarriers.

[0123] By introducing S601-S603, microcarriers can be cleaned, dispersed and counted efficiently to obtain accurate microcarrier number data, which is the prerequisite for determining the optimal cell inoculation amount. At the same time, these steps also avoid problems such as microcarrier agglomeration during subsequent culture.

[0124] S502, determining the target culture cell number of the corresponding target culture cells according to the target microcarrier number.

[0125] It is understandable that the number of microcarriers determines the available cell growth area, so generally the more microcarriers there are, the more cells that can be inoculated. Based on the actual number of microcarriers and the expected composite ratio of cells to microcarriers (such as 1:10, 1:20, etc.), the target number of cells to be inoculated can be calculated, and a reasonable cell inoculation amount can be determined, so that the growth area provided by the microcarriers can be utilized to the maximum extent to avoid excess or insufficient materials.

[0126] Through S501 and S502, the entire cell culture process is made more controllable and efficient, and the advantages of microcarriers can be maximized to achieve high-density and high-yield cell preparation.

[0127] S303, allowing the target culture cell culture medium to settle for a certain period of time, removing the supernatant waste culture medium, and then adding a target microcarrier dissolving agent corresponding to the target microcarrier.

[0128] It is understandable that after the cell culture is completed, the complex of cells and microcarriers can be dissolved by adding a specific solvent to achieve separation of cells and microcarriers. The mixed volume ratio of the target microcarrier and the target microcarrier solvent can be 1:10. Among them, different types of microcarriers need to be dissolved using different solvents due to differences in materials and cross-linking methods. For example, for microcarriers after gelatin cross-linking, proteases (such as trypsin) can be used as solvents; for sodium alginate microspheres, metal ion chelators such as EDTA or sodium citrate can be used as solvents, and the concentration of the chelator is 4mM / L to 20mM / L, and the concentration of the trypsin solution is 0.001% to 0.5%. In practical applications, the type and concentration of the solvent can be reasonably selected according to the characteristics of the specific microcarriers.

[0129] Figure 7 As shown in FIG. 7 , an experimental diagram of using a solvent to dissolve a controllably soluble surface-modified microcarrier provided by an embodiment of the present invention is shown. The corresponding solvent is added to the target culture cell culture medium obtained in step S302 in a certain proportion. After the solvent is added, the original complex formed by the microcarrier and the cell will gradually be dissolved and separated, releasing the target culture cells. In some embodiments, the microcarrier can be completely dissolved after adding the solvent for about 3 minutes.

[0130] Furthermore, in some embodiments, before S303, the following steps may be included:

[0131] An inverted optical microscope is used to obtain the confluence of the target cultured cells proliferating on the surface of the target microcarriers;

[0132] When the confluence exceeds a preset confluence threshold, it is determined that the proliferation of the target cultured cells is complete.

[0133] It is understood that confluence refers to the coverage or density of cells on the surface of the matrix. The cell growth on the surface of the microcarrier can be observed and photographed by an inverted optical microscope, and then the confluence density of cells on the surface of the microcarrier can be evaluated and calculated by using image analysis software or manual observation. When the confluence exceeds the preset confluence threshold, it is determined that the proliferation of the target cultured cells is complete. A reasonable confluence threshold can be set in advance, such as 70%-90%. When the calculated actual confluence exceeds this threshold, it is determined that the cells have reached a sufficiently dense proliferation state on the microcarrier. At this time, the number of cells has maximized the use of the growth area provided by the microcarrier, and the efficiency of continued culture will be greatly reduced. Therefore, once it is confirmed that this threshold has been reached, it can be decided to stop continuing the culture and proceed to the subsequent cell collection step.

[0134] By monitoring and judging the confluence density of cells, the best time for cell harvesting can be accurately controlled to obtain high-purity and high-activity target cultured cells.

[0135] Exemplarily, Vero and MSCs cells can be cultured on a large scale using 24-well plates statically and magnetic suspension stirring bottles to test the performance of surface-modified microspheres on adherent cell culture. After the cells and microcarriers are cultured for a certain period of time, the cell confluence on the microspheres is observed under a microscope to reach 90%. Live cells are counted with a hemocytometer. The cell proliferation multiple is calculated as the ratio of Xdaya / Xi, where Xdaya is the number of live cells attached to the microcarrier on day a.

[0136] Furthermore, after the proliferation is completed, the target cultured cells can be counted by a blood cell counting plate to determine the proliferation multiple of the cells, wherein the proliferation multiple = the number of live cells obtained by this counting / the number of cells initially inoculated. For example, the counting result is 1.2x10 7 Live cells, initial seeding 2x10 6 cells, then the proliferation multiple = 1.2x10 7 ÷2x10 6 =6 times.

[0137] Furthermore, by setting gelatin solutions of different concentrations, it can be found that as the gelatin concentration increases, the cell culture expansion multiple of the microcarrier increases first and then decreases. At a gelatin concentration of 8%, the cell culture reaches the maximum expansion multiple. For example, gelatin solutions of different concentrations (such as 4%, 6%, 8%, 10%, etc.) are prepared, and a variety of microcarriers are prepared using these different gelatin concentrations. Cells are cultured using these microcarriers, and cells are harvested when a preset confluence threshold is reached. The proliferation multiples of each group of cells are accurately counted by a blood cell counting plate or the like. It is found that as the gelatin concentration increases, the cell proliferation multiple does not continue to increase, but presents a trend curve of first increasing and then decreasing. At a gelatin concentration of 8%, the microcarrier's support performance for cells reaches the best, and the cell proliferation multiple reaches a peak value.

[0138] In some specific implementations, the cell proliferation is as follows: after culturing Vero cells for 4 days, the maximum expansion times reached is 9.42. After culturing MSCs cells for 6 days, the maximum expansion times reached is 5.80.

[0139] Furthermore, the target cultured cells can be stained with calcein and propidium iodide; wherein the target cultured cells include living cells and dead cells, the living cells are stained with calcein and exhibit green fluorescence under microscopic observation, and the dead cells are stained with propidium iodide and exhibit red fluorescence under microscopic observation.

[0140] It is understandable that Calcein-AM is a cell activity fluorescent probe that can be hydrolyzed by esterases in living cells to produce green fluorescence, while Propidium Iodide is a nucleic acid dye that can enter the cell nucleus and bind to DNA to emit red fluorescence only after the cell membrane of dead cells is destroyed. Figure 8 or Fig. 9 As shown, Figure 8 This is a staining experiment diagram of live cells and dead cells of Vero cells provided in one embodiment of the present invention. Fig. 9 The staining experiment diagram of live cells and dead cells of MSCs cells provided in one embodiment of the present invention is observed under an inverted fluorescence microscope. Calcein-AM is used to stain live cells, showing green fluorescence, and propidium iodide PI is used to stain dead cells, showing red fluorescence. It is observed that live cells with green fluorescence can be clearly observed around the microcarrier, and the proportion of dead cells with red fluorescence is very small, indicating that when Vero cells or MSCs cells are co-cultured with surface-modified microspheres, the cell activity is high, and the surface-modified microspheres can support cell adhesion, survival, growth and proliferation. Among them, Vero cells are a continuous aneuploid cell widely used in viral vaccines and viral vector research, and continuous cell lines can go through many division cycles without aging. Aneuploidy refers to the difference between the number of chromosomes in the cell and the usual number; MSCs refer to pluripotent stem cells (Mesenchymal Stem Cells), which are a type of adult stem cells derived from various tissues of the human body (such as bone marrow, fat, etc.), with multidirectional differentiation potential, and can differentiate into various cell types such as skeletal muscle, cartilage, and fat.

[0141] S304, after complete dissolution, complete culture medium is added to terminate the trypsin reaction and the culture medium of the target cultured cells after the target microcarriers are dissolved is centrifuged to separate the proliferated target cultured cells.

[0142] It is understandable that centrifugal force can be used to separate cell particles in the liquid from the solution. The cell particles have a density greater than that of the solution and will settle at the bottom of the centrifuge tube under high-speed centrifugation. The mixed liquid containing the target cultured cells obtained in S303 is transferred to a centrifuge tube. After centrifugation at an appropriate centrifugal speed (e.g., 1000-1500 rpm) and a centrifugal time (e.g., 5-10 minutes), a layer of target cultured cell particle precipitation will gather at the bottom of the tube. Carefully remove the upper clear liquid, leaving only the cell precipitation part at the bottom of the tube, and resuspend the cells with sterile PBS or other suitable buffer. If further purification is required, the cells can be washed by centrifugation several times. After each centrifugation, the supernatant is discarded and the cells are resuspended with fresh buffer.

[0143] Through the physical separation method of centrifugation, pure and active target cells can be efficiently separated from the mixed solution containing dissolved microcarrier residues. This not only removes the influence of microcarriers, but also largely removes the contamination of dead cells and other impurities, thereby obtaining a high-purity target cell product.

[0144] In some embodiments, after step 304, the method further includes counting the proliferated target cultured cells to obtain the number of proliferated target cultured cells and harvesting the cells, specifically including:

[0145] After the proliferation, the target culture medium containing cells and microcarriers removes the supernatant and discards the culture medium, and then washes the target microcarriers once or multiple times with a phosphate buffer;

[0146] adding a dissolving agent to decompose the matrix between cells of the target microcarrier and completely dissolve the target microcarrier;

[0147] Complete medium was added to terminate the reaction, and the cells were resuspended after centrifugation;

[0148] The proliferated target culture cells are counted using a blood cell counting plate to obtain the number of proliferated target culture cells, the proliferation multiples, and the harvested cells.

[0149] It is understandable that after completing the large-scale proliferation culture of target cells on soluble microcarriers, it is necessary to separate and purify the highly active target cells from the mixed system. First, the culture liquid containing a large number of cells and microcarriers is centrifuged for a short time, and the supernatant is discarded, leaving only the cell / microcarrier mixed precipitate at the bottom. Then, the precipitate is resuspended and washed with a non-cytotoxic buffer such as PBS to remove the residual culture medium and metabolites, which is conducive to the subsequent reaction. Next, it is necessary to add specific chemical reagents as dissolving agents, such as proteases or EDTA, to degrade the microcarrier matrix structure, so that the cells attached to the microcarrier are separated from the microcarrier. In order to prevent the dissolving agent from causing damage to the cells, it is necessary to immediately add fresh complete culture medium to terminate the reaction and provide nutrition to the cells. After another centrifugation, the cells are separated from the dissolved liquid microcarrier components and precipitated at the bottom. The supernatant is discarded and the purified cell precipitate is resuspended with an appropriate amount of culture medium. Finally, the cells are counted using standard methods such as a hemocytometer to accurately obtain the final number of target cells after proliferation culture. By comparing with the number of cells initially inoculated, the actual cell proliferation multiple can be calculated.

[0150] Embodiment 3:

[0151] like Fig.10, which is a schematic diagram of the structure of an electronic device for implementing a method for preparing a surface-modified microcarrier with controllable dissolution or a method for culturing cells of a surface-modified microcarrier with controllable dissolution provided by an embodiment of the present invention.

[0152] The electronic device 1 may include a processor 10, a memory 11, a bus 12 and a communication interface 13, and may also include a computer program stored in the memory 11 and executable on the processor 10, such as a preparation program for controllably dissolving surface-modified microcarriers or a cell culture program for controllably dissolving surface-modified microcarriers.

[0153] Wherein, the memory 11 includes at least one type of readable storage medium, and the readable storage medium includes flash memory, mobile hard disk, multimedia card, card-type memory (for example: SD or DX memory, etc.), magnetic memory, disk, optical disk, etc. The memory 11 may be an internal storage unit of the electronic device 1 in some embodiments, such as a mobile hard disk of the electronic device 1. The memory 11 may also be an external storage device of the electronic device 1 in other embodiments, such as a plug-in mobile hard disk, a smart memory card (Smart Media Card, SMC), a secure digital (Secure Digital, SD) card, a flash card (Flash Card), etc. equipped on the electronic device 1. Further, the memory 11 may also include both an internal storage unit of the electronic device 1 and an external storage device. The memory 11 may not only be used to store application software and various types of data installed in the electronic device 1, such as the preparation program of the controllable dissolution surface-modified microcarrier or the code of the cell culture program of the controllable dissolution surface-modified microcarrier, but also may be used to temporarily store data that has been output or is to be output.

[0154] The processor 10 may be composed of an integrated circuit in some embodiments, for example, a single packaged integrated circuit, or a plurality of packaged integrated circuits with the same or different functions, including one or more central processing units (CPUs), microprocessors, digital processing chips, graphics processors, and combinations of various control chips, etc. The processor 10 is the control core (Control Unit) of the electronic device, and uses various interfaces and lines to connect various components of the entire electronic device, and executes or executes programs or modules stored in the memory 11 (such as a preparation program for controllably dissolving surface-modified microcarriers or a cell culture program for controllably dissolving surface-modified microcarriers, etc.), and calls data stored in the memory 11 to execute various functions of the electronic device 1 and process data.

[0155] The bus may be a peripheral component interconnect (PCI) bus or an extended industry standard architecture (EISA) bus, etc. The bus may be divided into an address bus, a data bus, a control bus, etc. The bus is configured to realize connection and communication between the memory 11 and at least one processor 10, etc.

[0156] Figure 2 Only an electronic device with components is shown, and those skilled in the art will understand that Figure 2 The structure shown does not constitute a limitation on the electronic device 1, and may include fewer or more components than shown in the figure, or combine certain components, or arrange the components differently.

[0157] For example, although not shown, the electronic device 1 may also include a power source (such as a battery) for supplying power to each component. Preferably, the power source may be logically connected to the at least one processor 10 through a power management device, so that the power management device can realize functions such as charging management, discharging management, and power consumption management. The power source may also include any components such as one or more DC or AC power sources, recharging devices, power failure detection circuits, power converters or inverters, power status indicators, etc. The electronic device 1 may also include a variety of sensors, Bluetooth modules, Wi-Fi modules, etc., which will not be repeated here.

[0158] Furthermore, the electronic device 1 may also include a network interface. Optionally, the network interface may include a wired interface and / or a wireless interface (such as a WI-FI interface, a Bluetooth interface, etc.), which is generally used to establish a communication connection between the electronic device 1 and other electronic devices.

[0159] Optionally, the electronic device 1 may further include a user interface, which may be a display, an input unit (such as a keyboard), or a standard wired interface or a wireless interface. Optionally, in some embodiments, the display may be an LED display, a liquid crystal display, a touch-sensitive liquid crystal display, and an OLED (Organic Light-Emitting Diode) touch device. The display may also be appropriately referred to as a display screen or a display unit, which is used to display information processed in the electronic device 1 and to display a visual user interface.

[0160] It should be understood that the embodiment is for illustration only and the scope of the patent application is not limited to this structure.

[0161] The preparation program of the controllably dissolvable surface-modified microcarrier or the cell culture program of the controllably dissolvable surface-modified microcarrier stored in the memory 11 of the electronic device 1 is a combination of multiple instructions. When running in the processor 10, the specific implementation method of the processor 10 for the above instructions can refer to Figures 1 to 6 The description of the relevant steps in the corresponding embodiments will not be repeated here.

[0162] Furthermore, if the module / unit integrated in the electronic device 1 is implemented in the form of a software functional unit and sold or used as an independent product, it can be stored in a computer-readable storage medium. The computer-readable storage medium can be volatile or non-volatile. For example, the computer-readable medium can include: any entity or device capable of carrying the computer program code, a recording medium, a USB flash drive, a mobile hard disk, a magnetic disk, an optical disk, a computer memory, and a read-only memory (ROM).

[0163] The present invention also provides a computer-readable storage medium, wherein the computer-readable storage medium stores a computer program, and when the computer program is executed by a processor of an electronic device, the computer program can implement the following Figures 1 to 6 The relevant steps in the corresponding embodiments are not described in detail here.

[0164] The modules described as separate components may or may not be physically separated, and the components shown as modules may or may not be physical units, that is, they may be located in one place or distributed on multiple network units. Some or all of the modules may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0165] In addition, each functional module in each embodiment of the present invention may be integrated into one processing unit, each unit may exist physically separately, or two or more units may be integrated into one unit. The above-mentioned integrated unit may be implemented in the form of hardware or in the form of hardware plus software functional modules.

[0166] It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention.

[0167] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing a controllably dissolvable surface-modified microcarrier, characterized in that: The method comprises: Based on a predetermined solution concentration, a gelatin solution is prepared; Obtaining a target microcarrier, and immersing the target microcarrier in the gelatin solution to obtain a first mixed solution; wherein the matrix of the target microcarrier is pectin and sodium alginate; Performing a first constant temperature shaking treatment on the first mixed solution; wherein the time of the first constant temperature shaking treatment is 1 to 5 hours; performing sedimentation treatment on the first mixed solution after the first constant temperature shaking treatment, and removing the supernatant to obtain the target microcarrier modified with gelatin; Obtaining a crosslinking agent solution, and immersing the target microcarrier modified with gelatin in the crosslinking agent solution to obtain a second mixed solution; wherein the crosslinking agent is a genipin solution or a glutaraldehyde solution; Performing a second constant temperature shaking treatment on the second mixed solution; wherein the time of the first constant temperature shaking treatment is 1 to 7 hours; The second mixed solution after the second constant temperature shaking treatment is subjected to sedimentation treatment, and the supernatant is removed to obtain the target microcarrier after gelatin cross-linking.

2. The method for preparing the controllably dissolvable surface-modified microcarrier according to claim 1, characterized in that: The step of preparing a gelatin solution based on a preset solution concentration comprises: Weighing a corresponding weight of gelatin according to a preset solution concentration; wherein the preset solution concentration ranges from 1% to 16%; Gelatin is added into deionized water, and stirred in a 60° C. water bath until completely dissolved, to obtain the gelatin solution.

3. The method for preparing the controllably dissolvable surface-modified microcarrier according to claim 1, characterized in that: The method further comprises: Using ultrapure water to wash the gelatin-modified target microcarrier once or multiple times to remove unreacted gelatin; or, Using ultrapure water to wash the target microcarrier after gelatin cross-linking once or multiple times to remove unreacted cross-linking agent; The target microcarrier has a blue or transparent spherical or spherical appearance; The diameter of the target microcarrier is 10-1000 μm; The density of the target microcarrier is 1.02-1.05 g / cm 3 .

4. A cell culture method for controllably dissolving surface-modified microcarriers, characterized in that: The controllably dissolvable surface-modified microcarrier used in the cell culture method is prepared using the preparation method described in any one of claims 1 to 3, and the cell culture method comprises: Obtaining target microcarriers and target cultured cells, and pretreating the target microcarriers; Inoculating the target cultured cells into the pretreated target microcarriers for proliferation, to obtain a target cultured cell culture medium containing the target cultured cells and the target microcarriers; The target culture cell culture medium is allowed to settle for a certain period of time, and after removing the supernatant waste culture medium, a target microcarrier dissolving agent corresponding to the target microcarrier is added; wherein the mixing volume ratio of the target microcarrier to the target microcarrier dissolving agent is 1:5-10, and the dissolution time is 30s to 5min; After complete dissolution, complete culture medium is added to terminate the trypsin reaction and the culture medium of the target cultured cells after the target microcarriers are dissolved is centrifuged to separate the proliferated target cultured cells.

5. The cell culture method of the controllably dissolvable surface-modified microcarrier according to claim 4, characterized in that: The target microcarrier is composed of pectin and sodium alginate as a matrix and gelatin as a surface modification. The target microcarrier dissolving agent is a mixed solution of trypsin solution and chelating agent, wherein the chelating agent is an EDTA solution or a sodium citrate solution, and the concentration of the chelating agent is 4mM / L to 20mM / L. M / L, the concentration of the trypsin solution is 0.001% to 0.5%, trypsin is used to hydrolyze gelatin, and the chelating agent is used to chelate calcium ions to dissolve the target microcarrier and completely separate it from the proliferated target cultured cells.

6. The cell culture method of the controllably dissolvable surface-modified microcarrier according to claim 4, characterized in that: The pretreatment of the target microcarrier comprises: Sterilize the target microcarrier using 75% alcohol; Wash the sterilized target microcarrier once or multiple times using sterile water; The washed target microcarriers are added to complete culture medium for incubation.

7. The cell culture method of the controllably dissolvable surface-modified microcarrier according to claim 4, characterized in that: Before inoculating the target cultured cells into the pretreated target microcarriers for proliferation, the method further comprises: Counting the target cultured cells and the target microcarriers to obtain the number of the target cultured cells and the target microcarriers; The target culture cell number of the target culture cells corresponding to the target microcarrier number is determined, and the ratio of the number of the target microcarriers to the number of the target culture cells is 1:10-20.

8. The cell culture method of the controllably dissolvable surface-modified microcarrier according to claim 4, characterized in that: After the target culture cell culture medium is allowed to settle for a certain period of time, the supernatant waste culture medium is removed, and before the target microcarrier dissolving agent corresponding to the target microcarrier is added, the method further comprises: Using an inverted optical microscope to obtain the confluence of the target cultured cells proliferating on the surface of the target microcarrier; When the confluence exceeds a preset confluence threshold, it is determined that the proliferation of the target cultured cells is complete.

9. The cell culture method of controlled dissolution surface modified microcarriers according to claim 4, characterized in that: The cell culture method further comprises: The target cultured cells are stained with calcein and propidium iodide; wherein the target cultured cells include living cells and dead cells, the living cells are stained with calcein and exhibit green fluorescence under microscopic observation, and the dead cells are stained with propidium iodide and exhibit red fluorescence under microscopic observation.

10. The cell culture method of controlled dissolution surface modified microcarriers according to claim 4, characterized in that: After separating the proliferated target cultured cells, the method further includes counting the proliferated target cultured cells to obtain the number of the proliferated target cultured cells and harvesting the cells, which specifically includes: After removing the supernatant and discarded culture medium from the target culture medium containing cells and microcarriers after proliferation, the target microcarriers are washed once or multiple times using a phosphate buffer; Adding a dissolving agent to decompose the matrix between cells of the target microcarrier and completely dissolve the target microcarrier; Complete medium was added to terminate the reaction, and the cells were resuspended after centrifugation; The proliferated target cultured cells are counted by a blood cell counting plate to obtain the number of the proliferated target cultured cells, the proliferation multiples, and the harvested cells.

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