Three-dimensional culture method for large-scale preparation of stem cells

Abstract

To provide a three-dimensional (3D) culture method for large-scale preparation of stem cells.SOLUTION: Provided is a method for cell attachment culture on a three-dimensional microcarrier for seeding cells on the three-dimensional microcarrier, the method for cell attachment culture includes: (B1) cell seeding: a step of mixing a cell suspension with a dry three-dimensional microcarrier to obtain a microcarrier mixed with the cell suspension; (B2) cell attachment: a step of incubating the microcarrier mixed with the cell suspension obtained in step (B1) so that the cells attach to the three-dimensional microcarrier; and (B3) cell culture: a step of adding a complete culture medium and carrying out culture after the cell attachment in step (B2). Furthermore, a set of a method for harvesting cells on a three-dimensional microcarrier for separating cells from the three-dimensional microcarrier and a method for cell culture based on a three-dimensional microcarrier is also provided.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the field of cell culture, in particular to a three-dimensional (3D) culture method for the large-scale preparation of stem cells. [Background technology]

[0002] Stem cells are a type of pluripotent cell with the ability to self-renew, and after induction under appropriate conditions, they can differentiate into various functional cells, such as nerve cells, cardiomyocytes, vascular endothelial cells, kidney cells, and hepatocytes. With the development of stem cell research, the medical value of stem cells is immeasurable, and the possibility of clinical applications has a great impact on the development of medicine. Several studies have proven that stem cells, as well as their secretions, play a role in the treatment of various diseases, such as tumors, spinal cord injury, multiple sclerosis, stroke, amyotrophic lateral sclerosis, Alzheimer's disease, osteoarthritis, femoral head necrosis, intervertebral disc degeneration, myocardial infarction, liver cirrhosis, Crohn's disease, interstitial lung disease, systemic lupus erythematosus, erectile dysfunction, and premature ovarian failure. Stem cells that can be used in regenerative medicine include, but are not limited to, mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), adipocyte stem cells (ADSCs), neural stem cells (NSCs), induced pluripotent stem cells (iPSCs), etc., among which MSCs are the most clinically used.

[0003] Stem cells are considered a "drug" for management and approval in many countries around the world. Currently, there are 14 types of stem cell therapy products available internationally, but none have been approved in China yet. However, since 2018, stem cell drugs in China have been regulated by the Drug Supervision and Administration, and the National Medical Products Administration of the People's Republic of China (NMPA) has approved some stem cell drugs, three of which have been approved to participate in clinical trials through IND (clinical trial application). At the same time, 51 stem cell clinical studies have been submitted to the National Health Commission of the People's Republic of China and are allowed to conduct clinical research. These figures mean that China will enter a rapid development stage of stem cell drug development and transformation.

[0004] Stem cells and cell products derived from them have long been considered difficult to be made into drugs due to their special properties such as heterogeneity and changes in activity. Compared with traditional "chemical drugs" and "proteins, nucleic acids" and other biological drugs, the production and preparation of new cell drugs is more complex and difficult. However, currently, the manufacturing processes of both stem cell products commercially available in foreign countries and stem cell products for domestic clinical trials or research still rely on the traditional two-dimensional preparation process of artificial culture. This preparation method relies on a large number of manual operations, cumbersome steps, and excessively labor-intensive open-end processes, which make it difficult to standardize the operation and produce the large amounts of cells required to meet the needs of various cell therapies. In addition to the high cost and large area of ​​production space required, the limited number of batches of cell products produced, and the safety issues caused by the unstable quality of stem cells per batch due to uncertainties and uncontrollable factors in each process all question the possibility of stem cells becoming drugs for large-scale application. Therefore, in the future, if cells are to be formulated into marketable commercial products, the scale will be 10 11 In some cases, a large number of cells may be required. In particular, when a cell product requires multiple high doses to achieve a therapeutic effect, such amounts cannot be provided consistently and timely by existing manufacturing processes. Therefore, as a key factor for promoting the stem cell therapy industry and enabling clinical grade stem cell preparation, there is an urgent need to establish a method that can simplify operations, reduce manual operations, and enable closed or semi-closed large-scale culture of stem cells. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention provides a three-dimensional culture method for the large-scale preparation of stem cells. [Means for solving the problem]

[0006] The present invention provides a set of three-dimensional microcarrier-based cell culture methods, including a three-dimensional microcarrier-based cell resuscitation method, a three-dimensional microcarrier-based in situ passaging method, and a three-dimensional microcarrier-based in situ cell cryopreservation method. The three-dimensional microcarrier-based cell resuscitation method comprises the steps of: inoculating a cell suspension of cryopreserved cells or cryopreserved cell microtissues after thawing onto the three-dimensional microcarriers for cell resuscitation and culture; The in situ passaging method based on three-dimensional microcarrier cell culture comprises the following steps: inserting a seed microcarrier into a new microcarrier; the seed microcarrier is the microcarrier that is cultured with the cells to be passaged; The in situ cell cryopreservation method based on three-dimensional microcarriers includes the following steps: (A1) centrifuging the three-dimensional microcarrier suspension of cells and discarding the supernatant to obtain cell-containing carriers; (A2) mixing the cell-containing carriers obtained in step (A1) with a cryopreservation solution and adding them to a cryogenic storage tube; (A3) cooling the cryogenic storage tube to which the cell-containing carriers were added in step (A2) and then transferring it to liquid nitrogen, allowing in situ cryopreservation of the cells on the three-dimensional microcarriers.

[0007] The method further includes a method of cell attachment culture on three-dimensional microcarriers, a method of harvesting cells on the three-dimensional microcarriers, and a method of sampling cells cultured on the microcarriers, where the method of cell attachment culture on three-dimensional microcarriers is used to seed cells on the three-dimensional microcarriers, the method of harvesting cells on the three-dimensional microcarriers is used to separate cells from the three-dimensional microcarriers, and the method of sampling cells cultured on the microcarriers is used to perform calculations of the total number and / or cell density of cells cultured on the microcarriers.

[0008] The method for cell attachment culture on three-dimensional microcarriers includes the following steps: (B1) cell inoculation: mixing a cell suspension with dry three-dimensional microcarriers to obtain microcarriers mixed with a cell suspension; (B2) cell attachment: incubating the microcarriers mixed with the cell suspension obtained in step (B1) so that the cells attach to the three-dimensional microcarriers; and (B3) cell culture: adding a complete culture medium after cell attachment in step (B2) and carrying out culture.

[0009] A method for harvesting cells on three-dimensional microcarriers includes the following steps: (C1) allowing the three-dimensional microcarriers to settle or be centrifuged to which cultured cells are attached, and removing the supernatant to obtain three-dimensional microcarriers containing cells; (C2) adding a dissolution solution to the three-dimensional microcarriers containing cells, and incubating and dissolving the three-dimensional microcarriers therein; (C3) after the three-dimensional microcarriers have been completely dissolved in step (C2), adding a stop solution to terminate the dissolution, or proceeding directly to step (C4) to terminate the dissolution; and (C4) centrifuging the system obtained in step (C3), discarding the supernatant, and harvesting the cells on the three-dimensional microcarriers.

[0010] The method for sampling cells cultured on microcarriers includes the following steps: (D1) weighing a predetermined weight of microcarriers for cell culture, putting them into a sampling tube, adding liquid to simulate the cell culture process, and soaking them at 4-60 °C for 0-24 h, and then marking a scale corresponding to the volume occupied by the microcarriers on the sampling tube as a standard scale; (D2) sampling the cells cultured on the microcarriers from the cell culture system using the sampling tube to make the volume of the microcarriers into the standard scale, as follows:

[0011] In the three-dimensional microcarrier-based cell resuscitation method, the cryopreserved cell microtissue is a cell cryopreserved in a three-dimensional microcarrier. 0.1-25×10 per mg of cryopreserved microtissue 6 In one embodiment of the invention, there are 0.25×10 cells per mg of microtissue. 6 There are cells. The three-dimensional microcarrier-based cell resuscitation method is a three-dimensional microcarrier-based dynamic cell resuscitation method or a three-dimensional microcarrier-based static cell resuscitation method.

[0012] The dynamic cell resuscitation method based on three-dimensional microcarriers includes the following steps: (E1) harvesting three-dimensional microcarriers, placing them in a cell culture vessel, adding cell culture medium, and immediately treating the three-dimensional microcarriers with the next step or with a treatment method of standing or stirring for at least 1 minute; (E2) thawing cryopreserved cells or cryopreserved cell microtissues to obtain a cell suspension, inoculating the cell culture vessel containing the microcarriers prepared in step (E1) at 37°C, adding cell culture medium to adjust the ratio of microcarriers to medium to 1 mg:1-1000 μL, placing the cell culture vessel on a stirrer, and stirring in an incubator; and (E3) continuing to stir the cells after completion of step (E2) until cell resuscitation is complete.

[0013] The static cell resuscitation method based on three-dimensional microcarriers includes the following steps: (F1) preparation of static culture vessel and microcarriers: harvesting three-dimensional microcarriers and placing them in a cell culture vessel; (F2) thawing and inoculation of cryopreserved cells: harvesting cryopreserved cells or cryopreserved cell microtissues, thawing them at 37°C, and then inoculating them into the static culture vessel containing the microcarriers prepared in step (F1) for 20-240 minutes; and (F3) after completion of step (F2), continuing to culture the cells until cell resuscitation is completed.

[0014] In step (E1), after the cell culture medium is added, the mixture can be left standing for 1 to 10 minutes. In one embodiment of the present invention, the standing time is 10 minutes. In step (E1), the volume of cell culture medium added during stirring and resuscitation in step (E2) is 0-50% of the volume of the medium. In an embodiment of the present invention, the volume of cell culture medium added during stirring and resuscitation in step (E2) is 1 / 6 of the volume of the medium. In step (E2), the volume of the cell suspension after thawing is 1-100% of the volume of the medium during stirring and resuscitation in step (E2). In an embodiment of the present invention, the volume of the cell suspension is 1 / 30 of the volume of the medium during stirring and resuscitation in step (E2). The cell concentration in the cell suspension is 5-500×10 5 In one embodiment, the cell concentration in the cell suspension is 1×10 6 In cells / mL. In step (E2), the cells or cell microtissues are 5 to 100 × 10 3 The cell culture vessel containing the microcarriers is inoculated with a cell number to microcarrier ratio of cells / mg of microcarriers. In one embodiment, the ratio of cell number to new microcarriers is 10 4 Cells / mg microcarrier; the microcarrier to medium ratio is adjusted to 1 mg:300 μL by adding cell culture medium.

[0015] In step (E2), the stirring is constant speed stirring or variable speed alternating stirring or variable speed cyclic stirring. The stirring may be clockwise stirring, counterclockwise stirring, or alternating stirring. The stirring time is 0.1 to 100 hours (for example, 96 hours). The stirring speed is 1 to 200 rpm, preferably 5 to 80 rpm, and more preferably 10 to 60 rpm. Alternatively, agitation resuscitation may be performed as follows: (a) or (b) or (c): (a) Allow to stand for 1-4 hours, then stir clockwise at periodic variable speed for 1-24 hours, then stir clockwise at a constant speed for up to 96 hours; (b) Stirring clockwise at periodic variable speed for 1 to 24 hours, followed by constant clockwise stirring for up to 96 hours; (c) Stir clockwise at a constant speed for 1 to 96 hours.

[0016] In one embodiment of the invention, stirring is performed by leaving for 4 hours, then stirring clockwise at a periodic variable speed of 40 rpm for 5 minutes and 20 rpm for 20 minutes for up to 24 hours, and stirring clockwise at a constant speed (60 rpm) for up to 96 hours. In one embodiment of the invention, stirring is performed by periodic variable speed clockwise stirring of 5 minute cycles at 40 rpm and 20 minute cycles at 20 rpm for up to 24 hours, followed by constant speed (60 rpm) clockwise stirring for up to 96 hours. In one embodiment of the present invention, stirring is performed by constant (60 rpm) clockwise stirring for up to 96 hours.

[0017] In step (E3), the stirring method can refer to the previous section. In one embodiment of the present invention, the stirring is performed at a constant speed (60 rpm) in a clockwise direction. In steps (E1) to (E3), the cell culture vessel is a vessel capable of stirring and suspending the microcarriers. The cell culture vessel used in one embodiment of the present invention is a cell culture flask with built-in impeller (Belco Glass, USA; catalog number: 1965-61001).

[0018] In step (F2), the ratio of microcarriers to cell number is 5 to 100 × 10 3 In one embodiment of the invention, the cell to fresh microcarrier number ratio is 10,000 cells / mg microcarrier. In step (F2), after the cells are inoculated, cell culture medium is added and the ratio of medium to microcarriers is adjusted to 100-400 μL:20 mg. In one embodiment of the present invention, the ratio of cell culture medium to microcarriers is 200 μL:20 mg. In one embodiment of the present invention, the incubation time is 240 minutes. In the above-mentioned cell resuscitation method based on three-dimensional microcarrier, the three-dimensional microcarrier is a three-dimensional porous microcarrier.The three-dimensional porous microcarrier can be specifically 3D TableTrix microcarrier (Beijing Huaguan Biological Technology Co., Ltd.; catalog number: F01-100).

[0019] In the in situ passaging method based on three-dimensional microcarrier cell culture, the new microcarriers are microcarriers that do not contain cells. The seed microcarriers have a cell density of 10,000-1,000,000 cells / mg microcarrier (e.g., 100,000-500,000 cells / mg microcarrier, or 120,000-500,000 cells / mg microcarrier). The ratio of seed microcarriers to new microcarriers is 0.0002-200 mg seed microcarrier / mg new microcarrier (e.g., 2-15 mg seed microcarrier / 100 mg new microcarrier). Further, the seed microcarriers have a cell density of 120,000-500,000 cells / mg microcarrier (particularly suitable for static in situ passaging as described below) or 150,000-500,000 cells / mg microcarrier (particularly suitable for dynamic in situ passaging as described below). The ratio of seed microcarriers to fresh microcarriers is 1-3 mg seed microcarrier / 20 mg fresh microcarrier (particularly suitable for static in situ passaging as described below), or 2-6.7 mg seed microcarrier / 100 mg fresh microcarrier (particularly suitable for dynamic in situ passaging as described below). The three-dimensional microcarrier cell culture-based in situ passaging method is a three-dimensional microcarrier cell culture-based dynamic in situ passaging method or a three-dimensional microcarrier cell culture-based static in situ passaging method.

[0020] The dynamic in situ passaging method based on three-dimensional microcarrier cell culture comprises the following steps: (G1) Preparation step of new microcarrier suspension: Place new microcarriers in an impeller-embedded cell culture flask, add cell culture medium at a ratio of 1-2,000 μL:1 mg of microcarrier, and leave or stir for more than 0 h to obtain a new microcarrier suspension; (G2) Preparation step of seed microcarrier suspension: Seed microcarriers have a cell density of 10,000-1 million cells / mg microcarrier, and seed microcarriers are adjusted to 0.1-50 mg / mL. resuspend in cell culture medium to obtain a seed microcarrier suspension; (G3) seeding step: mix the seed microcarrier suspension prepared in (G2) into the impeller-equipped cell culture flask containing the new microcarrier suspension prepared in (G1), the ratio of seed microcarriers to new microcarriers prepared in (G1) is 0.0002-200 mg seed microcarriers / mg new microcarriers, add cell culture medium to adjust the ratio of microcarriers to medium to 1 mg:1-1000 μL; place the impeller-equipped cell culture flask on a stirrer and place in an incubator with stirring for 0-100 h; (G4) culturing step: continue stirring to complete dynamic in situ passaging;

[0021] The static in situ passaging method based on three-dimensional microcarrier cell culture includes the following steps: (H1) preparation of seed microcarrier suspension: the cell density of the seed microcarrier is 10,000-1,000,000 cells / mg microcarrier, and the seed microcarrier is resuspended in cell culture medium to 0.1-50 mg / mL to obtain a seed microcarrier suspension; (H2) inoculation: the seed microcarrier suspension prepared in (H1) is dropwise added to new microcarriers with a ratio of seed microcarriers to new microcarriers of 0.0002-200 mg seed microcarriers / mg new microcarrier, mixed well, and placed in an incubator for 0.5-24 hours to incubate; (H3) culture step: adding cell culture medium and continuing the culture, i.e., completing the static in situ passaging.

[0022] In step (G1), the ratio of new microcarriers and cell culture medium placed in the impeller-equipped cell culture flask can be 100 mg microcarriers / 10 mL cell culture medium, and after being placed in the impeller-equipped cell culture flask, the microcarriers can be left for 0.1 to 24 hours (particularly 14 hours in a specific embodiment of the present invention). In step (G2), the cell density in the seed microcarriers can be 150,000 to 500,000 cells / mg microcarrier, and the content of seed microcarriers in the seed microcarrier suspension can be 1 to 10 mg / mL (in a specific embodiment of the present invention, the content of seed microcarriers in the seed microcarrier suspension is specifically 1 mg / mL). In one embodiment of the invention, the cell density in the seed microcarriers is typically 500,000 cells / mg microcarriers and the content of seed microcarriers in the seed microcarrier suspension is typically 1 mg / mL. In one embodiment of the invention, the cell density in the seed microcarriers is typically 150,000 cells / mg microcarriers and the content of seed microcarriers in the seed microcarrier suspension is typically 1 mg / mL.

[0023] In step (G3), the ratio of seed microcarriers to new microcarriers may be 1-10 mg seed microcarriers / 100 mg new microcarriers (e.g., 2-6.7 mg seed microcarriers / 100 mg new microcarriers); the ratio of microcarriers to medium may be adjusted to 1-10 mg:1 mL (e.g., 106.7-204 mg:60 mL) by adding cell culture medium; the stirring speed may be 1 rpm-200 rpm, the stirring may be by constant speed stirring or variable speed alternating stirring or variable speed cycle stirring, and the stirring direction may be clockwise stirring, counterclockwise stirring or alternating stirring, thereby transferring the cells on the seed microcarriers to the new microcarriers. In one embodiment of the invention, the seed microcarrier to fresh microcarrier ratio is specifically 2 mg seed microcarrier / 100 mg fresh microcarrier; the microcarrier to medium ratio is specifically adjusted to 204 mg:60 mL by adding cell culture medium; and the agitation is a variable speed cycle with specific parameters specifically 60 rpm for 5 minutes and 20 rpm for 20 minutes for a total cycle of 24 hours. In one embodiment of the invention, the seed microcarrier to fresh microcarrier ratio is specifically 6.7 mg seed microcarrier / 100 mg fresh microcarrier, the microcarrier to medium ratio is specifically adjusted to 106.7 mg:60 mL by adding cell culture medium, and the agitation is specifically a variable speed cycle clockwise agitation with the specified parameters of 60 rpm for 5 minutes and 20 rpm for 20 minutes for a total cycle of 24 hours.

[0024] In step (H1), the cell density in the seed microcarriers may be 120,000-500,000 cells / mg microcarriers; the content of seed microcarriers in the seed microcarrier suspension may be 5-15 mg / mL. In one embodiment of the invention, the cell density in the seed microcarriers is typically 500,000 cells / mg microcarriers and the content of seed microcarriers in the seed microcarrier suspension is typically 5 mg / mL. In one embodiment of the invention, the cell density in the seed microcarriers is typically 120,000 cells / mg microcarriers and the content of seed microcarriers in the seed microcarrier suspension is typically 15 mg / mL. Further, in step (H2), the ratio of seed microcarriers to fresh microcarriers may be 1-3 mg seed microcarriers / 20 mg fresh microcarriers; and the incubation time may be 0-24 hours. In an embodiment of the invention, the ratio of seed microcarriers to fresh microcarriers is specifically 1 mg seed microcarriers / 20 mg fresh microcarriers; and the incubation time is specifically 2 hours. In an embodiment of the invention, the ratio of seed microcarriers to fresh microcarriers is specifically 3 mg seed microcarriers / 20 mg fresh microcarriers; and the incubation time is specifically 2 hours. Here, the total incubation time of the method may depend on the cells, the ratio of seeds and new carriers provided, the desired number of cells finally obtained, etc.

[0025] In the in situ passaging method based on three-dimensional microcarrier cell culture, the microcarrier is 3D FloTrix microcarrier (Beijing Huaguan Biotechnology Co., Ltd.; catalog number: CNF-F01T-50). The impeller-integrated cell culture flask is a product of Belco Glass Co., Ltd., USA, catalog number: 1965-61001. The stirrer is a low-speed stirrer (3D FloTrix miniSpin low-speed stirrer, Beijing Huaguan Biotechnology Co., Ltd.; catalog number: 3D FTmS-2-2).

[0026] In the in situ cell cryopreservation method based on three-dimensional microcarriers, in step (A1), centrifugation is performed at a speed of 50-1610×g, specifically 400×g, for 1-10 minutes, specifically 2 minutes. In step (A2), the ratio of the volume of the cryopreservation solution to the mass of the cell-containing carrier is 1mL:0.1-50mg, specifically 1mL:10mg or 1mL:0.1-20mg. In the three-dimensional microcarrier-based in situ cell cryopreservation method, the cryopreservation solution includes 10% DMSO, 10-90% FBS and 0-80% basal medium, 10% DMSO and 0-80% basal medium, 10-90% FBS and 0-80% basal medium, or other commercially available cell cryopreservation solutions such as CF101 Cell Regene serum-free cryopreservation solution. In the three-dimensional microcarrier-based in situ cell cryopreservation method, the cryogenic storage tube is placed in a freezer and cooled to -20 to -196°C, specifically -20 to -80°C; the cooling is programmed or unprogrammed; the programmed cooling has a cooling rate of -1 to -15°C / min; the cooling time can be 1 to 24 hours, specifically 24 hours, 10 to 24 hours, or 15 to 24 hours. In the in situ cell cryopreservation method based on three-dimensional microcarriers, cooling is carried out in step (A2) by placing the cryogenic storage tube in a cell cooling box and then in a freezer within 1 to 10 minutes. In the three-dimensional microcarrier-based in situ cell cryopreservation method, 37 °C pre-warmed PBS or basal medium added to a cryogenic storage tube is diluted in a ratio that can be 1:5 to 30. In the in situ cell cryopreservation method based on three-dimensional microcarriers, step (A3) is followed by a cell resuscitation step: the cryopreservation tube after the process of step (A3) is placed in a water bath, and the cells are resuscitated by removing the water bath when the ice in the cryopreservation tube melts, and then the cells are diluted by adding pre-warmed PBS or basal medium at 37°C, and then centrifuged. In the in situ cell cryopreservation method based on three-dimensional microcarriers, the temperature of the water bath can be 35-40°C, and the time of the water bath can be 1-5 minutes. In the in situ cell cryopreservation method based on three-dimensional microcarriers, when resuscitating cells after step (A3), the centrifugation speed can be 50-1610×g, specifically 400×g, for 1-10 minutes, specifically 2 minutes.

[0027] In the cell attachment culture method on three-dimensional microcarriers, the cell suspension was 1 × 10 4 ~1×10 8 The cell suspension has a density of 1000 μL / mL, and the cell suspension is obtained by resuspending the cells in a culture medium or a liquid biological matrix material, where the material can be specifically selected from the group consisting of collagen, gelatin, gelatin derivatives, proteoglycan, glycoprotein, alginate, alginate derivatives, agar, matrix rubber, hyaluronic acid, fibronectin or laminin. The volume ratio of the cell suspension to the mass of the three-dimensional microcarrier is 1-1000 μL:1 mg, specifically 10 μL:1 mg, 1-10 μL:1 mg, 10-15 μL:1 mg, 5-15 μL:1 mg, or 250-350 μL:1 mg. In the method for cell attachment culture on three-dimensional microcarriers, in step (B1), the cell suspension is mixed with the three-dimensional microcarriers as follows: the cell suspension is added dropwise to the three-dimensional microcarriers. In step (B1), dry three-dimensional microcarriers are used and the cell suspension is mixed directly therewith, eliminating the need to pre-swell the three-dimensional microcarriers and thus simplifying the seeding of cells into the microcarriers. In the method of cell attachment culture on three-dimensional microcarriers, the incubation conditions can be as follows: temperature of 35-40°C, specifically 37°C, 37-40°C or 35-37°C; duration of 0.5-24 hours, specifically 2 hours or 24 hours; volume percentage concentration of carbon dioxide of 5-30%, specifically 5%-10% or 5-20%. The attachment method includes gravity attachment method, swelling attachment method, stirring rotary attachment method, centrifugal method, surface acoustic wave method or magnetic attachment method. In the cell attachment culture method on a three-dimensional microcarrier, the culture conditions may be 35 to 40°C, specifically 37°C, 37 to 40°C, or 35 to 37°C, and the volume percentage concentration of carbon dioxide may be 5 to 30%, specifically 5%, 5 to 10%, or 5 to 20%. In the method for culturing cells attached to a three-dimensional microcarrier, prior to step (B1), there is a step of gas sterilizing, radiation sterilizing or UV sterilizing the three-dimensional microcarrier. In the method of cell attachment culture on three-dimensional microcarriers, the three-dimensional microcarriers are porous microcarriers, specifically 3D FloTrix microcarriers with catalog number: CNF-F01T-50; Cytopore 1 and Cytopore 2 (commercially available from GE), catalog numbers: 17-0911 and 17-1271; CultiSpher (M9418) (commercially available from Sigma, as well as Beijing Huaguan Biotechnology Co., Ltd.).

[0028] In the method of harvesting cells on three-dimensional microcarriers, the lysis solution comprises an active ingredient for dissolving the microcarriers with the elimination of cell attachment.The active ingredient is selected from at least one aqueous solution of collagenase, pepsin, hyaluronidase, dispase, neutral protease, proteinase K, matrix metalloproteinase, sodium citrate, trypsin, deoxyribonuclease, trypsin substitute, protein hydrolase, ethylenediaminetetraacetic acid, lysozyme and glutathione.In one embodiment, the formulation of the lysis solution comprises 0.1% (weight percent) collagenase, 0.1% (weight percent) ethylenediaminetetraacetic acid, and 0.05% (weight percent) trypsin. In the method of harvesting cells onto three-dimensional microcarriers, the stopping solution is selected from at least one of the following, depending on the selected composition of the lysis solution: whole medium, PBS containing 10% serum albumin or serum, trypsin inhibitor, protease inhibitor, PBS, and an ionic chelating agent. The ionic chelating agent may be, in particular, ethylenediaminetetraacetic acid.

[0029] In the method for harvesting cells on three-dimensional microcarriers, in step (C1), the centrifugation can be at a speed of 50 to 1610 × g, specifically 200 × g, for 1 to 10 minutes, specifically 2 minutes. The steps for removing the supernatant are as follows: centrifugation and pipetting of the supernatant; then add an appropriate amount of PBS, shake gently by hand for 20 to 30 seconds, pipette the supernatant, and repeat the PBS washing once. In the method of harvesting cells on a three-dimensional microcarrier, the ratio of the mass of the three-dimensional microcarrier to the volume of the dissolution solution for attaching and culturing the cells can be 1 mg:0.01-5 mL, specifically 20 mg:3 mL, 1 mg:0.15-0.5 mL or 1 mg:0.1-2 mL; incubation can be at a temperature of 4° C.-40° C., specifically 37° C., for 10 seconds-24 hours, specifically 30 minutes. In a particular embodiment, during the described incubation period, blowing is performed gently several times using a 1 mL pipette at 10 minute intervals. In the method of harvesting cells onto a three-dimensional microcarrier, the ratio of the mass of the three-dimensional microcarrier for attaching and culturing the cells to the volume of the stopping solution can be 1 mg:0.01-50 mL, specifically 20 mg:3 mL or 1 mg:0.1-25 mL. In the method for harvesting cells on three-dimensional microcarriers, in step (C4), centrifugation is performed at a speed of 50 to 1610×g, specifically 400×g, for 1 to 10 minutes, specifically 2 minutes or 2 to 8 minutes.

[0030] The method for sampling cells cultured on microcarriers for calculation of the total number of cells cultured on the microcarriers and / or the cell density includes the following steps: (D1) weighing a certain weight of microcarriers for cell culture, placing them in a sampling tube, adding liquid to simulate a cell culture process, soaking them at 4-60 °C for 0-24 h, and then marking a scale corresponding to the volume occupied by the microcarriers on the sampling tube as a standard scale; (D2) sampling the microcarriers from the cell culture system with the sampling tube to bring the volume of the microcarriers to a standard scale; (D3) counting the cells in the microcarriers collected in (D2); (D4) calculating the total number of cells in the entire cell culture system based on the weight of the microcarriers weighed in (D1), the number of cells counted in (D3), and the total weight of the microcarriers in the entire cell culture system; thus, calculating the cell density. In step (D1), the method of adding liquid to the sampling tube containing the microcarriers and immersing them at 4 to 60 °C for 0 to 24 hours is a method of complete immersion and swelling of the microcarriers. Here, the liquid added can be the same or similar in viscosity, hydrophilicity, pH, and ionic concentration as the medium of the present invention used for cell culture on the microcarriers (e.g., the medium of the present invention used for cell culture or PBS). The temperature and time required to immerse and swell the microcarriers vary depending on the microcarrier. This can be done according to the instructions of the microcarriers from various manufacturers. For example, using the microcarrier Cytodex 1 available from GE, the dried microcarriers are swelled by immersing them in PBS at room temperature for at least 3 hours at a ratio of 30 to 50 mL / g. For example, 3D FloTrix microcarriers (CNF-F01T-50) available from Beijing Huaguan Biotechnology Co., Ltd. are immersed in PBS for 0.5 to 24 hours, specifically 6 hours, at a ratio of 10 to 1000 μL:1 mg, specifically 10 μL:1 mg, 10 to 15 μL:1 mg, 5 to 15 μL:1 mg, or 250 to 350 μL:1 mg, and allowed to swell. Step (D1) may also include a further step of natural or centrifugal sedimentation (e.g., centrifugation at 1,500 rpm for 2 minutes) in order to allow the microcarriers to settle as quickly as possible after immersion in the liquid. In step (D2), sampling the microcarriers from the cell culture system using a sampling tube can be carried out according to a method including the following steps: pipetting the suspension from the cell culture system and adding it to the sampling tube, allowing the microcarriers to settle either naturally or by centrifugation (to increase the sedimentation rate, the centrifugation is at a speed of 50 to 1610 x g, specifically 200 x g for 1 to 10 minutes, specifically 2 minutes), observing whether the standard scale calibrated in step (D1) is reached, and if not, continuing to pipette the suspension until the standard scale is reached, and if it is exceeded, removing the excess microcarriers from the sampling tube. In step (D3), counting of cells in the microcarriers collected in (D2) may be performed according to a method comprising the following steps: in (D2), centrifuging the sampling tube (e.g., at 200×g for 2 minutes), discarding the supernatant, adding a cell digestion solution to the precipitate to digest the cells from the microcarriers, and then counting. In step (D4), the total number of cells in the entire cell culture system is equal to the number of cells counted in (D3) multiplied by the total weight of the microcarriers relative to the weight of the microcarriers weighed in (D1) above. In step (D4), this total number is divided by the total weight or number of microcarriers, or the total volume of the culture to obtain cell density in different units, e.g. cells / mg microcarrier, cells / microcarrier, cells / mL culture.

[0031] In the method for sampling cells cultured on a microcarrier, the microcarrier is a 3D FloTrix microcarrier (catalog number: CNF-F01T-50) available from Beijing Huaguan Biological Technology Co., Ltd. The sampling tube is specifically a 1.5 ml microcentrifuge tube. In one embodiment, the weight of the microcarrier weighed in step (D1) is 5 mg, the sampling tube is a 1.5 ml microcentrifuge tube, the standard scale is 0.1 mL scale, the weight of the microcarrier in the cell culture system is 200 mg, the initial number of cells in the culture is 2 million, the medium in the culture system has a volume of 60 mL, and sampling is performed after 72 hours of cell culture. Any of the above methods further include a three-dimensional microcarrier-based method for large-scale growth of cells, comprising the steps of: achieving large-scale growth of cells on the three-dimensional microcarriers in a stirred bioreactor.

[0032] The stirred bioreactor may include, but is not limited to, a semi-automated, fully automated, or integrated bioreactor. The stirred bioreactor may specifically be either a 3D FloTrix™ mini-spin small-scale bioreactor or a 3D FloTrix™ vivaSpin bioreactor. One bioreactor alone or two bioreactors in combination may be used. The conditions under which the cells are cultured in the bioreactor can be controlled according to the growth of the cells. For example, the incubation pH can be 6.5-7.5, preferably 7.0-7.5, more preferably 7.2, the active pumping of CO2 can be about 3-10%, preferably about 5-8%, more preferably about 5%, the temperature can be 30°C-40°C, preferably 37°C, the stirring speed can be 10-150 rpm, preferably 40-80 rpm, and the frequency of gas delivery can be 1-40 ccm, more preferably about 5-20 ccm. In one example of the present invention, cells were seeded into three-dimensional microcarriers and then placed in a 3D FloTrix™ miniSpin small-scale bioreactor for culturing for 6 days at a constant clockwise speed of 60 rpm before being transferred to a 3D FloTrix™ vivaSpin bioreactor. The process parameters set for the 3D FloTrix™ vivaSpin bioreactor were controlled as follows: active pumping of 12 ccm of 5% CO2 throughout the process, and 37°C. At T=0 hours, 800 mL of complete medium was actively pumped at a rate of 10 mL / min, at T=96 hours, 500 mL of liquid was actively pumped at a rate of 10 mL / min followed by 500 mL of fresh medium at a rate of 10 mL / min, at T=144 hours, 500 mL of liquid was actively pumped at a rate of 10 mL / min followed by 50 tablets of 3D TableTrix™ microcarriers resuspended in 500 mL of fresh medium that was actively pumped into the reactor at 10 mL / min. The agitation speed from T=0-12 hours and T=144-156 hours was variable speed with cycles of 40 rpm for 5 minutes, 20 rpm for 20 minutes, and a constant speed of 60 rpm for the remaining time.

[0033] In another embodiment of the present invention, cells were inoculated into three-dimensional microcarriers and then directly inoculated into a 3D FloTrix™ vivaSpin bioreactor. The process parameters set for the 3D FloTrix™ vivaSpin bioreactor were controlled as follows: active pumping of 10 ccm of 5% CO2 throughout the entire process, and 37°C. At T=0 hours, 460 mL of complete medium was actively pumped at a rate of 10 mL / min, followed by 500 mL of liquid actively pumped at a rate of 10 mL / min at T=48 hours. Every 24 hours thereafter, 500 mL of liquid was actively pumped at a rate of 10 mL / min, followed by 500 mL of fresh medium actively pumped into the reactor at a rate of 10 mL / min until 120 hours. After actively pumping 500 mL of liquid n at 10 mL / min at T=120 h, 40 tablets of 3D TableTrix™ microcarriers resuspended in 500 mL of fresh medium were actively pumped into the reactor at a rate of 10 mL / min. The stirring speed was a constant 60 rpm from T=0 to 120 h, variable speed with cycles of 60 rpm for 5 min and 20 rpm for 20 min from T=120 to 136 h, and a constant 60 rpm from T=136 to 240 h (after 240 h, the stirring mode was a constant 60 rpm).

[0034] Any of the above three-dimensional microcarriers may specifically refer to microcarrier beads having a porous structure, where the cells to be cultured are cultured on the three-dimensional microcarriers in a cell culture vessel (e.g., cell culture flask, well plate or bioreactor). In microcarrier culture, cells may attach to the surface of the three-dimensional microcarrier and inside the pores connected thereto, while the microcarrier may be suspended in the culture medium in the culture vessel by gentle stirring. Exemplary three-dimensional microcarriers include, but are not limited to, 3D TableTrix™ microcarriers, 3D FloTrix™ microcarriers, etc.

[0035] Any of the above cells are adherent cells, which can be specifically stem cells.Stem cells include but are not limited to mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), adipose stem cells (ADSCs), neural stem cells (NSCs), induced pluripotent stem cells (iPSCs) and cells derived from stem cells from various tissue sources.Stem cells can be specifically mesenchymal stem cells.MSCs can be specifically adipose-derived MSCs or umbilical cord MSCs. [Brief description of the drawings]

[0036] [Figure 1] 1 shows a cell culture flask with a built-in impeller used in Example 1 of the present invention. [Diagram 2] 1 shows the number of cells gradually attached onto microcarriers and the attachment efficiency by the rotary attachment method of cells and microcarriers in Example 1 of the present invention. [Diagram 3] 1 shows a graph of the results of fluorescent staining after cell attachment according to the method in Example 2 of the present invention. [Figure 4] FIG. 2 shows an image of microcarrier lysis observed under bright field microscope and live cells observed under fluorescent microscope in Example 2 of the present invention. [Diagram 5] 1 shows a comparison of the activity of cells on three-dimensional microcarriers (3D FloTrix microcarriers) harvested from the lysate in Example 2 of the present invention, compared with the activity of cells in a culture flask obtained by conventional trypsin treatment. [Figure 6] This shows that 5 mg of microcarriers achieves a scale that exactly matches the 0.1 mL scale on a 1.5 ml microcentrifuge tube in Example 3 of the present invention. [Figure 7] This shows the state of 1 mL of suspension collected with a pipette by the suspension quantitative sampling method after natural precipitation in Example 3 of the present invention. A1 to A3 show three repetitions. [Figure 8] This shows the state of 1 mL of suspension collected with a pipette by the microcarrier quantitative sampling method after natural precipitation in Example 3 of the present invention. B1 to B3 show three repetitions. [Figure 9] 1 shows the results of comparing the total number of cells obtained by the suspension quantitative sampling method and the microcarrier quantitative sampling method in Example 3 of the present invention with the actual situation. [Figure 10] 1 shows the monitoring results of three-dimensional static resuscitated cells and control cells in Example 4 of the present invention. [Figure 11] 1 shows the proliferation of three-dimensional statically resuscitated cells and control cells in Example 4 of the present invention. [Figure 12] 1 shows the monitoring results of three-dimensional static resuscitated cell microstructure in Example 4 of the present invention. [Figure 13] 1 shows the results of cell monitoring using a three-dimensional dynamic resuscitated cell method in Example 4 of the present invention. [Figure 14] 1 shows cell proliferation by the three-dimensional dynamic resuscitation cell method in Example 4 of the present invention. [Figure 15] 1 shows a comparison of six cell resuscitation methods in Example 4 of the present invention. [Figure 16] 1 shows the monitoring results of three-dimensional static resuscitated cell microstructure in Example 4 of the present invention. [Figure 17] 1 shows cell proliferation in a three-dimensional dynamic resuscitated cell microstructure in Example 4 of the present invention. [Figure 18] In Example 4 of the present invention, when adipose-derived MSCs were seeded by the static in situ passaging method, only a few microcarriers (bright spots) had cells present, but cells were present on all microcarriers by day 4 after in situ passaging. [Figure 19] In Example 5 of the present invention, it is shown that two days after umbilical cord MSCs were seeded by the static in situ passaging method, only a few microcarriers (bright spots) had many cells present, whereas four days after in situ passaging, cells were present on all microcarriers. [Figure 20]In Example 5 of the present invention, it is shown that after adipose-derived MSCs were cultured by the static in situ subculture method, the number of cells increased from 500,000 at the time of inoculation to 2.1 million, which was not significantly different (p=0.32) from the number of cells (2.4 million) obtained by the conventional subculture method after digestion. [Figure 21] In Example 5 of the present invention, it is shown that after umbilical cord MSCs were cultured by static in situ subculture method, the cell number increased from 300,000 at the time of inoculation to 1.9 million. [Figure 22] In Example 5 of the present invention, it is shown that two days after adipose-derived MSCs were seeded by the dynamic in situ passaging method, only a few microcarriers (bright spots) had many cells present, whereas five days after in situ passaging, cells were present on all microcarriers. [Figure 23] In Example 5 of the present invention, when umbilical cord MSCs were seeded by the dynamic in situ passaging method, only a few microcarriers (bright spots) had many cells present, whereas 5 days after in situ passaging, cells were present on all microcarriers. [Figure 24] In Example 5 of the present invention, we show that adipose-derived MSCs can be grown and expanded by a dynamic in situ passaging method from as few as 2 million cells on the initial seed microcarriers to 50 million after 5 days of culture. [Diagram 25] In Example 5 of the present invention, it is shown that umbilical cord MSCs can be grown and expanded by a dynamic in situ passaging method from as few as 1 million cells on the initial seed microcarriers to 15.2 million after 7 days of culture. [Figure 26] 1 shows a graph of the resuscitation results of in situ cryopreserved HEK293 cells on microcarriers after different cryopreservation times in Example 6 of the present invention. [Figure 27] 13 is a graph showing the results of the percentage of viable cells revived before and after cryopreservation of HEK293 cells on microcarriers in Example 6 of the present invention. [Figure 28]1 shows histograms of the number of HEK293 cells on 3D FloTrix microcarriers and competitive microcarriers (GE Cytodex) after cryopreservation and resuscitation in Example 6 of the present invention. [Figure 29] 1 shows a graph of the resuscitation results of in situ cryopreserved AD-MSCs on microcarriers after different cryopreservation times in Example 6 of the present invention. [Diagram 30] 1 shows a comparison of the activity of AD-MSCs before and after cryopreservation in Example 6 of the present invention. [Diagram 31] 1 shows statistics on cell numbers before and after cryopreservation and cell resuscitation rate after cryopreservation of AD-MSCs in Example 6 of the present invention. [Diagram 32] 1 shows the statistical results of cell number proliferation in the first part of Example 7 of the present invention. [Diagram 33] 1 shows the automated bioreactor (3D vivaSPIN V1, Beijing Huaguan Biotechnology Co., Ltd.) used in Part 1 of Example 7 of the present invention. [Diagram 34] 1 shows the results of cell activity in Part 1 of Example 7 of the present invention. [Diagram 35] 1 shows the statistical results of cell number proliferation in the second part of Example 7 of the present invention. [Diagram 36] 1 shows the results of cell activity in Part 2 of Example 7 of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The following embodiments / examples facilitate a better understanding of the present invention, but are not intended to limit the present invention. All experimental methods in the following embodiments / examples were routine methods unless otherwise specified. All test materials used in the following embodiments were purchased from routine biochemical reagent stores unless otherwise specified. The quantitative tests in the following embodiments / examples were set up in triplicate experiments, and the average values ​​were obtained as results. EXAMPLES

[0038] Example 1: Method for three-dimensional microcarrier cell attachment culture The microcarriers used in the examples were 3D FloTrix microcarriers purchased from Beijing Huaguan Biotechnology Co., Ltd., with a catalog number of CNF-F01T-50. I. Method of three-dimensional microcarrier cell attachment using agitator rotation attachment; 1. Microcarrier preparation: 200 mg of microcarrier powder was weighed out for ultraviolet sterilization, and then poured into a sterile cell culture flask with impeller (as shown in Figure 1), and three groups were prepared; 2. Cell preparation: Prepare adipose-derived MSC suspension in advance and add 5 × 10 6 The cells were resuspended in 60 ml of complete medium for later use, and three groups were prepared; 3. Cell inoculation: The above cell suspension was mixed into a cell culture flask with built-in impeller and thoroughly mixed with 200 mg of microcarriers; 4. Cell attachment: The cell culture flask with an impeller was placed on a slow agitator and placed in a 37°C, 5% CO2 incubator with stirring at 80 rpm to allow the cells to spin-attach to the microcarriers; 5. In-situ counting to obtain deposition rate: a. Microcarrier harvesting: Three groups of microcarriers were mixed with the cell suspension after stirring for 2, 4 and 6 hours, respectively, then collected through a 70 μm cell sieve and washed once with PBS to wash away any cells not attached to the microcarriers. The cell-containing microcarrier suspension was then centrifuged at 1500 rpm for 2 minutes and the supernatant was discarded. b. Cell counting: 200 mg of microcarriers were added to 50 mL of 0.1% crystal violet solution (0.1 g crystal violet, 2.1 g citric acid, 20 μL Tween-80, 100 ml deionized water) for 2-5 h at 37°C, and the cells were counted in a cell counting plate to obtain the attachment rate after conversion by the initial inoculum number. c. Result analysis: Figure 2 shows that as the agitation time increased, cells could gradually attach to the microcarriers by rolling attachment, and the attachment efficiency reached 67% at 2 hours of agitation, 81% at 4 hours, and 92% at 6 hours.

[0039] II. A method for three-dimensional microcarrier cell attachment using small-volume, high-density swelling attachment 1. Microcarrier preparation: 20 mg of microcarrier powder was weighed out for UV sterilization for later use; 2. Cell preparation: Prepare adipose-derived MSC suspension at 2.5 × 10 6 It was prepared at a density of cells / mL, and 200 μL was prepared for every 20 mg of three-dimensional microcarriers; 3. Cell inoculation: 200 μL of cell suspension was pipetted and dropped onto 20 mg of microcarriers, and the cell suspension was thoroughly mixed with the microcarriers; 4. Cell attachment: The microcarriers thoroughly mixed with the cell suspension were placed in a 37℃, 5% CO2 incubator and incubated for 2 hours to allow the cells to attach to the microcarriers; 5. Cell culture: After the cells attached, 3ml of complete medium was added, and the cells were incubated at 37℃ in a 5% CO2 incubator for 24 hours, then observed; 6. Control experiment: Steps 3 to 5 were adopted for the small-volume, high-density attachment method of the present invention; in addition, a control experiment group for the general attachment method (large-volume, low-density) was prepared, that is, 200 μL of cell suspension was mixed with 3 mL of complete medium, and 20 mg of microcarriers was directly added, and then observed for 24 hours; 7. Cell Observation: 1) Kit: Animal Cell Killing Viability / Cytotoxicity Assay Kit, Catalog Number: KGAF001; 2) 50-100 μL of the cell-containing three-dimensional microcarrier suspension was added to a 96-well plate. 3) The supernatant was removed as much as possible, PBS was added, washed once, and after 2 minutes, the PBS was removed as much as possible. Each well was stained by adding 100 μL of staining solution made up according to the kit's instructions. After staining for 20 to 30 minutes at room temperature and protected from light, the results of observation under a fluorescent microscope are shown in Figure 3. As can be seen from Figure 3, the live cells were stained with the staining solution and observed to emit light by fluorescence, and the luminous cells were live cells. This figure shows that by the large-volume, low-density attachment method, a small number of cells were attached to the microcarriers, mainly on the surface of the microcarriers, whereas by the small-volume, high-density attachment method, a large number of cells could be attached to the microcarriers, and the cells were not only on the surface of the microcarriers but also in the pores of the porous microcarriers, thus indicating that the small-volume, high-density attachment method of the present invention is more effective in attaching cells to the microcarriers and can better utilize the large area of ​​the porous microcarriers.

[0040] Example 2 Method for harvesting cells on three-dimensional microcarriers The microcarriers used in this example were 3D FloTrix microcarriers purchased from Beijing Huaguan Biotechnology Co., Ltd., with a catalog number of CNF-F01T-50. 1. Lysis solution formulation: 0.1% (mass%) collagenase (Sigma-Aldrich (Shanghai) Trading Co., Ltd., C0130), 0.1% (mass%) EDTA (Beijing Solar Biotechnology Co., Ltd., E1170), 0.05% (mass%) trypsin (M&C Gene Technology Co., Ltd. (Beijing), CC017); Stop solution: complete medium; 2. 20 mg of three-dimensional microcarriers with adipose-derived MSCs from the culture dish were transferred to a centrifuge tube and centrifuged at 400 × g for 2 minutes, the supernatant was removed by pipetting, an appropriate amount of PBS was added, and the mixture was gently shaken by hand for 20 to 30 seconds, the supernatant was removed by pipetting as much as possible, and the PBS washing was repeated once. 3. 3 mL of lysis solution was added and the cells were incubated at 37°C in a cell incubator for 30 minutes, during which the cells were gently blown several times with a 1 mL pipette every 10 minutes. After 4.30 minutes the microcarriers were completely dissolved and 3 mL of complete medium was added to stop the dissolution process. 5. Centrifuge at 200 x g for 5 minutes, discard the supernatant and resuspend the cells as required for subsequent applications.

[0041] 6. Cell detection methods: (1) In situ calcein-AM / PI staining of live cells: 1) Kit: Animal Cell Killing Viability / Cytotoxicity Assay Kit, Catalog Number: KGAF001; 2) 20 mg of the cell-containing 3D microcarrier suspension was added to the centrifuge tube separation tube and centrifuged at 400×g for 2 minutes; 3) Remove as much of the supernatant as possible, add PBS, wash once, remove as much PBS as possible after 2 minutes, and add 100 μL of staining solution composed according to the kit's instructions for staining. After staining for 20-30 minutes at room temperature and protected from light, the tube was centrifuged at 400 × g for 2 minutes; 4) The supernatant was discarded, 3 mL of lysis solution was added, microcarrier lysis was observed under an optical microscope, and live cells were observed under a fluorescent microscope at 0 min, 10 min, and 30 min; the results are shown in Figure 4. As seen in Figure 4, the microcarriers were observed in the bright field of an optical microscope and gradually dissolved and disappeared over time, leaving only the cells behind. Observation of the stained cells under a fluorescent microscope showed that as the lysis solution dissolved the microcarriers, the cells were separated from the microcarriers into single cells, but the number of dead cells did not increase and the cells remained active. This indicated that the lysis solution for the microcarriers did not affect the activity of the microcarriers and cells could be resuscitated from the microcarriers.

[0042] (2) Analysis method for determining apoptosis rate by flow cytometry: 1) Cell harvesting: 20 mg of three-dimensional microcarrier suspension containing cells was centrifuged at 400×g for 2 min and the supernatant was discarded. 3 mL of 0.1% collagenase type IV solution (1 g collagenase type IV (Gibco, 17104019) dissolved in 1 L PBS) was added, the solution was incubated at 37°C for 30 min in a cell incubator, centrifuged at 400×g for 5 min and the supernatant was discarded. For harvesting of two-dimensional (2D) cells, a conventional trypsin digestion method was used, i.e., the medium in the cell culture flask was discarded, an appropriate amount of PBS was added, washed twice, an appropriate amount of 0.25% trypsin solution was added and the cells were incubated at 37°C for 2 min or until detached from the flask. After stopping by adding an equal amount of complete medium, the suspension was removed into a centrifuge tube and centrifuged at 400×g for 5 min and the supernatant was discarded. 2) Cell washing: The cells were resuspended once with PBS pre-cooled at 4° C. and centrifuged at 400×g for 5 minutes, and then the cells were washed. 3) Cell staining: Cells were stained using Annexin V-FITC / PI Apoptosis Detection Kit (HaiGene Biotech Co., Ltd, S0185), i.e., add 300 μL binding buffer, followed by 5 μL Annexin V-FITC, mix, and incubate at room temperature for 15 min, protected from light; then add 5 μL PI and stain for 5 min; replenish with 200 μL binding buffer. 4) Cell flow cytometry: Flow cytometry of stained cells was performed using a BD FACSAria II flow cytometer and cell sorting analysis reports were generated by the instrument.

[0043] 5) Interpretation of Cell Flow Cytometry Reports: A) Annexin-V negative-PI negative represents normal cells, i.e. the bottom left group in the 4-frame cell classification diagram, and the percentage of cells is the percentage of live cells. B) Annexin-V positive-PI negative represents cells in the early stage of apoptosis, i.e., the lower right group in the four-frame cell classification diagram, and the percentage of cells is the percentage of cells in the early stage of apoptosis. C) Annexin-V positive-PI positive represents cells in the late stages of apoptosis or necrosis, i.e., the top right group of the 4-frame cell classification diagram, and the percentage of cells is the percentage of cells in the late stages of apoptosis. As can be seen from the results in Figure 5, the activity of cells collected on three-dimensional microcarriers (3D FloTrix microcarriers) by the lysis solution was similar to or slightly higher than that of cells obtained in culture flasks by conventional trypsin digestion, demonstrating that the present invention is effective in collecting cells with high activity by targeting the microcarriers with the lysis solution, thereby collecting the cells on the microcarriers.

[0044] Example 3 Method for sampling and counting cells cultured on microcarriers The microcarriers used in this example were 3D FloTrix microcarriers purchased from Beijing Huaguan Biotechnology Co., Ltd., with a catalog number of CNF-F01T-50. The cells involved in this example were mesenchymal stem cells. Design concept: In this invention, the standard scale is calibrated by measuring the volume of a given weight of microcarrier. When using microcarriers for cell culture, the volume of microcarriers (independent of the volume of culture medium) needs to be harvested to reach the standard scale, and then the cells are counted and converted to the whole culture system to obtain the total number of cells, which is called the microcarrier quantitative harvesting method.

[0045] Examples of specific protocols include: 1. 5 mg of microcarriers were added to a 1.5 mL sampling tube (microcentrifuge tube), dissolved in 1 mL of PBS, fully soaked and swollen, and then centrifuged at 1500 rpm for 2 minutes to calibrate the volume occupied by the microcarriers in the sampling tube. In this example, it was found that 5 mg of microcarriers were precipitated to a scale that exactly matched the 0.1 mL scale on the 1.5 mL sampling tube (Figure 6). 2. After resuspending 2 million cells in 60 ml of medium, 200 mg of microcarriers were added and incubated at 37° C. in a 5% CO2 incubator. 3. After 72 hours, two sampling methods were used: (a) The microcarriers were suspended as uniformly as possible in 60 mL of medium by shaking the culture vessel, and 1 mL of the suspension was pipetted as quickly as possible and added to a 1.5 mL sampling tube. A total of three tubes were sampled with a pipette (see Figure 7). This was the suspension quantitative sampling method. (b) The microcarriers were suspended in the medium by shaking the culture vessel, and the suspension was pipetted into a 1.5 mL sampling tube. After waiting a few minutes for the microcarriers to settle, it was observed whether the volume of the microcarriers reached the standard scale calibrated in step 1, and in this example, a standard scale of 0.1 mL was used (corresponding to 5 mg of microcarriers, FIG. 6). If not, the suspension was continued to be pipetted until the scale was reached, and if exceeded, the excess microcarriers were removed from the sampling tube. A total of three tubes were sampled with a pipette (see FIG. 8). This was the microcarrier quantitative sampling method. 4. The above sampling tube was centrifuged at 1500 rpm for 2 minutes and the supernatant was discarded. The cells were digested from the microcarriers by adding cell digestion solution, then counted, and the total number of cells in the culture system was estimated according to the corresponding formula that depends on the sampling method. (a) Quantitative suspension sampling method: Number of cells in 1 mL of suspension sample × total suspension volume of culture system (60 mL) = total number of cells. The results are shown in Table 1.

[0046] [Table 1]

[0047] (b) Quantitative microcarrier sampling method: number of cells in a 5 mg microcarrier (i.e., 0.1 mL microcarrier volume) sample × total microcarrier weight in the culture (i.e., 200 mg) = total cell number. The results are shown in Table 2.

[0048] [Table 2]

[0049] 5. Remove all microcarrier suspension, 60 mL of medium culture system from 200 mg microcarriers, centrifuge at 1500 rpm for 2 minutes, discard the supernatant, digest the cells from the microcarriers by adding cell digestion solution, count the actual total number and compare with the data from the sampling count in step 4. The results are shown in Figure 9. The results indicate that the three samples using the microcarrier quantitative sampling method were more reproducible (less variance) and closer to the actual total.

[0050] Example 4 Three-dimensional microcarrier-based cell resuscitation method The microcarriers used in this example were 3D FloTrix microcarriers purchased from Beijing Huaguan Biotechnology Co., Ltd., with a catalog number of CNF-F01T-50. The adipose mesenchymal stem cell medium used in this example was a human mesenchymal stem cell medium (Wuhan Vinosai Biotechnology Co., Ltd., catalog number: M001).

[0051] (I) Three-dimensional microcarrier-based cell resuscitation and static culture I. Resuscitation and static culture of adipose-derived mesenchymal stem cells 1. Preparation of static culture vessels and microcarriers Sterile and dry microcarriers were placed into the culture vessel; the culture vessels available for use were well plates, Petri dishes, etc. without adhesion treatment. The appropriate size should be selected according to the number of cells to be resuscitated, and attention should be paid to the airtightness of the vessel according to the environment required by the cells. The required microcarriers were aseptically taken into the selected culture vessel, ensuring that the microcarriers were kept at room temperature and dried until inoculation was completed. For the experiment, one tablet (20 mg) of microcarriers was used in a non-adherent treated 6-well plate. 2. Resuscitation of cryopreserved cells Cryopreserved adipose-derived MSCs were harvested and quickly thawed in a constant temperature water bath at 37° C. When the ice was gone, they were quickly transferred to an ultraclean bench for subsequent routine resuscitation procedures. 3. Transplantation of thawed cryopreserved cells After completion of step 2, the thawed cells are washed and centrifuged to remove the supernatant, and the cells are resuspended in culture medium, preferably at 5 to 500 × 10 5 A cell suspension of 1 × 10 cells / mL was obtained. The cell concentration in the suspension was 1 × 10 6 cells / mL. 4. Inoculation After completion of step 3, the cell suspension is diluted with 5–100 × 10 3 Six-well plates with microcarriers prepared in step 1 were seeded with a cell-to-microcarrier ratio of 100-400 μL:20 mg of cells / mg, and a medium volume-to-microcarrier ratio of 100-400 μL:20 mg. In the study, 200 μL of the cell suspension (total cell count of 2 × 10 5 ) was inoculated.

[0052] 5. Incubation After the completion of step 4, incubation for 20 to 240 minutes was required, but it was necessary to pay attention to conditions such as temperature, humidity, sterility, and gas to maintain a proper environment throughout the incubation process. A normal incubator for normal cell culture was sufficient. If the incubator does not have a humidity setting, some sterile wetting agent (PBS, culture medium, distilled water, and some other liquids that provide a moist environment but do not affect or benefit the cells) can be added manually around the inoculated microcarriers. When incubating in small dishes, the small dishes can be placed in a larger container, and similarly for well plates, a moist box made around the small dishes can be placed in the larger container. In the test, a 6-well plate was selected and incubated in a CO2 incubator at 37°C for 2 hours, and 2mL of PBS was added between the 6-well plates. 6. Medium supplementation After the completion of step 5, the replenishment amount was selected according to the inoculated cell number, medium exchange cycle, and medium type. 1 ml of serum medium was usually sufficient for 620,000 cells per day. In the experiment, 8 ml of medium was replenished. 7. Culture and Resuscitation After completion of step 6, the resuscitated cells were placed in a suitable culture environment for subsequent cell resuscitation and culture. In this test, the 6-well plate was placed in a CO2 incubator at 37°C and cultured. 8. Monitoring Cell culture time, period of medium replenishment and period of cell status detection can be set according to the needs of the experiment. Cell status indicators included medium color, microcarrier status, cell staining analysis, cell lysis count, etc. Cells were cultured for a total of 5 days in the experiment, and medium was replenished on the 4th day. Cell detection using the kit was performed on the 1st and 4th days after inoculation (step 10), and cell counting was performed on the 5th day (step 11).

[0053] 9. Fresh cell control group (1) Cryopreserved adipose-derived MSCs were harvested and quickly thawed in a 37°C thermostatic water bath. When the ice ran out, they were quickly transferred to an ultraclean bench for subsequent regular resuscitation operations. The thawed cells were washed, centrifuged to remove the supernatant, and resuspended using culture medium to obtain a cell suspension, which was then plated at 15,000 cells / cm in a T75 cell culture flask supplemented with 15 ml of culture medium. 2 and incubated for 3 days to allow complete growth. (2) After step (1) is completed, the fully grown cells are subjected to standard cell digestion, centrifuged to remove the supernatant, and then diluted with preferably 5 to 500 × 10 5 The cells were used in a medium with a cell concentration of 1 × 10 cells / mL. The cell concentration in the cell suspension was 1 × 10 6 cells / mL. (3) After completion of step (2), the operation was continued by inoculating the cells into a 6-well plate containing microcarriers according to steps 4 to 8. Cell detection using the kit was performed on the 1st and 4th days after inoculation (step 10), and cell counting was performed on the 5th day (step 11).

[0054] 10. Cell monitoring (1) Kit: Animal Cell Killing Viability / Cytotoxicity Assay Kit, Catalog Number: KGAF001; (2) 50–100 μL of the microcarrier suspension containing the cells was added to a 96-well plate; (3) Remove as much of the supernatant as possible, add PBS, wash once, remove as much PBS as possible after 2 minutes, and stain each well by adding 100 μL of staining solution made according to the kit's instructions. Stain for 20-30 minutes at room temperature, protect from light, and then observe under a fluorescent microscope.

[0055] The results are shown in Figure 10. The results showed that cryopreserved adipose-derived MSCs resuscitated onto microcarriers for static culture ranged from 1-2 cells initially on each microcarrier sphere to many more cells on all microcarriers after 4 days, indicating that cryopreserved adipose-derived MSCs can be directly resuscitated onto microcarriers for static culture, whereas when comparing the number of cells on microspheres seeded with resuscitated cells to the number of cells on microspheres seeded with fresh cells, there was almost no difference, indicating that by resuscitating cells directly onto microcarriers, cell growth is the same as when traditionally obtained from culture flasks and seeded onto microcarriers, thus eliminating the need for tedious operations to resuscitate cells in culture flasks and then digest them to obtain fresh cells for seeding onto microcarriers.

[0056] 11. Cell counting to measure cell proliferation On the fifth day of cell culture, the cells were harvested according to the method described in the patent application “Method for harvesting cells on three-dimensional microcarriers” (Chinese Patent Application No. 201910101736.0) and counted by means of a cell counting plate. The results are shown in Figure 11. As a result, when adipose-derived MSCs were resuscitated in static culture on microcarriers (resuscitation group), the number of cells increased from 200,000 at the time of inoculation to 50.27 million, which was not significantly different from the number of cells (5.187 million) obtained by the conventional post-digestion static culture method (fresh group) (p = 0.42).

[0057] II. Resuscitation and static culture of adipose-derived mesenchymal stem cell microcarriers Cryopreserved adipose-derived MSC microcarriers were prepared according to the method described in the patent application “In situ cell cryopreservation method on three-dimensional microcarriers” (Chinese Application No. 201910097650.5).

[0058] 1. Preparation of static culture vessels and microcarriers Same as step 1 in Part I. 2. Resuscitation of cryopreserved cells Cryopreserved adipose-derived MSC microcarriers (hereafter referred to as adipose-derived MSC microtissues) were harvested and rapidly thawed in a constant temperature water bath at 37° C. Because the microtissues may affect the observation of the lysis results, the cryogenic storage tubes needed to be tilted and gently shaken during observation, and when the ice was gone, they were quickly transferred to an ultraclean bench for subsequent routine resuscitation operations. 3. Transplantation of thawed cryopreserved cells After completion of step 2, the thawed cells were washed, centrifuged to remove the supernatant, and the cells were resuspended in culture medium to obtain a cell suspension. The number of cells in the microtissues in the cell suspension ranged from 5 to 500 × 10 5 It is preferred that the cell count is in cells / ml. However, the actual microcarriers themselves can occupy a large volume, or insufficient supernatant removal can result in a somewhat larger resuspension volume than expected, which can be scaled to harvest the required cells if an accurate count is required. To remove the supernatant, the centrifugal force can be increased and the supernatant pipetted as far as possible, while for the microcarriers themselves, the occupied volume can be eliminated with little effect on subsequent experiments. 4. Inoculation After completion of step 3, 200 μl of the cell suspension was inoculated into the 6-well plate containing the microcarriers prepared in step 1. 5. Incubation Same as step 5 in Part I. 6. Medium supplementation Same as step 6 in Part I. 7. Culture and Resuscitation Same as step 7 in Part I. 8. Monitoring Same as step 8 in Part I. 9. Cell monitoring Same as step 10 in Part I. The results are shown in Figure 12. The results show that the cryopreserved adipose-derived MSC microtissues were resuscitated onto new microcarriers in static culture. On the first day after resuscitation, only a few microcarriers had many cells on them (bright spots), which are resuscitated microtissues, and other microcarriers with fewer cells are new microcarriers, while on the fourth day after culture, all microcarriers had cells on them, indicating that the cryopreserved microtissues could be resuscitated onto new microcarrier tablets for static culture.

[0059] (II) Three-dimensional microcarrier-based cell resuscitation and dynamic culture I. Resuscitation and dynamic culture of adipose-derived mesenchymal stem cells 1. Preparation of dynamic culture vessels and microcarriers An appropriate amount of microcarriers was placed into a cell culture flask equipped with an impeller or another clean, sterile, endotoxin-free container, where the microcarriers were agitated and suspended; the microcarriers were resuspended by adding 0-50% (v / v) final culture medium and left undisturbed or agitated for >0 h (no time limit). In the experiment, 10 tablets of 200 mg microcarriers were added to a 125 ml cell culture flask equipped with an impeller containing 10 ml medium and left for 10 min. 2. Resuscitation of cryopreserved cells Cryopreserved adipose-derived MSCs were harvested and quickly thawed in a constant temperature water bath at 37° C. When the ice was gone, they were quickly transferred to an ultraclean bench for subsequent routine resuscitation procedures. 3. Transplantation of thawed cryopreserved cells After completion of step 2, the thawed cells are washed, centrifuged to remove the supernatant, and the cells are resuspended in culture medium to obtain a cell suspension. 5 The cell concentration in the suspension was 1 × 10 cells / mL in a total of 2 mL. 6 cells / mL. 4. Inoculation After completing step 3, the above cell suspension was mixed with new microcarriers in the cell culture flask equipped with an impeller prepared in step 1. The ratio of cells to new microcarriers was 5 to 100 × 10 3 The ratio of cells / mg of microcarriers to be inoculated was 1 mg:1,000 µL, and after mixing, cell culture medium was added to adjust the ratio of microcarriers to medium to 1 mg:1,000 µL. In the experiment, 2 million resuscitated cells were inoculated into 200 mg of new microcarriers and the medium was replenished to 60 mL.

[0060] The impeller-equipped cell culture flask was placed on a low-speed stirrer and placed in an incubator for stirring. The stirring could be performed by constant-speed stirring or variable-speed alternating or variable-speed cyclic stirring, the stirring direction could be clockwise, counterclockwise, or alternating stirring to transfer the cells from the seed microcarriers to new microcarriers, the stirring time was 0.1-100 h, and the stirring speed was 1-200 rpm. In the experiments, the variable-speed cyclic clockwise stirring method was compared with the constant-speed clockwise stirring method, and three sets of experiments were set up as follows: Group 1 (static inoculation): Cell culture flasks with built-in impellers were placed on a slow-speed stirrer and placed in an incubator for 4 h, then stirred at variable speed cycles of 40 rpm clockwise for 5 min and 20 rpm for 20 min up to 24 h, then stirred at a constant speed (60 rpm) clockwise up to 96 h. Group 2 (intermittent inoculation): Cell culture flasks with impellers were placed on a slow-speed stirrer and placed in an incubator and periodically stirred clockwise at 40 rpm for 5 min, 20 rpm for 20 min for 24 h, followed by constant clockwise stirring (60 rpm) for up to 96 h. Group 3 (constant speed inoculation): Cell culture flasks with impellers were placed on a slow speed stirrer and placed in an incubator at a constant clockwise speed (60 revolutions / min) for up to 96 hours. Each group was sampled for cell detection every 4 hours (step 6). Cultivation was continued. After completion of step 4, the culture was continued with constant clockwise stirring (60 rpm) until day 6. Samples were taken on days 1, 2, 3, 4, 5 and 6 for cell counting (step 7).

[0061] 6. Cell monitoring (1) Kit: Animal Cell Killing Viability / Cytotoxicity Assay Kit, Catalog Number: KGAF001; (2) 50–100 μL of the microcarrier suspension containing the cells was added to a 96-well plate; (3) Remove as much of the supernatant as possible, add PBS, wash once, remove as much PBS as possible after 2 minutes, and stain each well by adding 100 μL of staining solution made according to the kit's instructions. Stain for 20-30 minutes at room temperature, protect from light, and then observe under a fluorescent microscope. The results are shown in Figure 13. We compared the effect of different stirring methods and speed settings during resuscitation of cryopreserved adipose-derived MSCs in dynamic culture on cell distribution in the microcarriers, while constant-speed culture after resuscitation promoted uniform distribution of cells. 7. Cell counting to measure cell proliferation After various days of cell inoculation, the cells were harvested according to the method described in the patent application “Method for harvesting cells on three-dimensional microcarriers” (Chinese Patent Application No. 201910101736.0) and counted using a cell counting plate. The results are shown in Figure 14. The results show that the cryopreserved cells resuscitated on the microcarriers proliferated from the initial 2 million to approximately 17.13 million after 3 days of culture, and there was no significant difference in the cell number among these three speed adjustments (groups 1 to 3 in step 4), indicating that resuscitation of cryopreserved adipose-derived MSCs on microcarriers for dynamic culture did not affect the cell resuscitation and proliferation process regardless of the stirring speed for inoculation (a moderate speed suitable for the cells).

[0062] Comparison of methods Three-dimensional dynamic resuscitation cell group: group 3 of step 4. Three-dimensional dynamic fresh cell population: (1) Cryopreserved adipose-derived MSCs were harvested and quickly thawed in a constant temperature water bath at 37°C. When the ice ran out, they were quickly transferred to an ultraclean bench for subsequent regular resuscitation operations. The thawed cells were washed, centrifuged to remove the supernatant, and the cells were resuspended using culture medium to obtain a cell suspension, which was then plated at 15,000 cells / cm in a T75 cell culture flask supplemented with 15 ml of culture medium. 2 (2) After step (1) is completed, the fully grown cells are subjected to a conventional cell digestion, centrifuged to remove the supernatant, and then cultured at a concentration of preferably 5 to 500 × 10 5 The cells were used in a medium with a cell concentration of 1 × 10 cells / mL. The cell concentration in the cell suspension was 1 × 10 6 cells / mL. (3) After completion of step (2), the cell suspension was placed into the prepared impeller-equipped cell culture flask and mixed with the microcarriers after preparing the vessel and microcarriers according to step 1 of part (II). After mixing, cell culture medium was added and the ratio of microcarriers to medium was adjusted to 1 mg:1-1,000 μL. In the experiment, 2 million fresh cells were inoculated into 200 mg of new microcarriers and the medium was replenished to 60 mL. The impeller-equipped cell culture flask was placed on a low-speed stirrer and placed in an incubator with constant clockwise stirring at 60 rpm until the 6th day, and samples were taken on the 1st, 2nd, 3rd, 4th, 5th and 6th days for cell counting.

[0063] Three-dimensional static resuscitated cell population: Three-dimensional static resuscitated cell population in step 1 of part (I). Three-dimensional static fresh cell population: Three-dimensional static fresh cell population in step 1 of part (I). 2D resuscitation group: Cryopreserved adipose-derived MSCs were harvested and quickly thawed in a 37°C constant temperature water bath. When the ice ran out, they were quickly transferred to an ultraclean bench for subsequent regular resuscitation operations. The thawed cells were washed, centrifuged to remove the supernatant, and the cells were resuspended using culture medium to obtain a cell suspension, which was seeded into a 6-well plate at a density of 100,000 cells / well, 4 mL of medium was added, and one well was taken for cell counting on days 1, 2, 3, and 4. Secondary passage group: A T75 filled with MSCs was harvested, fresh cells were digested from the T75 according to conventional cell digestion methods, the supernatant was removed by centrifugation, the cells were resuspended using culture medium to obtain a cell suspension, which was seeded into a 6-well plate at a density of 100,000 cells / well, 4 mL of medium was added, and one well was harvested for cell counting on days 1, 2, 3, and 4, respectively.

[0064] The results are shown in Figure 15. FIG. 15A shows that adipose-derived MSCs were dynamically cultured after resuscitation into microcarriers and expanded from the initial 2 million resuscitated cells to 76.33 million after 6 days of culture. Adipose-derived MSCs were resuscitated to full expansion in culture flasks and then passaged into microcarriers for dynamic culture and expanded from the initial 2 million cells to 58.93 million cells after 6 days of culture. There was no significant difference between the two types of cells in the first 3 days, and on days 4 and 5, the passaged cells grew faster than the resuscitated cells, but on day 6, the resuscitated cells were more numerous than the passaged cells. FIG. 15B shows the 2D control of the corresponding cells, from which it can be seen that the passaged cells grew slower than the resuscitated cells in the first 3 days, and by day 4, both cell groups were space-limited and decreased in number, indicating that the resuscitated cells grew slower. FIG. 15C shows a comparison of the growth times of resuscitated cells seeded into static and dynamic cultures of microcarriers. There was no significant difference between the two, but subtle differences showed that static resuscitation was slightly slower than dynamic resuscitation, suggesting that resuscitated cells are more suitable for moderate agitation speeds. Figure 15D showed that passaged cells were more resistant to shear forces, and there was no significant difference between the two, and overall, cryopreservation was very damaging to cells and required some time to resuscitate; Figure 15E and Figure 15F compared cells in both dynamic and 2D cultures, with no significant difference in the first 3 days, and 2D on the 4th day showed a significant difference due to space limitations.

[0065] II. Resuscitation and dynamic culture of adipose-derived mesenchymal stem cell microcarriers Cryopreserved adipose-derived MSC microcarriers were prepared according to the method described in the patent application “In situ cell cryopreservation method on three-dimensional microcarriers” (Chinese Application No. 201910097650.5).

[0066] 1. Preparation of dynamic culture vessels and microcarriers An appropriate amount of microcarriers was placed into a cell culture flask with built-in impeller or another clean, sterile, endotoxin-free container where the microcarriers were agitated and suspended; the microcarriers were resuspended by adding 0-50% (v / v) final culture medium and left stationary or agitated for >0 h (no time limit). In the experiment, ten tablets of 200 mg microcarriers were added to a 125 ml cell culture flask containing 10 ml medium and with a built-in impeller and left for 10 min.

[0067] 2. Resuscitation of cryopreserved microtissues Cryopreserved adipose-derived MSC microcarriers (hereafter referred to as adipose-derived MSC microtissues) were harvested and rapidly thawed in a 37°C constant temperature water bath. Because the microtissues may affect the observation of the lysis results, the cryogenic storage tubes needed to be tilted and gently shaken during observation, and when the ice ran out, they were quickly transferred to an ultraclean bench for subsequent regular resuscitation operations. 0.1–25×10 MSCs per mg of frozen tissue were obtained. 6 There are 5–100 × 10 cells in total. 3 0.25 × 10 cells / mg of microcarriers were intended for inoculation. 6 There were 2 × 10 cells in total. 6 The cells were

[0068] 3. Transplantation of thawed cryopreserved cells After completion of step 2, the thawed cells are washed, centrifuged to remove the supernatant, and the cells are resuspended in culture medium to obtain a cell suspension. 5 The cell concentration in the suspension was 1 × 10 cells / mL in a total of 2 mL. 6 cells / mL.

[0069] 4. Inoculation After completing step 3, the above microtissue suspension was mixed into the impeller-equipped cell culture flask prepared in step 1 and mixed with new microcarriers. The number of cells on the resuscitated microtissue ranged from 5 to 100 × 10 3 cells / mg of new microcarriers, and the ratio of microcarriers to medium was adjusted to 1 mg:1,000 µL by adding medium after mixing. In the experiment, 2 million resuscitated cells were inoculated into 200 mg of new microcarriers and supplemented with medium to 60 mL. The impeller-equipped cell culture flask was placed on a slow-speed agitator and placed in an incubator for agitation. Agitation can be performed by constant speed agitation or variable speed alternating or variable speed cyclic agitation, and the agitation direction can be clockwise, counterclockwise, or alternating agitation to perform the culture. The experiment was performed using a constant clockwise speed of 60 rpm for 96 hours, and then continued at a constant clockwise speed of 60 rpm for 4 days. Samples were taken on the fourth day for cell detection (step 5) and cell counting (step 6).

[0070] 5. Cell monitoring (1) Kit: Animal Cell Killing Viability / Cytotoxicity Assay Kit, Catalog Number: KGAF001; (2) 50–100 μL of the microcarrier suspension containing the cells was added to a 96-well plate; (3) Remove as much of the supernatant as possible, add PBS, wash once, remove as much PBS as possible after 2 minutes, and stain each well by adding 100 μL of staining solution made according to the kit's instructions. Stain for 20-30 minutes at room temperature, protect from light, and then observe under a fluorescent microscope. The results are shown in Figure 16. The results show that when cryopreserved adipose-derived MSC microcarriers were resuscitated onto new carriers, they gradually resumed expansion from fewer cells initially to more cells on each microcarrier sphere, indicating that cryopreserved microtissues can be directly resuscitated onto new carriers.

[0071] 6. Cell counting to measure cell proliferation After various days of cell inoculation, the cells were harvested according to the method described in the patent application "Method for harvesting cells on three-dimensional microcarriers" (Chinese Patent Application No. 201910101736.0) and counted using a cell counting plate. The results are shown in Figure 17. The results showed that there was no significant difference between cryopreserved umbilical cord MSC microtissues after resuscitation into culture microcarriers and cryopreserved cells after resuscitation seeding into culture microcarriers, with a P value of 0.73. The microtissue population expanded from the initial seed microcarriers with only 2 million cells to 42.82 million after 4 days of culture, indicating that microtissues can be resuscitated in microcarriers after cryopreservation.

[0072] Example 5: In situ passaging method based on three-dimensional microcarrier cell culture The microcarriers used in this example were 3D FloTrix microcarriers purchased from Beijing Huaguan Biotechnology Co., Ltd., with a catalog number of CNF-F01T-50. (I) Static in situ passaging based on three-dimensional microcarrier cell culture Test cells: adipose-derived MSCs and umbilical cord MSCs. I. Static in situ passaging method 1. Preparation of novel microcarriers Sterile and dried microcarriers were placed into the culture vessel; in this embodiment, 20 mg of microcarriers were used in a 6-well plate. 2. Preparation of seed microcarrier suspension The seed microcarriers were microcarriers that were cultured with the test cells. The seed microcarriers were cultured according to Patent Application No. 201910098003.6 (Method of Cell Attachment Cultivation on Three-Dimensional Microcarriers). Microcarriers with a cell density of 10,000-1,000,000 cells / mg microcarrier were used as seed microcarriers, and the seed microcarriers were resuspended to 0.1-50 mg / mL (i.e., 0.5-25 million cells / mL volume) using cell culture medium. Here, for adipose-derived MSCs: cell density of 500,000 cells / mg microcarrier and resuspension density of 5 mg / mL; for umbilical cord MSCs: cell density of 120,000 cells / mg microcarrier and resuspension density of 15 mg / mL. 3. Inoculation After thoroughly mixing the seed microcarrier suspension prepared in step 2 above, an appropriate amount of the seed microcarrier suspension was dropped by a pipette into the 6-well plate prepared in step 1 containing 20 mg of new microcarriers, and the seed microcarrier suspension was thoroughly mixed with the new microcarriers; the ratio of seed microcarriers to new microcarriers was 0.0002-200 mg seed microcarriers / mg new microcarriers. The mixed microcarriers were incubated in an incubator for an appropriate time (0.5-24 hours, especially 2 hours or 24 hours); the cells on the seed microcarriers were passaged onto the new microcarriers. For adipose-derived MSCs: 1 mg of seed microcarriers inoculated into 20 mg of fresh microcarriers; for umbilical cord MSCs: 3 mg of seed microcarrier suspension inoculated into 20 mg of microcarriers; both groups of cells were incubated for 2 hours in the incubator. 4. Culturing: After incubation, cell culture was continued by adding cell culture medium according to conventional cell culture methods, and then static in situ passaging was completed.

[0073] II. Control Experiments For the control experiment, cells were digested and then inoculated onto passaging microcarriers, i.e., cells on seed microcarriers were digested and harvested (Patent Application No. 201910101736.0 "Method for harvesting cells on three-dimensional microcarriers") and then resuspended in a cell-only suspension, which was mixed with 20 mg of microcarriers at the same cell concentration and inoculation volume as in step 3, incubated equally for 2 hours, and then added to cell culture medium for further incubation.

[0074] III. Results 1. Cell Observation (1) Kit: Animal Cell Killing Viability / Cytotoxicity Assay Kit, Catalog Number: KGAF001; (2) 50–100 μL of the microcarrier suspension containing the cells was added to a 96-well plate; (3) Remove as much of the supernatant as possible, add PBS, wash once, remove as much PBS as possible after 2 minutes, and stain each well by adding 100 μL of staining solution made according to the kit's instructions. Stain for 20-30 minutes at room temperature, protect from light, and then observe under a fluorescent microscope.

[0075] Result analysis: FIG. 18 shows that when adipose-derived MSCs were seeded by the static in situ passaging method, only a few microcarriers had cells on them (highlighted), which were the seed microcarriers, while the others without cells were new microcarriers, whereas on day 4 after in situ passaging, all the microcarriers had cells on them, and the cells on the seed microcarriers could be transferred and expanded into the new microcarriers. FIG. 19 shows that 2 days after seeding by static in situ passaging, only a few microcarriers had many cells (bright spots) on them, which were the seed microcarriers, and the others with few cells were new microcarriers, while 4 days after in situ passaging, all the microcarriers had cells on them, and the cells on the seed microcarriers could be transferred and expanded on the new microcarriers.

[0076] 2. Cell counting to determine if cells have completed passage expansion This will be carried out in accordance with the patent application "Method for harvesting cells on three-dimensional microcarriers" (application number 201910101736.0). After 4 days of incubation, 20 mg of cell growth microcarriers were transferred from the culture well plate to a centrifuge tube, centrifuged at 400 × g for 2 minutes, the supernatant was transferred by pipetting, an appropriate amount of PBS was added, gently shaken by hand for 20-30 seconds, the supernatant was transferred by pipetting as much as possible, the PBS washing was repeated once, 3 mL of lysis solution (lysis solution formulation: 0.1% collagenase, 0.1% ethylenediaminetetraacetic acid, 0.05% trypsin; % indicated / 100 mL) was added, and the mixture was incubated in an incubator for 30 minutes, during which the cells were gently extracted several times with a 1 mL pipette every 10 minutes; after 30 minutes, the microcarriers were completely dissolved, and 3 mL of total medium was added to terminate the dissolution process; the tube was centrifuged at 200 × g for 5 minutes, the supernatant was discarded, and the cells were resuspended and counted through a cell counting plate. Result analysis: Figure 20 shows that the number of adipose-derived MSCs increased from 0.5 million at inoculation to 2.1 million cells by the static in situ passaging method, which was not significantly different from the number of cells obtained by the conventional digestion-passaging method (2.4 million) (p=0.32). Figure 21 shows that the number of umbilical cord MSCs cultured by the static in situ passaging method increased from 0.3 million at inoculation to 1.9 million cells, indicating that the cells underwent proliferation and expansion.

[0077] (II) Dynamic in situ passaging based on three-dimensional microcarrier cell culture Test cells: adipose-derived MSCs and umbilical cord MSCs. I. Dynamic in situ passaging 1. Preparation of fresh microcarrier suspension: Weigh out 200 mg of fresh microcarrier powder and pour into an impeller-equipped cell culture flask (Bellco Glass, USA; Catalog No.: 1965-61001; schematic diagram shown in Figure 1); add cell culture medium in a ratio of 1-2000 μL:1 mg of microcarrier and leave or stir for 0 hours (excluding 0 hours, no time limit). Here, for adipose-derived MSCs: 20 ml of cell culture medium was added for 200 mg of new microcarriers and left for 14 hours. For umbilical cord MSCs: 10 ml of cell culture medium was added for 100 mg of new microcarriers and left for 14 hours. 2. Preparation of seed microcarrier suspension The seed microcarriers were microcarriers that were cultured with the test cells. The seed microcarriers were cultured according to Patent Application No. 201910098003.6 (Method of Cell Attachment Cultivation on Three-Dimensional Microcarriers). Microcarriers with a cell density of 10,000-1,000,000 cells / mg microcarrier (e.g., 100,000-500,000 cells / mg microcarrier) were used as seed microcarriers, and the seed microcarriers were resuspended to 0.1-50 mg / mL (i.e., 0.5-25 million cells / mL volume). Here, for adipose-derived MSCs: seed microcarriers with a cell density of 500,000 / mg and a resuspension density of 1 mg / mL. For umbilical cord MSCs: seed microcarriers with a cell density of 150,000 / mg and a resuspension density of 1 mg / mL. 3. Inoculation The seed microcarrier suspension prepared in step 2 above was mixed well with the fresh microcarrier suspension in the impeller-equipped cell culture flask in step 1; the ratio of seed microcarriers to fresh microcarriers was 0.0002-200 mg seed microcarriers / mg fresh microcarriers; cell culture medium was added to adjust the ratio of microcarriers to culture medium to 1 mg:1-1000 µL. Here, for adipose-derived MSCs: 4 mg of seed microcarriers (total of 2 million cells) were used to inoculate 200 mg of new microcarriers and supplemented with cell culture medium to 60 mL. For umbilical cord MSCs: 6.7 mg of seed microcarriers (total of 1 million cells) were used to inoculate 100 mg of new microcarriers and supplemented with cell culture medium to 60 mL. The cell culture flask with built-in impeller was placed on a slow-speed agitator (3D FloTrix miniSpin slow-speed agitator, Beijing Huaguan Biotechnology Co., Ltd., Catalog No.: 3D FTmS-2-2) and placed in an incubator for agitation. Agitation could be performed by constant speed agitation or variable speed alternating or variable speed cyclic agitation, the agitation direction could be clockwise, counterclockwise, or alternating agitation to move the cells from the seed microcarriers to new microcarriers, and the agitation time ranged from 0.1 to 100 h. Here, a variable speed periodic clockwise stirring regime was used for both MSCs, specifically, a cycle of 60 rpm for 5 minutes and 20 rpm for 20 minutes, for a total of 24 hours. 4. Culture medium After inoculation, the culture was allowed to continue with agitation until a growth effect was achieved. II. Control Experiments A control experiment was performed using adipose-derived MSCs, which were digested and then inoculated into microcarriers for passage. That is, the cells on the seed microcarriers were digested and harvested (according to Patent Application No. 201910101736.0 "Method for harvesting cells on three-dimensional microcarriers") and then resuspended in a cell-only suspension. Two million cells were resuspended in 4 mL and inoculated into 200 mg of new microcarriers in an impeller-equipped cell culture flask, and the cell culture medium was supplemented to 60 mL. The impeller-equipped cell culture flask was placed on a low-speed agitator (3D FloTrix Spminix low-speed agitator, Beijing Huaguan Biotechnology Co., Ltd., Catalog No.: 3D FTmS-2-2) and placed in an incubator for 24 hours with a cycle of 60 rpm for 5 minutes and 20 rpm for 20 minutes, specifically, the agitation culture was continued until a proliferation effect was obtained. III. Results 1. Cell Observation: (1) Kit: Animal Cell Killing Viability / Cytotoxicity Assay Kit, Catalog Number: KGAF001; (2) 50–100 μL of the microcarrier suspension containing the cells was added to a 96-well plate; (3) Remove as much of the supernatant as possible, add PBS, wash once, remove as much PBS as possible after 2 minutes, and stain each well by adding 100 μL of staining solution made according to the kit's instructions. Stain for 20-30 minutes at room temperature, protect from light, and then observe under a fluorescent microscope. Result analysis: Figure 22 shows that on the second day after inoculation of adipose-derived MSCs by dynamic in situ passaging, only a few microcarriers had many cells on them as seen in the fluorescent image (bright dots), which were seed microcarriers, but from the bright field, it was clear that other microcarriers were present and others with few cells were new microcarriers, proving that some cells were transferred to new microcarriers by in situ passaging, and on the fifth day after in situ passaging, cells were present on all microcarriers, indicating that the cells that were passaged onto new microcarriers had proliferated and expanded. Figure 23 shows that when inoculated with umbilical cord MSCs by dynamic in situ passaging method, only a few microcarriers had many cells on them (bright dots), as seen in the fluorescent image, but from the bright field, it was clear that other microcarriers were present and others without cells were new microcarriers, proving that on the fifth day after in situ passaging, cells were present on all microcarriers, indicating that the cells on the seed microcarriers were transferred to new microcarriers and could proliferate and expand. 2. Cell counting to determine if cells have completed passage expansion This will be carried out in accordance with the patent application "Method for recovering cells on three-dimensional microcarriers" (application number 201910101736.0). On different days after inoculation, 3 mL of microcarrier suspension was transferred from the flask to a centrifuge tube, centrifuged at 400 × g for 2 minutes, the supernatant was transferred by pipetting, an appropriate amount of PBS was added, gently shaken by hand for 20-30 seconds, the supernatant was transferred by pipetting as much as possible, the PBS washing was repeated once, 3 mL of lysis solution (lysis solution formulation: 0.1% collagenase, 0.1% ethylenediaminetetraacetic acid, 0.05% trypsin; % indicated / 100 mL) was added, and the cells were incubated in a 37 ° C incubator for 30 minutes, during which the cells were gently blown several times every 10 minutes with a 1 mL pipette; after 30 minutes, the microcarriers were completely dissolved, and 3 mL of total medium was added to end the dissolution process; the tube was centrifuged at 200 × g for 5 minutes, the supernatant was discarded, and the cells were resuspended according to the requirements of the subsequent application, and the cells were counted through a counting plate. Result analysis: Figure 24 shows that adipose-derived MSCs can be grown and expanded by the dynamic in situ passaging method from seed microcarriers initially containing only 2 million cells to 59.77 million after 5 days in culture, which was not significantly different (p=0.14) from the cell number obtained by the conventional post-digestion passaging method (51.4 million). Figure 25 shows that umbilical cord MSCs can be grown and expanded by the dynamic in situ passaging method from seed microcarriers initially containing only 1 million cells to 15.2 million after 7 days in culture.

[0078] Example 6: Three-dimensional microcarrier-based in situ cell cryopreservation method I. The cells in the sections were HEK293T cells purchased from the Cell Resource Center of Cellular Basic Medicine, Peking Union Medical College, with the catalog number of CBP60439; the three-dimensional microcarriers were 3D FloTrix microcarriers purchased from Beijing Huaguan Biotechnology Co., Ltd., with the catalog number of CNF-F01T-50. In the examples, three-dimensional microcarrier suspensions of cells were prepared according to the following steps. 1. Microcarrier preparation: 200mg of microcarrier powder was weighed out for UV sterilization, then poured into a sterile cell culture flask with impeller (as shown in Figure 1), and three groups were prepared; 2. Cell preparation: Adipose-derived MSC suspension was prepared in advance, with 5 × 10 6 The cells were resuspended in 60 ml of complete medium for later use; three groups were prepared; 3. Cell inoculation: The above cell suspension was mixed into a cell culture flask with an impeller and thoroughly mixed with 200 mg of microcarriers; 4. Cell attachment: The cell culture flask with impeller was placed on a slow agitator and placed in a 37°C, 5% CO2 incubator with agitation at 80 rpm, and the cells were allowed to attach to the microcarriers by rotation; 1. The three-dimensional microcarrier suspension containing HEK293T cells was centrifuged at 400×g for 2 minutes and the supernatant was discarded. 2. Cryopreservation solution (90% FBS + 10% DMSO) was added and distributed into cryogenic storage tubes (1 ml of cryopreservation solution contained 10 mg of cell-laden microcarriers, 0.5-1 ml per tube). 3. The cryogenic storage tube should be loaded into the cell programmed cooling box, placed in a -80°C freezer within 5 minutes, and transferred to liquid nitrogen after 24 hours. 4. Resuscitation of cells attached to microcarriers: For resuscitation, a pre-warmed basal medium at 37°C was prepared, then the cryogenic storage tube was quickly placed in a 37°C water bath with constant gentle shaking during this time, and the tube was quickly removed from the water bath when a single grain of rice-sized ice remained (approximately 1-2 min in the water bath). The thawed three-dimensional microcarrier suspension containing the cells was then immediately diluted with basal medium at a dilution ratio of 1:5, followed by centrifugation at 400×g for 2 min, and then resuspended for later use.

[0079] 5. Cell detection methods: (1) In situ calcein-AM / PI staining of live cells: 1) Kit: Animal Cell Killing Viability / Cytotoxicity Assay Kit, Catalog Number: KGAF001; 2) 50–100 μl of the three-dimensional microcarrier suspension containing cells resuscitated before or after cryopreservation was added to a 96-well plate, respectively; 3) The supernatant was removed as much as possible, PBS was added, washed once, and after 2 minutes, the PBS was removed as much as possible. Each well was stained by adding 100 μL of staining solution made according to the kit's instructions. After staining for 20 to 30 minutes at room temperature and protecting from light, the results of observation under a fluorescent microscope are shown in Figure 26.

[0080] As can be seen from FIG. 26, live cells were stained with the dye solution and observed to emit light by fluorescence, and the cells that emitted light were live cells. The irradiated spherical microcarriers proved that live cells were still attached to the microcarriers. FIG. 26A demonstrates that live cells could be cryopreserved in situ and resuscitated on 3D FloTrix microcarriers, even after 2 weeks of cryopreservation, successfully. However, in FIG. 26B, after cryopreservation and resuscitation of the competitive microcarriers (GE Cytodex, Catalog No.: 17-1271-01, purchased from Beijing Syncfer Technology Co., Ltd.), it was found that the cells had detached from the microcarriers, and fluorescence observation revealed that the cells were not attached to the microcarriers, but were scattered and scattered in the liquid.

[0081] (2) Analytical method for detection of apoptosis rate by flow cytometry: 1) Cell harvesting: 20 mg of the cell-containing three-dimensional microcarrier suspension was centrifuged at 400 x g for 2 min and the supernatant was discarded. 3 mL of 0.1% collagenase type IV solution (1 g of collagenase type IV (Gibco, 17104019) dissolved in 1 L of PBS) was added and the solution was incubated at 37 °C for 30 min in a cell incubator, centrifuged at 400 x g for 5 min and the supernatant was discarded. 2) Cell washing: The cells were resuspended once with PBS pre-cooled at 4° C. and centrifuged at 400×g for 5 minutes, and then the cells were washed. 3) Cell staining: Cells were stained using Annexin V-FITC / PI Apoptosis Detection Kit (HaiGene Biotech Co., Ltd, S0185), i.e., add 300 μL binding buffer, followed by 5 μL Annexin V-FITC, mix, and incubate at room temperature for 15 min, protected from light; then add 5 μL PI and stain for 5 min; replenish with 200 μL binding buffer. 4) Cell flow cytometry: Flow cytometry of stained cells was performed using a BD FACSAria II flow cytometer, and a report of cell classification analysis was generated by the instrument. 5) Interpretation of Cell Flow Cytometry Reports: A) Annexin-V negative-PI negative represents normal cells, i.e., the lower left group in the 4-frame cell classification diagram, and the percentage of cells is the percentage of live cells. B) Annexin-V positive-PI negative represents cells in the early stage of apoptosis, i.e., the lower right group in the 4-frame cell classification diagram, and the percentage of cells in this group is the percentage of cells in the early stage of apoptosis. C) Annexin-V positive-PI positive represents late stage apoptotic or necrotic cells, i.e., the top right group of the 4-frame cell classification diagram, and the percentage of cells is the percentage of cells in the late stage of apoptosis. FIG. 27 shows that the percentage of viable cells on 3D FloTrix microcarriers before cryopreservation was 90.4%, while the percentage of viable cells after cryopreservation and then resuscitation was 89.6%, a decrease of just under 1%, thus indicating that cells on 3D FloTrix microcarriers used in the present invention can be preserved in situ with the microcarriers and can maintain their activity after resuscitation.

[0082] (3) In situ cell counting method: 1) Microcarrier harvesting: 20 mg of the three-dimensional microcarrier suspension containing cells was harvested through a 70 μm cell sieve and washed once with PBS to wash off any cells not attached to the microcarriers. The cell-containing microcarrier suspension was then centrifuged at 1500 rpm for 2 minutes and the supernatant was discarded. 2) Cell counting: Incubate at 37°C for 2-5 hours in 3mL of 0.1% crystal violet solution (0.1g crystal violet, 2.1g citric acid, 20μL Tween-80, 100ml deionized water), and count the cells in a cell counting plate. 3) Result analysis: The results in Figure 28 show that after cryopreservation and resuscitation, the number of cells on 3D FloTrix microcarriers remained consistent with that before cryopreservation, while the number of cells on the competitive microcarriers (GE Cytodex, Cat. No.: 17-1271-01, purchased from Beijing SynchroFur Technology Co., Ltd.) was greatly reduced due to microcarrier rupture and cell scattering; therefore, in this invention, the use of 3D FloTrix microcarriers for cell culture, followed by cryopreservation and subsequent resuscitation, enables in situ three-dimensional cell freezing, and maintains the whole chain of cell culture and preservation methods in a three-dimensional environment.

[0083] II. The cells in the sections were AD-MSC cells (from Tsinghua University School of Medicine); the three-dimensional microcarriers were 3D FloTrix microcarriers purchased from Beijing Huaguan Biotechnology Co., Ltd., with a catalog number of CNF-F01T-50. 1. The three-dimensional microcarrier suspension containing AD-MSC cells was centrifuged at 400 x g for 2 minutes and the supernatant was discarded. 2. Cryopreservation solution (90% FBS + 10% DMSO) was added and distributed into cryogenic storage tubes (1 ml of cryopreservation solution contained 10 mg of cell-laden microcarriers, 0.5-1 ml per tube). 3. The cryogenic storage tubes were loaded into a cell programmed cooling box, placed in a -80°C freezer within 5 minutes, and transferred to liquid nitrogen after 24 hours. 4. Resuscitation of cells attached to microcarriers: For resuscitation, a pre-warmed basal medium at 37°C was prepared, then the cryogenic storage tube was quickly placed in a 37°C water bath with constant gentle shaking during this time, and the tube was quickly removed from the water bath when a single grain of rice-sized ice remained (approximately 1-2 min in the water bath). The thawed three-dimensional microcarrier suspension containing the cells was then immediately diluted with basal medium at a dilution ratio of 1:5, followed by centrifugation at 400×g for 2 min, and then resuspended for later use.

[0084] 5. Cell detection methods: (1) In situ calcein-AM / PI staining of live cells: 1) Kit: Animal Cell Killing Viability / Cytotoxicity Assay Kit, Catalog Number: KGAF001; 2) 50–100 μl of the three-dimensional microcarrier suspension containing cells resuscitated before or after cryopreservation was added to a 96-well plate, respectively; 3) The supernatant was removed as much as possible, PBS was added, washed once, and after 2 minutes, the PBS was removed as much as possible. Each well was stained by adding 100 μL of staining solution made according to the kit's instructions. After staining for 20 to 30 minutes at room temperature and protecting from light, the results of observation under a fluorescent microscope are shown in Figure 29. As can be seen from Figure 29, live cells were stained with the dye and observed to emit light by fluorescence, and the cells that emitted light were live cells. The irradiated spherical microcarriers demonstrated that the live cells were still attached to the microcarriers. Figure 29 shows the resuscitation results of AD-MSCs after 1-2 days of cryopreservation on 3D FloTrix microcarriers, and the luminescent live cells were still attached to the microcarriers, indicating that 3D FloTrix aids in the cryopreservation of MSCs. Figure 30 shows that MSCs on 3D FloTrix microcarriers have a viable cell ratio of more than 90% before and after cryopreservation, and the difference in cell activity before and after cryopreservation was not significant. Therefore, the MSCs on 3D FloTrix microcarriers used in the present invention can be preserved in situ with the microcarriers and can maintain their activity after recovery.

[0085] (3) Lysed cell counting method: 1) Microcarrier harvesting: 20 mg of the three-dimensional microcarrier suspension containing cells was harvested through a 70 μm cell sieve and washed once with PBS to wash off any cells not attached to the microcarriers. The cell-containing microcarrier suspension was then centrifuged at 1500 rpm for 2 minutes and the supernatant was discarded. 2) Cell counting: 3 mL of lysis solution (lysis solution formulation: 0.1% collagenase, 0.1% ethylenediaminetetraacetic acid, 0.05% trypsin; % indicated g / 100 mL) was added and incubated in an incubator for 30 minutes, during which the cells were gently blown several times with a 1 mL pipette every 10 minutes; after 30 minutes, the microcarriers were completely dissolved and 3 mL of total medium was added to terminate the lysis process; the tube was centrifuged at 200 × g for 5 minutes, the supernatant was discarded, and the cells were resuspended and counted through a cell counting plate. 3) Result analysis: The results in Figure 31 show that after cryopreservation and resuscitation, the number of cells on the 3D FloTrix microcarriers remained consistent with the number before cryopreservation. Thus, in the present invention, the use of 3D FloTrix microcarriers for MSC culture allows for long-term cryopreservation, maintains the entire chain of cell culture, and preserves in a three-dimensional environment.

[0086] Example 7: Large-scale expansion of stem cells using three-dimensional culture methods I. Continuous cell growth culture in stirred bioreactors 1. Ten tablets (total 200 mg) of 3D TableTrix microcarriers (Beijing Huaguan Biological Technology Co., Ltd.; catalog number: F01-100) were placed into a cell culture flask with built-in impeller (Belco Glass, USA; catalog number: 1965-61001) and 10 ml of cell culture medium was added. 2. 50 mL of adipose-derived MSC cell suspension (containing 2 million cells) was inoculated into the impeller-equipped cell culture flask containing the microcarriers prepared in step 1. 3. The impeller-equipped cell culture flask from step 2 was placed on a 3D FloTrix™ mini-spin small-scale bioreactor (Beijing Huaguan Biotechnology Co., Ltd.; 3D miniSPIN M1) in a cell culture incubator and incubated at a constant speed of 60 rpm clockwise for 6 days (replenished with 60 mL of fresh medium on day 3). Cell sampling and counting were performed daily to obtain data for cell expansion curves. On day 6, microtissues containing 2 million cells (i.e., cell-containing microcarriers) harvested from cell culture flasks were resuspended in 50 mL of fresh medium using an electric pipette and transferred to a 3D FloTrix™ vivaSpin bioreactor (Beijing Huaguan Biological Technology Co., Ltd.; 3D vivaSPIN V1, FIG. 33) and 50 tablets (total 1 g) of 3D TableTrix microcarriers (Beijing Huaguan Biological Technology Co., Ltd.; Catalog No.: F01-100) pre-resuspended (left for 0-30 min) in 50 mL of medium were added. Cells were sampled and counted daily to obtain data on cell growth curves and also to measure cell activity. The process parameters set for the 3D FloTrix™ vivaSpin bioreactor were controlled as follows: active pumping of 12 ccm of 5% CO2 throughout the process, and 37° C. At T=0 hours, 800 mL of complete medium was actively pumped at a rate of 10 mL / min, 500 mL of liquid was actively pumped at a rate of 10 mL / min, followed by 500 mL of fresh medium at T=96 hours, 500 mL of liquid was actively pumped at a rate of 10 mL / min, followed by 50 tablets of 3D TableTrix™ microcarriers resuspended in 500 mL of fresh medium that was actively pumped into the reactor at 10 mL / min at T=144 hours. The stirring speed for T = 0–12 h and T = 144–156 h was variable speed with cycles of 40 rpm for 5 min and 20 rpm for 20 min, and a constant speed of 60 rpm for the remaining time. Cell sampling and counting method: All samples were taken in 3 replicates of 1 mL samples by pipetting through the sampler of the 3D FloTrix™ vivaSpin bioreactor, transferred to centrifuge tubes, centrifuged at 400×g for 2 min, and the supernatant was pipetted off. 1 mL of lysis solution (lysis solution formulation: 0.1% collagenase, 0.1% ethylenediaminetetraacetic acid, 0.05% trypsin; % indicated g / 100 mL) was added and incubated in an incubator for 30 min until the microcarriers were completely dissolved (during which the cells were gently blown several times with a 1 mL pipette every 10 min), and 1 mL of total medium was added to terminate the lysis process; centrifuged at 200×g for 5 min, the supernatant was discarded, and the cells were suspended and counted through a cell counting plate. Cell activity assay: 50μL of cell suspension was mixed with 50μL of 0.2% trypan blue staining solution, and an appropriate amount of cells was taken for counting. Either an automatic cell counter (e.g. Countstar® Shanghai Ruichu Biotechnology Co., Ltd.) or a cell counting plate can be used. The number of clear cells and the number of blue-stained cells were recorded separately, and the number of cells in the cell suspension was extrapolated from the corresponding dilution ratio. Cell activity ratio (%) = number of unstained cells / (number of unstained cells + number of blue-stained cells) × 100%. The results of the cell count assay are shown in Figure 32. The entire culture process started with 2 million cells inoculated directly onto 10 tablets of Microcarriers™ in a cell culture flask of a 3D FloTrix™ Minispin for 6 days, followed by the addition of new Microcarriers™ and growth in a 3D FloTrix™ viSpviin automated bioreactor for 8 days of further culture to reach a final harvest of 100 million cells, of which in the small-scale bioreactor from day 0 to day 6, after 6 days of culture, 49.2 million cells were resuscitated from the initial 2 million, representing an increase in proliferation of about 24.6 times. Using them as seeds, after 8 days of culture in the automated bioreactor, a total of about 2.59 billion cells were resuscitated, representing an expansion of about 52.7 times, i.e., a total expansion of about 1,295 times in 14 days of culture. The results of the cell viability assay are shown in Figure 34. The results showed that the cell viability was maintained at over 90% throughout the culture process. All culture methods were performed with only one person and 0.25 ml of 0.2 ml of 0.25 ... 2 It required less than 1000 sq. m. space, a culture period of 14 days, 2 liters of culture medium and 110 microcarriers, resulting in significant savings in human, material and financial resources.

[0087] II. Continuous Cell Growth in 3D FloTrix™ vivaSpin Bioreactors (1000 mL) 1. 40 tablets of 3D TableTrix™ microcarriers were added to a sterile 3D FloTrix™ vivaSpin bioreactor and 40 mL of a suspension of 6.6 million adipose-derived MSCs were resuspended on a sterile work bench. 2. The bioreactor was assembled and process parameters were set to control as follows: active pumping of 10 ccm of 5% CO2 throughout the process, and 37°C. At T=0 hours, 460 mL of complete medium was actively pumped at a rate of 10 mL / min, followed by 500 mL of liquid actively pumped at a rate of 10 mL / min at T=48 hours. Every 24 hours thereafter, 500 mL of liquid was actively pumped at a rate of 10 mL / min, followed by 500 mL of fresh medium actively pumped into the reactor at a rate of 10 mL / min until 120 hours. After 500 mL of liquid n was actively pumped at 10 mL / min at T=120 hours, 40 tablets of 3D TableTrix™ microcarriers resuspended in 500 mL of fresh medium were actively pumped into the reactor at a rate of 10 mL / min. From T = 0 to 120 h, the stirring speed was a constant speed of 60 rpm, from T = 120 to 136 h, the stirring speed was variable speed with cycles of 60 rpm for 5 min and 20 rpm for 20 min, and from T = 136 to 240 h, the stirring speed was a constant speed of 60 rpm (after 240 h, the stirring mode was a constant speed of 60 rpm). Cells were sampled and counted daily to obtain data for cell growth curves and to measure cell activity. The results are shown in Figures 35 and 36. The results show that adipose-derived MSCs grew at 2.24 x 10 cells in a 3D FloTrix™ vivaSpin bioreactor from 6.6 million cells seeded onto 40 microcarriers. 9 The cells were expanded and an additional 40 microcarriers (resuspended in medium) were pumped in on day 6, resulting in approximately 340-fold expansion after 10 days of fully contained culture. The activity of adipose-derived MSCs in the 3D FloTrix™ vivaSpin bioreactor was consistently maintained above 90%.

[0088] III. Partial characterization of MSCs cultured in 2D flasks and 3D microcarriers using flow cytometry analysis The microtissues cultured in steps 1 and 2 were harvested from the cell suspension according to the patent application "Method for harvesting cells on three-dimensional microcarriers" (Chinese Patent Application No. 201910101736.0), centrifuged at 200 × g for 5 minutes, the supernatant was discarded, and divided equally into 9 centrifugation tubes, and they were mixed with PE-CD19 (Biolegend, USA, Catalog No.: 302207), APC-CD14 (Biolegend, USA, Catalog No.: 301803), FITC-CD34 (Biolegend, USA, Catalog No. The cells were stained with fluorescently labeled antibodies of MSCs: APC-CD45 (Biolegend, USA, Catalog No. 343503), APC-CD45 (Biolegend, USA, Catalog No. 368511), FITC-HLADR (Biolegend, USA, Catalog No. 307603), FITC-CD90 (Biolegend, USA, Catalog No. 328107), FITC-CD105 (Biolegend, USA, Catalog No. 800505), and FITC-CD73 (Biolegend, USA, Catalog No. 344015), with blank buffer as a negative control. After incubation in a 4°C freezer for 0.5 h, the cells were washed twice with PBS, resuspended in PBS, and transferred to a flow tube. The stained cells were subjected to flow cytometry using a BD FACSAria II flow cytometry sorter, and the expression ratios of MSC characteristic surface markers were reported by the instrument for cell sorting analysis.

[0089] [Table 3]

[0090] The data showed that MSCs cultured at large scale on 3D microcarriers had a similar expression profile to those cultured in traditional 2D, and both met qualification criteria. The above data suggested that the use of three-dimensional microcarriers for large-scale culture of MSCs is feasible, cost-effective and can be carried out in a closed system with a high degree of process control and minimal space occupation. [Industrial Applicability]

[0091] The present invention provides a set of three-dimensional microcarrier-based cell culture methods, including a three-dimensional microcarrier-based cell resuscitation method, a three-dimensional microcarrier cell culture-based in situ passaging method, a three-dimensional microcarrier-based method for large-scale expansion of cells, a three-dimensional microcarrier-based in situ cell cryopreservation method, a cell attachment culture method on three-dimensional microcarriers, a method for harvesting cells on three-dimensional microcarriers, and a method for sampling cells cultured on microcarriers.

[0092] The three-dimensional microcarrier-based cell resuscitation method provided by the present invention uses three-dimensional microcarriers as a substrate, which allows the seeding of resuscitated cells or microtissues directly onto new carriers (microcarrier resuscitation method), and then the process of cell attachment, adhesion and growth on the microcarriers can be completed, avoiding the complicated operation of first having to recover the cells to a two-dimensional substrate for full growth and harvesting before the cells are seeded onto a new culture substrate. Microcarriers have high elasticity, they are gentler during dynamic incubation, which buffers the irritation caused by the collision of fragile and freshly resuscitated cells with the microcarriers, and porous microcarriers have a larger specific surface area that provides more space for wall attachment. The porous structure also allows fragile cells to be embedded in the pores, which are less susceptible to the shear force of the flow medium. This method allows cell passaging to be performed without interrupting the three-dimensional culture process, thereby allowing fully enclosed cell culture and continuous growth, with the aim of maintaining the cells in a three-dimensional state throughout the preparation of three-dimensional cultured cell products or tissue engineering products, and providing some innovation or complementation or experience in the field of cell resuscitation. Advantages of the cell resuscitation method based on three-dimensional microcarriers: (1) streamlining the process when the resuscitated cells are to be recultured in three dimensions, this method eliminates the cumbersome process and reagent consumables required for the process from single cell resuscitation and cryopreservation after the cells have grown to full size on the two-dimensional culture substrate that has been passaged and digested before being inoculated into the microcarriers for three-dimensional culture, which can save a lot of time and cost; (2) the obtained cells are close to in vivo cells, the cryopreservation and resuscitation for the culture and subsequent reculture of the cells are maintained similarly to the in vivo three-dimensional environment, therefore the obtained cells are close to in vivo cells in terms of physiology and function.

[0093] The in situ passaging method based on three-dimensional microcarrier cell culture provided by the present invention inoculates the cultured microcarriers (i.e., seed microcarriers) into new microcarriers to achieve "in situ passaging". Cells can attach, spread, grow, and migrate on the three-dimensional microcarriers, and then attach to the new microcarriers by shedding the cells on the seed microcarriers, and then grow and grow on the new microcarriers, or the new microcarriers and the seed microcarriers come into contact, and the cells on the seed microcarriers migrate, so that the cells on the seed microcarriers are transferred to the new microcarriers to continue growing, which can be judiciously utilized to achieve "in situ passaging" of cells on the three-dimensional microcarriers. The advantages of the in situ passaging method based on three-dimensional microcarrier cell culture: (1) it avoids the traditional two-dimensional cell digestion and passaging, which not only reduces the damage to cells by the digestion solution, but also eliminates the tedious operation method of digestion and passaging, thus saving considerable labor and resources in large-scale culture expansion and obtaining high-quality cells; (2) it is time-saving, it takes time to digest cells and inoculate them, but the passaging method saves time; (3) the choice of in situ passaging eliminates the need for reagents and consumables such as digestion solution, streamlining the method and reducing costs.

[0094] The in situ cell cryopreservation method based on three-dimensional microcarriers provided by the present invention allows for in situ cryopreservation of cell cultures on three-dimensional microcarriers without the need to separate the cells from the microcarriers and then cryopreserve them, but allows the cells to be stored directly in situ on the three-dimensional microcarriers. This method allows for cryopreservation of cells without interrupting the three-dimensional culture process, since the microcarriers are not destroyed or deformed during cryopreservation (-20 to -196°C liquid nitrogen storage) and resuscitation (i.e., warming and thawing from liquid nitrogen or -80°C), while the cells remain attached to the three-dimensional microcarriers, thus maintaining the cells in a three-dimensional state throughout the preparation of the three-dimensional cultured cell product or tissue engineered product, and facilitating the storage, transportation and resuscitation of the expanded three-dimensional cell product by the user. This method is suitable for long-term storage of cells cultured on three-dimensional microcarriers with high viability and unchanged cellular properties after resuscitation.

[0095] The method of cell attachment culture on three-dimensional microcarriers provided by the present invention uses a high-density, low-volume method in which the three-dimensional microcarriers are directly swelled with a cell suspension. The cell suspension is absorbed into the microcarriers immediately after being added to the dry three-dimensional microcarriers, facilitating the direct drawing of cells into the pores of the microcarriers during inoculation, dispersing the cells into the pores of the microcarriers and then attaching for regrowth, providing more growth areas for the cells, and on the other hand, saving the step of needing to swell the microcarriers in advance, thus simplifying the operation of inoculating the cells into the microcarriers. The aseptic operation of the culture is easier and the risk of contamination is avoided. This method is suitable for large-scale bioreactor culture, and the unique swelling-absorption property of the three-dimensional microcarriers allows a large number of cells to enter inside the connected openings of the carriers, better forming a bionic three-dimensional growth pattern, and promoting the function during in vitro cell culture.

[0096] The method of harvesting cells on three-dimensional microcarriers provided by the present invention is directed to harvesting cells on three-dimensional microcarriers. As cells are cultivated on the three-dimensional microcarriers, the present invention allows for gentle harvesting of cells on the carriers for subsequent application; the present invention targets the microcarriers for lysis, which dissolves the microcarriers from a solid to a liquid state, thereby releasing the cells on the carriers into a solution, which is separated from the cells by means of centrifugation, thus obtaining the cells. The lysis solution of the present invention is designed for the microcarriers and can be tailored to the composition of the microcarriers; the lysis solution dissolves the microcarriers, and is therefore gentle on the cells, maintaining cell activity and preserving proteins secreted by the cells themselves.

[0097] Comparing the method for sampling cells cultured on microcarriers provided by the present invention with conventional suspension quantitative sampling methods, triplicate samples of cells counted in a microcarrier culture system using the quantitative microcarrier sampling method provided by the present invention are reproducible (small variation) and very close to the actual total. The three-dimensional microcarrier-based method for large-scale expansion of cells provided by the present invention demonstrates that large-scale culture of MSCs using three-dimensional microcarriers is feasible, cost-effective, and can be carried out in a closed system with a higher degree of process control and minimal space occupancy, which reduces the cost and time of producing cells and allows cell therapy products to be prepared on a large scale for clinical use. The present invention is of great importance for the three-dimensional culture of cells, especially stem cells.

Claims

1. A set of three-dimensional microcarrier-based cell culture methods, including a three-dimensional microcarrier-based cell resuscitation method, a three-dimensional microcarrier-based in situ passaging method, and a three-dimensional microcarrier-based in situ cell cryopreservation method; The three-dimensional microcarrier-based cell resuscitation method comprises the steps of: inoculating a cell suspension of cryopreserved cells or cryopreserved cell microtissues after thawing onto the three-dimensional microcarriers for cell resuscitation and culture; The in situ passaging method based on three-dimensional microcarrier cell culture comprises the following steps: inserting a seed microcarrier into a new microcarrier; where the seed microcarrier is the microcarrier that is cultured with the cells to be passaged; The three-dimensional microcarrier-based in situ cell cryopreservation method comprises the following steps: (A1) centrifuging the three-dimensional microcarrier suspension of cells and discarding the supernatant to obtain cell-containing carriers; (A2) mixing the cell-containing carriers obtained in step (A1) with a cryopreservation solution and adding them to a cryogenic storage tube; (A3) cooling the cryogenic storage tube to which the cell-containing carriers were added in step (A2) and then transferring it to liquid nitrogen, allowing in situ cryopreservation of the cells on the three-dimensional microcarriers.

2. 10. The method of claim 1, wherein the method further comprises a method of cell attachment culture on three-dimensional microcarriers, a method of harvesting cells on the three-dimensional microcarriers, and a method of sampling cells cultured on the microcarriers, wherein the method of cell attachment culture on three-dimensional microcarriers is used to seed cells on the three-dimensional microcarriers, the method of harvesting cells on the three-dimensional microcarriers is used to separate cells from the three-dimensional microcarriers, and the method of sampling cells cultured on the microcarriers is used to perform calculations of the total number and / or cell density of cells cultured on the microcarriers; The method for cell attachment culture on three-dimensional microcarriers includes the following steps: (B1) cell inoculation: mixing a cell suspension with dry three-dimensional microcarriers to obtain microcarriers mixed with a cell suspension; (B2) cell attachment: incubating the microcarriers mixed with the cell suspension obtained in step (B1) so that the cells attach to the three-dimensional microcarriers; (B3) cell culture: adding a complete culture medium after cell attachment in step (B2) and carrying out culture; The method for harvesting cells on three-dimensional microcarriers includes the following steps: (C1) allowing the three-dimensional microcarriers attached to the cultured cells to settle naturally or by centrifugation, and removing the supernatant to obtain three-dimensional microcarriers containing cells; (C2) adding a dissolution solution to the three-dimensional microcarriers containing cells, and incubating and dissolving the three-dimensional microcarriers therein; (C3) after the three-dimensional microcarriers are completely dissolved in step (C2), adding a stop solution to complete the dissolution or proceeding directly to step (C4) to complete the dissolution; and (C4) centrifuging the system obtained in step (C3), discarding the supernatant, and harvesting the cells on the three-dimensional microcarriers; The method for sampling cells cultured on microcarriers includes the steps of: (D1) weighing a predetermined weight of microcarriers for cell culture, placing them in a sampling tube, adding liquid to simulate a cell culture process, soaking them at 4-60°C for 0-24 hours, and then marking a scale corresponding to the volume occupied by the microcarriers on the sampling tube as a standard scale; (D2) sampling the microcarriers from the cell culture system using the sampling tube to make the cells cultured on the microcarriers into the standard scale as follows:

3. 3. The method according to claim 1 or 2, wherein the three-dimensional microcarrier-based cell resuscitation method is a three-dimensional microcarrier-based dynamic cell resuscitation method or a three-dimensional microcarrier-based static cell resuscitation method; The dynamic cell resuscitation method based on three-dimensional microcarriers includes the following steps: (E1) collecting three-dimensional microcarriers, placing them in a cell culture vessel, adding cell culture medium, and immediately treating them with the next step or a treatment method of standing or stirring for 1 minute or more; (E2) thawing cryopreserved cells or cryopreserved cell microtissues to obtain a cell suspension, inoculating the cell culture vessel containing the microcarriers prepared in step (E1) at 37°C, then adding cell culture medium to adjust the ratio of microcarriers to medium to 1 mg:1-1000 μL, then placing the cell culture vessel on a stirrer and stirring in an incubator; (E3) continuing to stir the cells after step (E2) is completed until cell resuscitation is completed; The static cell resuscitation method based on three-dimensional microcarriers includes the following steps: (F1) preparation of static culture vessel and microcarriers: harvesting three-dimensional microcarriers and placing them in a cell culture vessel; (F2) thawing and inoculation of cryopreserved cells: harvesting cryopreserved cells or cryopreserved cell microtissues, thawing them at 37°C, and then inoculating them into the static culture vessel containing the microcarriers prepared in step (F1) for 20-240 minutes; and (F3) after completion of step (F2), continuing to culture the cells until cell resuscitation is completed.

4. 4. The method according to claim 3, wherein the stirring is constant speed stirring or variable speed alternating stirring or variable speed periodic stirring, the stirring is clockwise stirring, counterclockwise stirring or alternating stirring, the stirring time is 0.1-100 hours, and the stirring speed is 1-200 rpm.

5. 5. The method according to claim 4, wherein the stirring is as follows: (a) or (b) or (c): (a) Allow to stand for 1-4 hours, then stir clockwise at variable periodic speed for 1-24 hours, then stir clockwise at constant speed for up to 96 hours; (b) Periodically varying clockwise stirring for 1-24 hours, followed by constant clockwise stirring for up to 96 hours; (c) Stir constantly clockwise for 1 to 96 hours.

6. In step (E2) or (F2), the ratio of the number of cells to the number of microcarriers is 5 to 100 x 10 ３ The method according to any one of claims 3 to 5, wherein the microcarrier is a cell / mg microcarrier.

7. 7. The method of any one of claims 1 to 6, wherein in the three-dimensional microcarrier cell culture based in situ passaging method, the seed microcarriers have a cell density of 10,000 to 1,000,000 cells / mg of microcarrier and the ratio of seed microcarriers to fresh microcarriers is 0.0002 to 200 mg seed microcarriers / mg fresh microcarriers.

8. The method according to any one of claims 1 to 6, wherein the three-dimensional microcarrier cell culture-based in situ passaging method is a three-dimensional microcarrier cell culture-based dynamic in situ passaging method or a three-dimensional microcarrier cell culture-based static in situ passaging method; The dynamic in situ passaging method based on three-dimensional microcarrier cell culture comprises the following steps: (G1) preparing a new microcarrier suspension: placing new microcarriers in an impeller-equipped cell culture flask, adding cell culture medium at a ratio of 1-2,000 μL:1 mg of microcarrier, and standing or stirring for more than 0 hours to obtain a new microcarrier suspension; (G2) preparing a seed microcarrier suspension: the seed microcarriers have a cell density of 10,000-1 million cells / mg of microcarrier, and resuspending the seed microcarriers in cell culture medium to 0.1-50 mg / mL to obtain a seed microcarrier suspension; (G3) inoculation: (G1 (G2) mixing the seed microcarrier suspension prepared in (G3) with the impeller-equipped cell culture flask containing the fresh microcarrier suspension prepared in (G4), wherein the ratio of seed microcarriers to fresh microcarriers prepared in (G1) is 0.0002-200 mg seed microcarriers / mg fresh microcarriers, adding cell culture medium to adjust the microcarrier to medium ratio to 1 mg:1-1000 μL, placing the impeller-equipped cell culture flask on a stirrer and placing in an incubator for stirring for 0-100 hours; (G5) culturing: continuing stirring to complete dynamic in situ passaging; The static in situ passaging method based on three-dimensional microcarrier cell culture includes the following steps: (H1) preparing a seed microcarrier suspension: the cell density of the seed microcarriers is 10,000-1,000,000 cells / mg microcarrier, and the seed microcarriers are resuspended in cell culture medium to 0.1-50 mg / mL to obtain a seed microcarrier suspension; (H2) seeding: the seed microcarrier suspension prepared in (H1) is dropped into new microcarriers, the ratio of seed microcarriers to new microcarriers is 0.0002-200 mg seed microcarriers / mg new microcarriers, mixed thoroughly, and placed in an incubator for incubation for 0.5-24 hours; (H3) culturing: adding cell culture medium and continuing the culturing, i.e. completing the static in situ passaging.

9. 9. The method of claim 8, In step (G1), the ratio of new microcarriers and cell culture medium placed in the impeller-embedded cell culture flask is 100 mg microcarriers / 10 mL cell culture medium, and the microcarriers are left for 0.1 to 24 hours after being placed in the impeller-embedded cell culture flask; In step (G2), the cell density in the seed microcarriers is between 150,000 and 500,000 cells / mg microcarriers; and / or the content of seed microcarriers in the seed microcarrier suspension is between 1 and 10 mg / mL; Step (G3), the seed microcarrier to fresh microcarrier ratio is 1-10 mg seed microcarrier / 100 mg fresh microcarrier; and / or the microcarrier to medium ratio is adjusted to 1-10 mg:1 mL by adding cell culture medium; and / or the stirring speed is 1 rpm to 200 rpm, the stirring is by constant speed stirring or variable speed alternating stirring or variable speed periodic stirring, and the stirring direction is clockwise stirring, counterclockwise stirring or alternating stirring.

10. 10. The method of claim 8 or 9, A method in which in step (H1), the cell density in the seed microcarriers is 120,000-500,000 cells / mg microcarriers and the content of seed microcarriers in the seed microcarrier suspension is 5-15 mg / mL; A method wherein in step (H2), the ratio of seed microcarriers to fresh microcarriers is between 1 and 3 mg seed microcarriers / 20 mg fresh microcarriers; and / or the incubation time is between 0 and 24 hours.

11. The method according to any one of claims 1 to 10, wherein in the three-dimensional microcarrier-based in situ cell cryopreservation method, in the step (A1), the centrifugation is carried out at a speed of 50 to 1610 xg for a time of 1 to 10 minutes, and in the step (A2), the ratio of the volume of the cryopreservation solution to the mass of the cell-containing carrier is 1 mL:0.1 to 50 mg.

12. The method according to any one of claims 1 to 11, wherein the three-dimensional microcarrier based in situ cell cryopreservation method comprises a cryopreservation solution consisting of 10% DMSO, 10-90% FBS and 0-80% basal medium, 10% DMSO and 0-80% basal medium, 10-90% FBS and 0-80% basal medium, or other commercially available cryopreservation solutions, and the cryogenic storage tube is placed in a freezer and cooled to -20 to -196°C, the cooling being programmed or unprogrammed, the programmed cooling having a cooling rate of -1 to -15°C / min, and the freezing time being 1 to 24 hours.

13. The method according to any one of claims 1 to 12, wherein in the three-dimensional microcarrier-based in situ cell cryopreservation method, in step (A2), the cooling is performed by placing the cryogenic storage tube in a cell cooling box and placing it in a freezer for cooling within 1 to 10 minutes, and the pre-heated PBS or basal medium at 37°C added to the cryogenic storage tube is diluted at a ratio of 1:5 to 30.

14. The method according to any one of claims 1 to 13, wherein the three-dimensional microcarrier-based in situ cell cryopreservation method further comprises, after the step (A3), a cell resuscitation step: placing the cryogenic storage tube after the treatment of the step (A3) in a water bath, and removing the water bath when the ice in the cryogenic storage tube melts, thereby resuscitating the cells; then adding pre-heated PBS or basal medium at 37°C to dilute the cells, and then centrifuging; the water bath has a temperature of 35 to 0°C and a time of 1 to 5 minutes; and the centrifugation after the step (A3) is performed at a speed of 50 to 1610 x g for 1 to 10 minutes.

15. In the method for culturing cells on a three-dimensional microcarrier, the cell suspension is 1×10 ４ ~1×10 ８ A method for culturing cells on a three-dimensional microcarrier according to any one of claims 2 to 14, characterized in that the cell suspension has a density of 1000 μL / mL, the cell suspension is obtained by resuspending the cells in culture medium or liquid biomatrix raw material, the ratio of the volume of the cell suspension to the mass of the three-dimensional microcarrier is 1 to 1000 μL:1 mg, and in step (B1), the cell suspension is mixed with the three-dimensional microcarrier and the cell suspension is dropped onto the three-dimensional microcarrier.

16. The method according to any one of claims 2 to 15, wherein the incubation conditions for the method of cell attachment culture on a three-dimensional microcarrier are a temperature of 35 to 40°C, a time of 0.5 to 24 hours, and a volume percentage concentration of carbon dioxide of 5 to 30%, and the attachment method includes a gravity attachment method, a swelling attachment method, a stirring rotation type attachment method, a centrifugal method, a surface acoustic wave method, or a magnetic attachment method.

17. The method according to any one of claims 2 to 16, wherein the culture conditions in the method for cell attachment culture on a three-dimensional microcarrier are a temperature of 35 to 40°C and a percentage concentration of carbon dioxide of 5 to 30% by volume; the three-dimensional microcarrier is a porous microcarrier; and prior to step (B1), there is a step of gas sterilization, radiation sterilization or UV sterilization of the three-dimensional microcarrier.

18. 18. The method for harvesting cells on three-dimensional microcarriers according to any one of claims 2 to 17, wherein the lysis solution comprises an active ingredient for eliminating cell attachment and lysing the microcarriers, the active ingredient being selected from an aqueous solution of at least one of collagenase, pepsin, hyaluronidase, dispase, neutral protease, proteinase K, matrix metalloproteinase, sodium citrate, trypsin, deoxyribonuclease, trypsin substitutes, protein hydrolase, ethylenediaminetetraacetic acid, lysozyme and glutathione.

19. The method for harvesting cells on a three-dimensional microcarrier, characterized in that in the step (C1), the centrifugation is carried out at a speed of 50 to 1610 xg for 1 to 10 minutes, and the step of removing the supernatant comprises centrifugation, pipetting the supernatant, adding an appropriate amount of PBS, gently shaking by hand for 20 to 30 seconds, pipetting the supernatant, and repeating the PBS washing once.

20. The method according to any one of claims 2 to 19, wherein the ratio of the mass of the three-dimensional microcarrier for attaching and culturing the cells to the volume of the dissolution solution is 1 mg:0.01 to 5 mL, the incubation is performed at a temperature of 4°C to 40°C for 10 seconds to 24 hours, the ratio of the mass of the three-dimensional microcarrier for attaching and culturing the cells to the volume of the dissolution solution is 1 mg:0.01 to 5 mL, and in step (C4), centrifugation is performed at a speed of 50 to 1610 x g for 1 to 10 minutes.

21. 21. The method of claim 2, wherein the method for sampling cells cultured on microcarriers for calculation of the total number of cells cultured on the microcarriers and / or the cell density comprises the following steps: (D1) weighing a predetermined weight of microcarriers for cell culture, placing them in a sampling tube, adding liquid to simulate a cell culture process, and soaking them at 4-60°C for 0-24 hours, and then marking a scale corresponding to the volume occupied by the microcarriers as a standard scale on the sampling tube; (D2) sampling the microcarriers from a cell culture system using the sampling tube to bring the volume of the microcarriers to the standard scale; (D3) counting the cells in the microcarriers collected in (D2); (D4) calculating the total number of cells in the entire cell culture system based on the weight of the microcarriers weighed in (D1); and calculating the number of cells counted in (D3) and the total weight of the microcarriers counted in (D3) for the entire cell culture system and calculating the cell density.

22. The method according to any one of claims 2 to 21, wherein in step (D1), the liquid added has the same or similar viscosity, hydrophilicity, pH, and ion concentration as a medium used for cell culture on the microcarriers; in step (D1), the liquid and the microcarriers are in a ratio of 10 to 1000 μL:1 mg; and further comprising natural precipitation or centrifugal precipitation after immersing the microcarriers in the liquid.

23. 23. The method according to any one of claims 2 to 22, wherein in step (D2), the sampling of the microcarriers from the cell culture system using a sampling tube is carried out according to a method comprising the following steps: pipetting the suspension from the cell culture system, adding it to the sampling tube, and allowing the microcarriers to settle naturally or by centrifugation, and observing whether the volume of the microcarriers has reached the standard scale calibrated in step (D1); if not, continuing to pipette the suspension until the standard scale is reached, and if it has been exceeded, removing the excess microcarriers from the sampling tube.

24. 24. The method of any one of claims 2 to 23, wherein in step (D4), the total number of cells in the entire cell culture is equal to the number of cells counted in (D3) multiplied by the total mass of microcarriers related to the weight of the microcarriers weighed in (D1) above; and in step (A4), the total number is divided by the total mass or number of microcarriers, or the total volume of the culture to obtain different units of cell density in cells / mg microcarrier, cells / microcarrier, cells / mL culture.

25. 25. The method according to any one of claims 1 to 24, further comprising a method for large scale growth of cells based on three-dimensional microcarriers, said method comprising the following steps: achieving large scale growth of cells on three-dimensional microcarriers in a stirred bioreactor.

26. The method according to any one of claims 1 to 25, wherein the cells are adherent cells.

27. The method of claim 26, wherein the adherent cells are stem cells.

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