Preparation method of clinical-grade human umbilical cord mesenchymal stem cells

Abstract

The invention discloses a preparation method of clinical-grade human umbilical cord mesenchymal stem cells. According to the method, animal serum and antibiotics are not used, the size of tissue blocks is controlled to be 3-5mm * 3-5mm, the tissue blocks are flatly laid in a cell culture dish, and the distance between the tissue blocks is uniform, so that cells can climb out of the tissues, the number of initial cells is increased, and cells in a main cell bank and cells in a working cell bank are stably passaged and produced. Generally, about (0.5-1.5) * 10 < 9 > main cell bank cells and about (0.5-1.5) * 10 < 12 > working cell bank cells can be obtained by culturing one umbilical cord tissue; according to the method, more human umbilical cord mesenchymal stem cells can climb out of tissues after being cultured for 7 days, and a large number of human umbilical cord mesenchymal stem cells with high purity and good state can be harvested after being cultured for 12 days, so that the primary culture period of the human umbilical cord mesenchymal stem cells is shortened, the time and the cost are saved, and finally the human umbilical cord mesenchymal stem cells with high purity and good uniformity are obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological product cell separation and culture, and in particular, relates to a method for preparing clinical-grade human umbilical cord mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of adult pluripotent stem cells derived from the early developmental mesoderm and ectoderm. They can be expanded and cultured in vitro. Because of their self-renewal and multiple differentiation potentials, as well as their immunomodulatory and anti-inflammatory effects, they are widely used in the treatment of various refractory diseases such as clinical hematopoietic support, graft-versus-host disease and lupus erythematosus.

[0003] Human umbilical cord mesenchymal stem cells (UC-MSCs) are derived from the umbilical cord tissue of healthy pregnant women after childbirth. They are usually discarded as medical waste, have no impact on the health of pregnant women and infants, and do not involve any ethical controversy. UC-MSCs exist in large quantities in the Wharton's jelly of the human umbilical cord. Compared with relatively mature mesenchymal stem cells such as bone marrow and adipose, UC-MSCs have the advantages of abundant sources, convenient collection, and strong proliferation ability. They do not express MHC-II class antigens, have low immunogenicity, and do not require matching for clinical use. They can also secrete a large number of cytokines to participate in the body's immune regulation, and have broad prospects for clinical application.

[0004] At present, the main methods for isolating UC-MSCs are enzymatic digestion and tissue block adhesion. Enzymatic digestion mainly uses one or more enzymes (trypsin, collagenase, hyaluronidase, etc.) to digest umbilical cord tissue to obtain target cells. However, the degree of enzymatic digestion is not easy to control. Insufficient digestion cannot obtain a sufficient number of cells. If the digestion time is too long, the interstitial protein and cell membrane protein will be destroyed, thereby damaging the cells and causing poor activity. In addition, the umbilical cord tissue after enzymatic digestion is a viscous liquid that needs to be filtered. It is not easy to separate cells during centrifugation, increasing the risk of contamination. More importantly, the various enzymes used to digest the umbilical cord introduce animal-derived components or exogenous factors, which is not conducive to subsequent clinical applications. The tissue adhesion method is simple to operate to obtain human umbilical cord mesenchymal stem cells, has low economic cost, does not introduce exogenous factors, and is easy to standardize the preparation method, which is particularly suitable for subsequent new drug development and clinical applications. In the traditional shearing and adhesion method, Wharton's jelly is not easy to adhere to the wall or is easy to fall off, resulting in a long time for cells to crawl out and a low acquisition rate, which seriously limits its mass application.

[0005] At present, many institutions at home and abroad are conducting storage and clinical trials of human umbilical cord mesenchymal stem cells, which require a high number of human umbilical cord mesenchymal stem cells to be used. According to the intravenous infusion of 1.0×10^6 / kg body weight of P5 human umbilical cord mesenchymal stem cells each time, three infusions are a course of treatment. A body weight of 50kg-100kg requires (1.5-3.0)×10^8 cells / course. If the clinical trial study meets the human umbilical cord mesenchymal stem cell treatment dose of at least 300 people per course of treatment, the total amount of P5 mesenchymal stem cells should be no less than 4.5×10^10. However, most institutions now process an umbilical cord tissue about 20 cm long, and the number of P0 human umbilical cord mesenchymal stem cells obtained is between (0.5-1.0)×10^7; the number of P2 human umbilical cord mesenchymal stem cells obtained is between (2.0-5.0)×10^8 (the passage ratio is about 1:6-1:7); the number of P5 human umbilical cord mesenchymal stem cells obtained is between (3.0-4.5)×10^10 (the passage ratio is about 1:3-1:5); the cell yield is obviously insufficient. The main reason is that the human umbilical cord tissue separation and culture method is improper and the cell doubling rate and doubling time after passage are low, resulting in insufficient number of cells in the P2 daughter (master) cell bank and the P5 working cell bank. How to maximize the number of human umbilical cord mesenchymal stem cells required for clinical trial research and even direct clinical application in the future is an urgent problem to be solved. Summary of the invention

[0006] The purpose of the present invention is to provide a method for separating and culturing human umbilical cord mesenchymal stem cells which is fast, efficient and free of animal-derived additives, so as to meet the increasing clinical research and application.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for isolating and preparing clinical-grade human umbilical cord mesenchymal stem cells, comprising the following steps:

[0009] S1, screen and obtain human umbilical cord tissue with no family history of genetic diseases and negative results of four infectious diseases and pathogenic microorganism tests;

[0010] S2, collecting human umbilical cord tissue and storing the human umbilical cord tissue in a tissue protection solution, wherein the tissue protection solution is physiological saline, does not contain any antibiotics, and is sealed and refrigerated for transportation;

[0011] S3, after taking the tissue protection solution for pathogenic microorganism detection, the umbilical cord is cut into small segments of 1-3 cm, and the umbilical cord is repeatedly cleaned with the tissue protection solution to remove blood on the surface of the umbilical cord;

[0012] S4, cutting the cleaned human umbilical cord tissue longitudinally along the venous opening, peeling off the umbilical vein and umbilical artery of the umbilical cord, and removing the umbilical cord vein membrane and the outer surface amniotic membrane to obtain Wharton's jelly, and cutting the Wharton's jelly into square tissue pieces of 3-5 mm×3-5 mm;

[0013] S5, the tissue pieces are spread flat on the cell culture dish, the spacing between the pieces should be uniform, the spacing between the pieces is 4-7mm, and after being fixed at 37°C for 2h, 2mL of human umbilical cord mesenchymal stem cell serum-free culture medium is slowly added, and the tissue pieces cannot be impacted during the addition, and the tissue pieces cannot float. The culture is cultured in a 37°C, 5% CO2 incubator, and 8mL of culture medium is added after 24h of culture. The culture dish is not moved during this period, and the cell growth condition is not observed. On the 7th day, a full medium change is performed and the cell growth condition is observed. Subsequently, a full medium change is performed every 3 days, and the cell growth condition is observed;

[0014] S6, when the fusion degree of the cell clones reached about 90%, they were rinsed with PBS, digested with digestive enzymes, and the cells were collected to obtain the primary cells P0 of human umbilical cord mesenchymal stem cells;

[0015] S7, inoculating the primary cells P0 into a culture flask, and subculturing the cells when the cell confluence reaches about 90% to obtain first-generation P1 cells; continuing to subculture the first-generation P1 cells to obtain second-generation P2 cells, which are defined as master cell bank cells and cryopreserved in liquid nitrogen;

[0016] S8, after the master cell bank cells have passed the comprehensive quality inspection, the second generation P2 cells are revived and cultured for three consecutive generations to obtain the fifth generation (P5) cells, which are defined as working cell bank cells and frozen in liquid nitrogen.

[0017] Preferably, the blood test results detected in S1 are maternal peripheral blood and umbilical cord blood.

[0018] Preferably, the four infectious diseases in S1 include four human-specific viruses, namely, hepatitis B virus (HBV), hepatitis C virus (HCV), Treponema pallidum (TP), and human immunodeficiency virus (HIV), and the pathogenic microorganisms include bacteria, fungi, mycoplasmas, and endotoxins.

[0019] Preferably, the sealed refrigerated transportation in S2 is carried out using a medical refrigerated transport box, maintaining the temperature at 2-8°C for no more than 24 hours.

[0020] Preferably, the human umbilical cord mesenchymal stem cell serum-free culture medium is a mesenchymal stem cell serum-free culture medium, which does not contain animal-derived components.

[0021] Preferably, the PBS does not contain calcium ions and magnesium ions.

[0022] Preferably, the digestive enzymes are mild digestive enzymes and do not contain animal-derived components.

[0023] Preferably, the cell freezing solution used for freezing the cells of the master cell bank and the working cell bank is a dedicated cell freezing solution that does not contain serum components.

[0024] Preferably, in said S3, before cutting said umbilical cord into small segments of 1-3 cm, it also includes cutting off 2 cm from each of the ligatures at both ends of the umbilical cord and discarding them.

[0025] Preferably, in said S4, the operation process for obtaining Wharton's jelly is carried out in a culture dish containing the serum-free culture medium of human umbilical cord mesenchymal stem cells; and the square tissue block is rectangular or square.

[0026] Preferably, in S5, the tissue pieces are spread into a 10 cm cell culture dish, preferably 15-25 tissue pieces are spread into each dish.

[0027] Preferably, in S6, when the fusion degree of the cell clones is about 90%, the small pieces of tissue are removed, PBS is added to rinse the cells and then removed, digestive enzymes are slowly added, and the cells are observed to become round and begin to fall off in a sandy state under a microscope, and human umbilical cord mesenchymal stem cell serum-free culture medium is added to terminate the digestion, the cell suspension is collected and centrifuged at 1300 rpm for 5 min, the supernatant is discarded, and the volume is fixed with human umbilical cord mesenchymal stem cell serum-free culture medium, and then samples are counted to obtain primary cells P0.

[0028] Preferably, in the subculture of the S7 cells, when the cell fusion degree is about 90%, PBS is added to rinse the cells and then removed, digestive enzymes are slowly added, and it is observed under a microscope that the cells become round and begin to fall off in a sandy state, human umbilical cord mesenchymal stem cell serum-free culture medium is added to terminate the digestion, the cell suspension is collected and centrifuged at 1300 rpm for 5 min, and the supernatant is discarded, and the volume is fixed with human umbilical cord mesenchymal stem cell serum-free culture medium, and then samples are counted, and the cell suspension is divided into several culture bottles, mixed and placed in an incubator for culture, and after repeated digestion and subculture, the master cell bank cells are obtained.

[0029] Preferably, in S7, the cell cryopreservation specification of the master cell bank is 2.0×10^ 6 cells / tube.

[0030] Preferably, during the cell recovery culture in S8, the cells are taken out from the main cell bank and quickly transferred to a 37°C water bath for cell recovery. When the cells are completely dissolved, they are transferred to a biosafety cabinet, washed with PBS, centrifuged, fixed to volume with human umbilical cord mesenchymal stem cell serum-free culture medium, and then sampled and counted. The cell suspension is divided into several culture bottles, mixed, and then cultured in an incubator. After repeated digestion and subculture, the working cell bank cells are obtained.

[0031] Preferably, in S8, the cryopreservation specification of the working cell bank is 2.5×10^ 7 cells / tube or 5.0×10^ 6 cells / tube.

[0032] Preferably, the cell culture flasks used in S7 and S8 are T175 cell culture flasks. When performing subculture, the cell seeding density is 0.9-1.2×10^ 6 cells.

[0033] Preferably, at each stage of cell culture, including the tissue preservation solution used to collect human umbilical cord tissue, the full volume of medium replacement on the 7th day of umbilical cord tissue culture, the harvesting of cells from the master cell bank, and the harvesting of cells from the working cell bank, bacteria, fungi, mycoplasma, and endotoxin testing are required. If the test result is negative, the cells can continue to be cultured or frozen for later use.

[0034] Preferably, the method further comprises the following steps:

[0035] S7', when the master cell bank is frozen in S7, the cells are resuspended in a cell freezing solution, and then the cell freezing suspension is dispensed into a plurality of cryopreservation tubes according to the freezing specifications, 20-30 μL of the cell freezing suspension is retained and inoculated into a T25 cell culture flask, and the cell growth state is further cultured and observed to indirectly determine the quality of the frozen cells.

[0036] S8', when the working cell bank is frozen in S8, the cells are resuspended in a cell freezing solution, and then the cell freezing suspension is dispensed into a plurality of cryopreservation tubes according to the freezing specifications, 20-30 μL of the cell freezing suspension is retained and inoculated into a T25 cell culture flask, and the cell growth state is further cultured and observed to indirectly determine the quality of the frozen cells.

[0037] Further preferably, in step S7', the quality of the cryopreserved cells is determined by taking a suitable amount of cells for phenotypic testing when the cells in the master cell bank are harvested and cryopreserved, in addition to the cell morphology and viability meeting the quality standards, and the phenotypic testing results meet the basic standards set by the International Society for Cellular Therapy (ISCT).

[0038] Table 1. Criteria for determining the quality of human umbilical cord mesenchymal stem cells in the present invention

[0039]

[0040]

[0041] Further preferably, in step S8', the quality of the frozen cells is determined when the working cell bank is harvested and frozen. In addition to the cell morphology and viability meeting the quality standards, an appropriate amount of cells need to be taken for phenotypic detection, multidirectional differentiation ability, and immunoregulatory ability detection. The results of the phenotypic detection and multidirectional differentiation ability detection meet the basic standards set by the International Society for Cellular Therapy (ISCT), and the immunoregulatory ability meets the quality standards.

[0042] Further preferably, in step S7' or S8', the standard for determining the quality of the frozen cells is that the cell morphology is long spindle cells that grow adherently and have uniform morphology; and the positive expression rate of CD73, CD90 and CD105 is greater than or equal to 95%, and the positive expression rate of CD11b, CD19, CD34, CD45 or HLA-Ⅱ class molecules should not exceed 2%; under in vitro differentiation induction conditions, it has the ability to differentiate into osteoblasts, chondrocytes and adipocytes; the infectious disease and pathogenic microorganism test results are all negative, and the endotoxin result is less than 0.25EU / mL.

[0043] More preferably, in step S8', the quality standard for determining the immune regulation ability of the frozen cells is that the inhibition rate of the pro-inflammatory lymphocytes Th1 of the cells is greater than 50%, the inhibition rate of Th17 is greater than 40%, the promotion rate of the anti-inflammatory lymphocytes Treg is greater than 40%; and the PBMC inhibition rate is greater than 50%.

[0044] The method provided by the present invention uses physiological saline without any antibiotics to store and transport the collected human umbilical cord tissue. When separating human umbilical cord mesenchymal stem cells, only Wharton's jelly is left after the blood vessels, vascular membranes and the amniotic membrane layer on the outer surface are stripped and discarded, which can improve the purity and uniformity of human umbilical cord mesenchymal stem cells. The Wharton's jelly is then cut into small pieces of tissue with uniform size and fixed volume, which can avoid the problem that the tissue pieces are too small to float and the tissue pieces are too large to crawl out of the cells. The small pieces of tissue are spread on the cell culture dish at a certain ratio and spacing, which can ensure that the cells in each piece of Wharton's jelly have space to crawl out, and also reserve growth space for subsequent cell clones. The small pieces of tissue are fixed and then cultured, and the culture dish is not moved in the early stage of culture, which is conducive to the adhesion of the Wharton's jelly cut into small pieces, promotes the cells to crawl out, and shortens the culture time.

[0045] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0046] 1. The human umbilical cord tissue screening process of the present invention can effectively reduce the contamination of human-borne viruses; the reagents used for the collection, preservation, culture and cryopreservation of human umbilical cord tissue do not contain animal-derived components such as antibiotics and serum; the obtained human umbilical cord mesenchymal stem cells are safer, providing effective protection for their subsequent new drug development and clinical application.

[0047] 2. The method for separating and preparing human umbilical cord mesenchymal stem cells provided by the present invention controls the size of tissue blocks, which is conducive to the cells crawling out of the tissue, greatly increasing the number of initial cells, making the cells of the main cell bank and the working cell bank stable for passage and production, and increasing the cell yield; improving the culture efficiency, more human umbilical cord mesenchymal stem cells can be seen crawling out of the tissue on the 7th day of culture, and a large number of human umbilical cord mesenchymal stem cells with high purity and good condition can be harvested on the 12th day of culture, greatly shortening the primary culture cycle of human umbilical cord mesenchymal stem cells, saving time and cost, and finally obtaining human umbilical cord mesenchymal stem cells with high purity and good uniformity. Generally, one umbilical cord tissue can be cultured to obtain about (0.5-1.5)×10^ 9 master cell bank cells and approximately (0.5-1.5)×10^ 12 Working cell bank cells.

[0048] 3. The primary human umbilical cord mesenchymal stem cells obtained by the method provided by the present invention are inoculated with 0.9-1.2×10^ 6 The cells can be subcultured every 3 days, which can maximize the use of the space in the cell culture bottle and promote cell proliferation. The cells can be subcultured every 3 days, with a short cell doubling time and good stability.

[0049] 4. The method provided by the present invention establishes cell banks of human umbilical cord mesenchymal stem cells at various levels, performs hierarchical management, facilitates cell management, facilitates reasonable production arrangements, and provides sufficient time for cell quality testing.

[0050] 5. The method provided by the present invention is to retain a certain number of cells for re-cultivation when the working cell bank is frozen to observe their growth status. This process can be used as a self-check of the cell culture process and as a review method for cell microbial detection. In addition, the cell multiplication capacity after the P5 generation can be judged by the adhesion and proliferation of the cells during re-cultivation, which can be used as a preliminary experiment for safety evaluation and pre-screening for large-scale expansion of cells to the P10 or P15 generation, saving a lot of costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Morphological diagrams of human umbilical cord mesenchymal stem cells P0 cells (7th day), P2 cells (72h), and P5 cells (72h) cultured in a specific embodiment of the present invention;

[0052] Figure 2The morphological diagram of the human umbilical cord mesenchymal stem cells cultured in the working cell bank according to the specific embodiment of the present invention after being cryopreserved and then cultured for 24h, 48h and 72h;

[0053] Figure 3 This is a flow cytometry diagram of the phenotype of human umbilical cord mesenchymal stem cells cultured in a specific embodiment of the present invention;

[0054] Figure 4 This is a flow cytometry diagram of immunoregulation of human umbilical cord mesenchymal stem cells cultured in a specific embodiment of the present invention;

[0055] Figure 5 This is a flow cytometry diagram showing that human umbilical cord mesenchymal stem cells cultured in a specific embodiment of the present invention inhibit the proliferation of peripheral blood mononuclear cells;

[0056] Figure 6 This is a diagram of the adipogenic, osteogenic and chondrogenic differentiation of cultured human umbilical cord mesenchymal stem cells according to a specific embodiment of the present invention;

[0057] Figure 7 This is a picture of endothelial cells or non-target cells crawling out by the traditional tissue adhesion method described in Comparative Example 1;

[0058] Figure 8 This is a comparison chart of the number of cells cultured in the example and the comparative example according to the specific embodiment of the present invention. DETAILED DESCRIPTION

[0059] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0060] The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels or can be prepared by known methods.

[0061] Example 1: Isolation and preparation process of human umbilical cord mesenchymal stem cells

[0062] 1. Isolation and culture of human umbilical cord mesenchymal stem cells

[0063] 1.1 After informed consent from the donor and qualified infectious disease test results, about 20 cm of umbilical cord tissue from a healthy fetus is collected aseptically and transported to the laboratory in a sealed and refrigerated manner.

[0064] 1.2 Under a sterile environment, take the tissue preservation fluid from the umbilical cord collection bottle for pathogenic microorganism testing.

[0065] 1.3 Take out the umbilical cord with both ends tied and rinse it with saline solution, then cut it about 2cm inside the ligated part and discard the ligated umbilical cord tissue; cut the remaining umbilical cord tissue into 1-3cm small sections and place it in saline solution to wash repeatedly until the blood stains are basically washed away.

[0066] 1.4 Transfer the small segment of umbilical cord tissue to a culture dish containing culture medium, use surgical scissors to reach into the umbilical vein, cut the small segment of umbilical cord, use forceps to remove the umbilical artery, venous membrane and outer surface amniotic membrane, obtain Wharton's jelly and cut it into 3-5mm×3-5mm square tissue pieces.

[0067] 1.5 Spread the Wharton's jelly tissue pieces evenly in multiple 10 cm cell culture dishes, with a spacing of 4-7 mm between each piece, and 20 tissue pieces per dish.

[0068] 1.6 Mark the culture dishes, place them in an incubator at 37°C and 5% CO2 for 2 hours, then slowly add 2 mL of human umbilical cord mesenchymal stem cell serum-free culture medium (Ucom Biotech, NC0103) to each cell culture dish (do not impact small tissue pieces during addition), and culture them in an incubator at 37°C, 5% CO2 and saturated humidity.

[0069] 1.7 After 24 hours of culture of human umbilical cord tissue, slowly remove the cell culture dish and slowly add 8 mL of human umbilical cord mesenchymal stem cell serum-free medium to each dish. Culture for 6 days, during which the culture dish position is not moved and the cell growth is not observed. On the 7th day, a full volume of medium is replaced and the cell growth is observed. The full volume of medium is replaced every 3 days thereafter, and the cell growth is observed; the medium is replaced every 3 days in the later stage of culture, and the medium is taken for pathogenic microorganism detection when the medium is replaced on the 7th day. After about 10-12 days of culture, cells crawl out around more than 50% of the tissue blocks, and the local fusion degree reaches about 90%, and digestion treatment is performed for subculture.

[0070] 2. Establishment of human umbilical cord mesenchymal stem cell master cell bank

[0071] 2.1 Under a sterile environment, remove the original culture medium and small pieces of human umbilical cord tissue from the cell culture dish, add PBS to rinse the cells and then remove them, slowly add stem cell mild digestion enzyme (Youkang Biotechnology, NC1004.1), and digest at room temperature for about 2 minutes. Under a microscope, observe that the cells become round and begin to fall off in a sandy state. Add human umbilical cord mesenchymal stem cell serum-free culture medium to terminate digestion, collect the cell suspension, centrifuge at 1300rpm for 5 minutes, and discard the supernatant. Use human umbilical cord mesenchymal stem cell serum-free culture medium to make up the volume, then take samples for counting and viability to obtain human umbilical cord mesenchymal stem cell primary cells (P0).

[0072] 2.2 Inoculate each T175 cell culture flask with 0.9-1.2×10^ 6 The cells were added with human umbilical cord mesenchymal stem cell serum-free culture medium, and cultured in an incubator at 37° C., 5% CO 2 , and saturated humidity for 3 days before passage to obtain the first generation (P1) human umbilical cord mesenchymal stem cells.

[0073] 2.3 The first generation (P1) human umbilical cord mesenchymal stem cells were inoculated into each T175 cell culture flask at a rate of 0.9-1.2×10^ 6 The cells were added with human umbilical cord mesenchymal stem cell serum-free culture medium, and cultured in an incubator at 37° C., 5% CO 2 , and saturated humidity for 3 days before passage to obtain the second generation (P2) human umbilical cord mesenchymal stem cells.

[0074] 2.4 Collect the culture supernatant of the second generation (P2) human umbilical cord mesenchymal stem cells for microbial detection. After cell digestion, centrifuge and dilute with PBS to obtain samples for counting and viability. 6 The cells were used for flow cytometry phenotyping, and the cells were resuspended in serum-free freezing solution after centrifugation. 6 The cells were divided into cryopreservation tubes according to the specifications of cells / tube, and the remaining cryopreservation suspension was subjected to microbial testing to establish a master cell bank of human umbilical cord mesenchymal stem cells.

[0075] 3. Establishment of human umbilical cord mesenchymal stem cell working cell bank

[0076] 3.1 Take out qualified cells from the master cell bank and quickly transfer them to a 37°C water bath to thaw the cells. When the cells are completely dissolved, transfer them to a biosafety cabinet, wash the cells with PBS, centrifuge them, fix the volume with human umbilical cord mesenchymal stem cell serum-free medium, take samples and count them, and inoculate 0.9-1.2×10^ 6 The cells were added with human umbilical cord mesenchymal stem cell serum-free culture medium, cultured in an incubator at 37°C, 5% CO2, and saturated humidity, and subcultured every 3 days. After repeated digestion and subculture, the fifth generation (P5) human umbilical cord mesenchymal stem cells were obtained.

[0077] 3.2 Collect the culture supernatant of human umbilical cord mesenchymal stem cells at the fifth generation (P5) for microbial detection. After cell digestion, centrifuge and dilute with PBS to obtain samples for counting and viability. Take 1.0×10^ 6 Cells were used for flow cytometry phenotyping, and 2.0×10^ 6 Cells for cell immune regulation, 1.0×10^ 6 For immunosuppression detection, 5.0×10^ 6 After centrifugation again, add serum-free freezing solution to resuspend the cells and press 2.5×10^7 The cell suspension was divided into cryopreservation tubes according to the specifications of each cell / tube. 500 μL of the remaining cell cryopreservation suspension was taken for microbial detection. 20-30 μL was inoculated into a T25 cell culture bottle and placed in a CO2 incubator for re-cultivation. The supernatant and cell growth status were observed and photographed every day (the re-cultivated cells were discarded after 72 hours of culture). Figure 2 As shown. Thus, a human umbilical cord mesenchymal stem cell working cell bank is established. The cells are retained and cultured to observe the cell growth status. Before clinical application, the working cell bank cells can be subjected to genetic analysis, virus detection (including but not limited to detection of exogenous and exogenous viral factors, detection of known human-derived viruses, detection of bovine-derived viruses, detection of porcine-derived viruses, and detection of retroviruses) and other safety evaluations as required.

[0078] Table 2

[0079] Cell generation Doubling time (h) P1 cells 17.3 P2 cells 20.4 P3 cells 20.8 P4 cells 20.5 P5 cells 22.3

[0080] 4. Human umbilical cord mesenchymal stem cell detection method

[0081] 4.1 The cell phenotype flow cytometry detection method is as follows:

[0082] Take UC-MSCs 1.0×10 6 in two EP tubes (1.0×10 6 / tube), add 10 μL of UC-MSCs positive cell surface antigen (CD73, CD90 and CD105) antibody fluorescent dye, negative cell surface antigen (CD11b, CD19, CD34, CD45 and HLA-DR) antibody (Negative) mixed fluorescent dye, and incubate at room temperature in the dark for 30 minutes. Flow cytometry was used to detect the expression of UC-MSCs surface antigens.

[0083] Eligibility criteria: The positive expression rate of CD73, CD90 and CD105 is greater than or equal to 95%, and the positive expression rate of CD11b, CD19, CD34, CD45, and HLA-DR is less than 2%. Flow cytometry was used to identify the surface markers of human umbilical cord mesenchymal stem cells.

[0084] like Figure 3 As shown, the positive expression rates of CD73, CD90 and CD105 of UC-MSCs were all greater than 99%, and the positive expression rates of CD73, CD90 and CD105 were 0.00%, which met the qualification criteria.

[0085] 4.2 The cellular immune regulation detection method is as follows:

[0086] Take P5 generation UC-MSCs and calculate (0.5-1.0)×10^ 5 / well / mL was evenly inoculated into a 12-well plate, and cultured in a 37°C, 5% CO2 incubator until the confluence reached 80-90%, and mitomycin C was added at a final concentration of 10 μg / mL. After incubation in a 37°C incubator for 20 minutes, the cells were washed 1-2 times with human umbilical cord mesenchymal stem cell serum-free culture medium.

[0087] Peripheral blood mononuclear cells (PBMC) were collected and the cell concentration was adjusted to 1.0×10^ 6 / mL, set up PBMC culture group and PBMC+UC-MSCs co-culture group. After 3-4 days of culture, add 2μL Leukocyte Activation Cocktail to each well and co-culture for 4-5h.

[0088] The collected cells were subjected to flow cytometry detection according to the requirements of the Th1 / Th17 detection kit and the Treg detection kit.

[0089] Eligibility criteria: When UC-MSCs and PBMCs are co-cultured, the inhibition rate of Th1 is greater than 50%, and the inhibition rate of Th17 should be greater than 40%. Th1 / Th17 inhibition rate = (Th1 / Th17 ratio of PBMC culture group - Th1 / Th17 ratio of co-culture group) / Th1 / Th17 ratio of PBMC culture group × 100%.

[0090] Eligibility criteria: When UC-MSCs and PBMCs are co-cultured, the Treg promotion rate is greater than 40%. Treg promotion rate = (Treg ratio in co-culture group - Treg ratio in PBMC culture group) / Treg ratio in PBMC culture group.

[0091] like Figure 4 As shown, when UC-MSCs were co-cultured with PBMC, the inhibition rate of Th1 was 95.4%, the inhibition rate of Th17 was 90.6%, and the promotion rate of Treg was 75.6%.

[0092] 4.3 The peripheral blood mononuclear cell (PBMC) proliferation inhibition detection method is as follows:

[0093] Take P5 generation UC-MSCs and resuspend them in complete culture medium. 5 The cells were evenly inoculated into 12-well plates and cultured in a 37°C, 5% CO2 incubator until the confluence reached 90%. Mitomycin C was added at a final concentration of 10 μg / mL, and the cells were incubated in a 37°C incubator for 20 minutes, and then washed 1-2 times with human umbilical cord mesenchymal stem cell serum-free culture medium.

[0094] Take PBMC and adjust the cell concentration to 1.0×10^ 7 / mL. Add carboxyfluorescein diacetate succinimidyl ester (CFSE, final concentration 1 μM) to the suspension and incubate at 37°C in the dark for 1-3 minutes. Add 9 volumes of pre-cooled PBS, centrifuge at 1500rpm / min for 5 minutes, and discard the supernatant. Wash once with 10% FBS-RPMI 1640 by centrifugation to remove excess dye and terminate the staining reaction. Adjust the cell concentration to 1.0×10^ 6 / mL.

[0095] According to the following experimental groups, the treated PBMC suspension was added to the above 12-well plate at 1 mL / well, and 3 replicate wells were set up for each group. Group (1) added PBMC; Group (2) and (3) added PBMC, PHA (final concentration of 5 μg / mL), and IL-2 (final concentration of 200 IU / mL). The experimental groups are shown in Table 3:

[0096] Table 3

[0097]

[0098] After 48 hours, it was observed that the cells in the PHA group aggregated into clusters and proliferated massively.

[0099] After 72 h, the cells were collected, resuspended and filtered using 300 μL PBS, and proliferation was analyzed by flow cytometry.

[0100] Qualification criteria: When UC-MSCs and PBMC are co-cultured, the inhibition rate of PBMC should be greater than 50%. PBMC inhibition rate = (proliferation ratio of PHA stimulation group - proliferation ratio of co-culture group) / proliferation ratio of PHA stimulation group × 100%.

[0101] like Figure 5 As shown, when UC-MSCs were co-cultured with PBMCs, the inhibition rate of PBMCs was 80.8%.

[0102] 4.4 The cell differentiation induction detection method is as follows:

[0103] The adipogenic differentiation assay is as follows:

[0104] UC-MSCs were divided into 1.0×10^ 5 / well were evenly inoculated into 6-well plates and cultured in a 37°C, 5% CO2 incubator until the confluence reached 90%. The adipogenic conditioned medium was replaced every 2-3 days for the induced group. The adipogenic induction period was about 14 days. Oil Red O staining solution was used to identify the adipogenicity of the cells in the induced group and the control group, and images were collected. Qualified standard: There were red oil droplets in the cells of the adipogenic induction group, but no red oil droplets were seen in the control group. Figure 6 shown.

[0105] The osteogenic differentiation detection method is as follows:

[0106] UC-MSCs were divided into 1.0×10^ 5 The cells were evenly inoculated in 6-well plates and placed in a 37°C, 5% CO2 incubator until the confluence reached 80%. The osteogenic induction conditioned medium was replaced every 2-3 days for the induction group. The osteogenic induction was carried out for about 16 days. The cells in the induction group and the control group were stained with Alizarin red solution for osteogenic identification, and images were collected. Qualification criteria: Red calcified matrix deposition was observed in the osteogenic induction group, but no red deposition was observed in the cells in the control group. Figure 6 shown.

[0107] The chondrogenic differentiation assay method is as follows:

[0108] UC-MSCs were divided into 1.0×10^ 7 / mL resuspended, 15μL / drop inoculated into a 12-well plate, placed in a 37℃, 5% CO2 incubator for 2h, then slowly added 2mL of human umbilical cord mesenchymal stem cell serum-free medium along the wall, cultured overnight, allowing the cell droplets to shrink and form cell microspheres. The induction group was replaced with chondrogenic conditioned medium, and the medium was changed every 2-3 days, and chondrogenic induction lasted about 21 days. Alcian blue staining solution was used to identify chondrogenic staining of the induced group and control group cells, and images were collected. Qualification criteria: The chondrogenic induction group showed positive staining of blue cartilage proteoglycan, and no blue deposition was observed in the control group cells. Figure 6 shown.

[0109] Embodiment 2-7

[0110] Specific implementation method is the same as Example 1, except that the donor sources of human umbilical cord tissue are different, the lengths of human umbilical cord tissue are different, and the separation and preparation effects are shown in Table 4.

[0111] Table 4

[0112]

[0113]

[0114] Note: RSD is relative standard deviation, relative standard deviation (RSD) = standard deviation (SD) / arithmetic mean of calculated results (X) × 100%. This value eliminates the influence of data dimension and order of magnitude and can be used to compare the stability or consistency of data groups of different levels or scales. The smaller the RSD value, the better the stability or consistency; the larger the RSD value, the worse the stability or consistency.

[0115] As shown in Table 4, the total number of primary cell bank cells (P2) produced by one umbilical cord tissue according to the present invention is (0.5-1.5)×10^ 9 The total number of working cell bank cells (P5) produced is (0.5-1.5)×10^ 12 Each intravenous infusion of 1.0×10 ^6 / kg body weight of P5 human umbilical cord mesenchymal stem cells, three times as a course of treatment, calculated based on a body weight of 50kg, can meet the needs of 3,000 to 10,000 people for one course of treatment. The cell passage ratio from P0 to P2 is at least 1:11, and the cell passage ratio from P2 to P5 is about 1:10, both of which are higher than the current technical level. See Table 4.

[0116] like Figure 2 As shown, when the P5 cells were harvested and frozen into working cell bank cells, 20-30 μL of the cell freezing suspension was inoculated into a T25 cell culture flask for re-culture. The cell culture supernatant was observed every day to be clear, the cells were adherent and uniformly shaped in a long spindle shape, and the cells proliferated normally every day, which indirectly reflected that the cell viability and growth state were good when frozen.

[0117] Comparative Example 1. Preparation of human umbilical cord mesenchymal stem cells by traditional tissue adhesion method

[0118] Preparation of human umbilical cord mesenchymal stem cells by traditional tissue adhesion method: According to the human umbilical cord mesenchymal stem cell isolation and culture method of Example 1, healthy fetal umbilical cord tissue is collected, and the umbilical cord tissue is cut into 1-3 cm small segments after rinsing and washing. Without removing the umbilical artery, venous membrane and outer surface amniotic membrane, the umbilical cord tissue segments are directly cut into tissue pieces of about 1-2 mm × 1-2 mm, and spread in multiple 10 cm cell culture dishes, with the spacing between small pieces being 4-7 mm. The rest of the specific implementation methods are the same as in Example 1.

[0119] The human umbilical cord mesenchymal stem cell isolation and preparation method provided in Example 1 of the present invention was compared with the traditional tissue adhesion method. It was found that the human umbilical cord mesenchymal stem cells cultured by the method provided by the present invention had normal and good morphology and high uniformity ( Figure 1), the results of flow cytometry detection of its surface markers were better than those of comparative example 1, and some of the surface marker detection results of comparative example 1 exceeded the identification standard (Table 5). The traditional tissue adhesion method (comparative example 1) will have a large number of endothelial cells or non-target cells crawling out ( Figure 7 ), Figure 7 A is the cells that crawled out on the 10th day of tissue adherence culture. Its characteristics do not conform to the growth characteristics of human umbilical cord mesenchymal stem cells, that is, non-adherent growth, uniform long spindle-shaped cells; Figure 7 B is Figure 7 A cells were subcultured to the P2 generation (48 h), and it was found that some cells with non-human umbilical cord mesenchymal stem cell morphology still existed (framed part).

[0120] Table 5

[0121]

[0122] Comparative Example 2-4: Cut the umbilical cord tissue into small pieces of about 1-2 mm x 1-2 mm

[0123] According to the method for isolating and culturing human umbilical cord mesenchymal stem cells in Example 1, healthy fetal umbilical cord tissue was collected, and after washing, the umbilical cord tissue was cut into 1-3 cm small segments, and the umbilical artery, vein membrane and outer surface amniotic membrane were removed. Then, the umbilical cord tissue segments were cut into small tissue pieces of about 1-2 mm×1-2 mm, and spread flat in multiple 10 cm cell culture dishes, with the spacing between small pieces being 4-7 mm. The rest of the specific implementation methods were the same as in Example 1.

[0124] The difference between Comparative Examples 2-4 is that the donor sources of human umbilical cord tissue are different, and the lengths of human umbilical cord tissue are different. The separation and preparation effects are shown in Table 6.

[0125] Table 6

[0126]

[0127]

[0128] Comparative Example 5-7: Cut the umbilical cord tissue into small pieces of about 0.5-1.5 cm × 0.5-1.5 cm

[0129] According to the method for isolating and culturing human umbilical cord mesenchymal stem cells in Example 1, healthy fetal umbilical cord tissue was collected, and after washing, the umbilical cord tissue was cut into 1-3 cm small segments, and the umbilical artery, vein membrane and outer surface amniotic membrane were removed. Then, the umbilical cord tissue segments were cut into small tissue pieces of about 0.5-1.5 cm×0.5-1.5 cm, and spread in multiple 10 cm cell culture dishes, with the spacing between small pieces being 4-7 mm. The rest of the specific implementation methods were the same as in Example 1.

[0130] The difference between Comparative Examples 5-7 is that the donor sources of human umbilical cord tissue are different, and the lengths of human umbilical cord tissue are different. The separation and preparation effects are shown in Table 7.

[0131] Table 7

[0132]

[0133] Comparative Example 8-10: Moving the culture dish 6 days before culture

[0134] According to the human umbilical cord mesenchymal stem cell separation and culture method of Example 1, healthy fetal umbilical cord tissue is collected, and the umbilical cord tissue is cut into 1-3cm small segments after rinsing and cleaning, and the umbilical artery, vein membrane and outer surface amniotic membrane are removed. Then, the umbilical cord tissue segments are cut into small pieces of about 3-5mm×3-5mm, and are spread in multiple 10cm cell culture dishes, with a spacing of 4-7mm between small pieces, fixed for 2h, and slowly dripped 2mL of culture medium into each cell culture dish (the small pieces of tissue cannot be impacted during dripping), and placed in an incubator at 37°C, 5% CO2, and saturated humidity for culture. After culturing the human umbilical cord tissue for 24h, 8mL of culture medium is slowly added to each dish. Observe and replace the culture medium under a microscope every 3-4 days until the cell fusion reaches about 90%, and digest and culture. The remaining specific embodiments are the same as in Example 1.

[0135] The difference between Comparative Examples 8-10 is that the donor sources of human umbilical cord tissue are different, and the lengths of human umbilical cord tissue are different. The separation and preparation effects are shown in Table 8.

[0136] Table 8

[0137]

[0138] Comparative Example 11-14: No 2h fixation treatment at the beginning of culture

[0139] According to the method for isolating and culturing human umbilical cord mesenchymal stem cells in Example 1, healthy fetal umbilical cord tissue was collected, and after washing, the umbilical cord tissue was cut into 1-3 cm small segments, and the umbilical artery, vein membrane and outer surface amniotic membrane were removed. Then, the umbilical cord tissue segments were cut into small pieces of about 3-5 mm × 3-5 mm, and spread in multiple 10 cm cell culture dishes, with a spacing of 4-7 mm between small pieces. Without fixing for 2 hours, 2 mL of culture medium was slowly added to each cell culture dish (the small pieces of tissue should not be impacted during the addition), and the dish was placed in an incubator at 37°C, 5% CO2, and saturated humidity for culture. The remaining specific implementation methods are the same as in Example 1.

[0140] The difference between Comparative Examples 11-14 is that the donor sources of human umbilical cord tissue are different, and the lengths of human umbilical cord tissue are different. The separation and preparation effects are shown in Table 9.

[0141] Table 9

[0142]

[0143] In the comparative experiment, only a small number of cells crawled out of the umbilical cord tissue of some individuals on the 10th day of culture, and it still did not meet the requirements for subculture on the 13th day of culture. No subculture was performed and the data are not reflected.

[0144] The human umbilical cord mesenchymal stem cell separation and preparation method provided by the present invention was compared and analyzed with comparative examples 2-14. The time for the comparative examples to crawl out of the human umbilical cord tissue was longer, that is, the P0 culture time was prolonged, and the relative standard deviation (RSD) increased (except for comparative examples 5-7). The P0 culture time of the embodiments was 12 days (RSD = 0%, see Table 4), and the longest P0 culture time in the comparative examples was 17 days (RSD was 21.1%, 0%, 4.7%, and 4.6%, respectively, see Tables 6-9). Although the primary culture time of comparative examples 5-7 was not prolonged, the total number of P0 and P2 generation cells obtained in all comparative examples (2-14) was reduced by 1-2 orders of magnitude compared with that of embodiment 2-7, see Figure 8 The relative standard deviations (RSDs) of the number of P0 cells and the total number of P2 cells obtained per centimeter of umbilical cord in the embodiment were 16.6% and 34.1%, respectively (see Table 3), while the RSDs of the number of P0 cells obtained per centimeter of umbilical cord in the comparative example were 67.7%, 43.5%, 70.2%, and 56.9%, respectively, as shown in Tables 6-9 (increased by 51.1%, 26.8%, 53.5%, and 40.3%, respectively, compared with the embodiment), indicating that according to the method provided by the present invention, human umbilical cords from different sources can be obtained. The number of cells obtained from the umbilical cord tissue has small variability, good uniformity, and good experimental stability; the RSDs of the total number of P2 cells in the comparative examples were 81.5%, 35.7%, 73.3%, and 61.8%, respectively, as shown in Tables 6-9 (increased by 47.4%, 1.6%, 39.2%, and 27.7%, respectively, compared with the embodiments), indicating that if the method of the present invention is not used, the total number of cells obtained from human umbilical cord tissues from different sources has large variability and poor uniformity, and the method of the present invention has better stability and uniformity.

[0145] The present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for isolating and preparing clinical-grade human umbilical cord mesenchymal stem cells, characterized in that: The following steps are involved: S1, screen and obtain human umbilical cord tissue with no family history of genetic diseases and negative results of four infectious diseases and pathogenic microorganism tests; S2, collecting human umbilical cord tissue and storing the human umbilical cord tissue in a tissue protection solution, wherein the tissue protection solution is physiological saline, does not contain any antibiotics, and is sealed and refrigerated for transportation; S3, after taking the tissue protection solution for pathogenic microorganism detection, the umbilical cord is cut into small segments of 1-3 cm, and the umbilical cord is repeatedly cleaned with the tissue protection solution to remove blood on the surface of the umbilical cord; S4, cutting the cleaned human umbilical cord tissue longitudinally along the venous opening, peeling off the umbilical vein and umbilical artery of the umbilical cord, and removing the umbilical cord vein membrane and the outer surface amniotic membrane to obtain Wharton's jelly, and cutting the Wharton's jelly into square tissue pieces of 3-5 mm×3-5 mm; S5, the tissue pieces are spread flat in the cell culture dish, the spacing between the pieces should be uniform, the spacing between the pieces is 4-7mm, and after being fixed at 37°C for 2h, 2mL of human umbilical cord mesenchymal stem cell serum-free culture medium is slowly added, and the tissue pieces cannot be impacted during the addition, and the tissue pieces cannot float. The culture is cultured in a 37°C, 5% CO2 incubator, and 8mL of culture medium is added after 24h of culture. The culture dish is not moved during this period, and the cell growth condition is not observed. On the 7th day, a full medium change is performed and the cell growth condition is observed. Subsequently, the full medium change is performed every 3 days, and the cell growth condition is observed; S6, when the fusion degree of the cell clones reached about 90%, they were rinsed with PBS, digested with digestive enzymes, and the cells were collected to obtain the primary cells P0 of human umbilical cord mesenchymal stem cells; S7, inoculating the primary cells P0 into a culture flask, and subculturing the cells when the cell confluence reaches about 90% to obtain first-generation P1 cells; continuing to subculture the first-generation P1 cells to obtain second-generation P2 cells, which are defined as master cell bank cells and cryopreserved in liquid nitrogen; S8, after the master cell bank cells have passed the comprehensive quality inspection, the second generation P2 cells are revived and cultured for three consecutive generations to obtain the fifth generation (P5) cells, which are defined as working cell bank cells and frozen in liquid nitrogen.

2. The method for isolating and preparing clinical-grade human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The detection in S1 is the blood test results of maternal peripheral blood and umbilical cord blood; the four infectious diseases in S1 include four human-specific viruses: hepatitis B virus, hepatitis C virus, syphilis spirochete, and HIV; the pathogenic microorganisms include bacteria, fungi, mycoplasma and endotoxin; the sealed refrigerated transportation in S2 is the use of medical refrigerated transport boxes, maintaining the temperature at 2-8°C for no more than 24 hours.

3. The method for isolating and preparing clinical-grade human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The human umbilical cord mesenchymal stem cell serum-free medium is a mesenchymal stem cell serum-free medium and does not contain animal-derived components; The PBS does not contain calcium ions and magnesium ions; The digestive enzyme is a mild digestive enzyme and does not contain animal-derived components; The cell freezing solution used for freezing the cells of the master cell bank and the cells of the working cell bank is a special cell freezing solution that does not contain serum components.

4. The method for isolating and preparing clinical-grade human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: In said S3, before cutting the umbilical cord into 1-3 cm small segments, it also includes cutting off 2 cm from the ligatures at both ends of the umbilical cord and discarding them; In S4, the operation process for obtaining Wharton's jelly is carried out in a culture dish containing the serum-free culture medium of human umbilical cord mesenchymal stem cells; the square tissue block is rectangular or square.

5. The method for isolating and preparing clinical-grade human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: In S5, the tissue pieces are spread into a 10 cm cell culture dish, preferably 15-25 tissue pieces are spread into each dish; In the S6, when the fusion degree of the cell clone group is about 90%, the small tissue pieces are removed, PBS is added to rinse the cells and then removed, digestive enzymes are slowly added, and it is observed under a microscope that the cells become round and begin to fall off in a sandy state, human umbilical cord mesenchymal stem cell serum-free culture medium is added to terminate the digestion, the cell suspension is collected and centrifuged at 1300 rpm for 5 minutes, the supernatant is discarded, and the volume is fixed with human umbilical cord mesenchymal stem cell serum-free culture medium, and then samples are counted to obtain primary cells P0.

6. The method for isolating and preparing clinical-grade human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: In the subculture of the S7 cells, when the cell confluence is about 90%, PBS is added to rinse the cells and then removed, digestive enzymes are slowly added, and the cells are observed to become round and begin to fall off in a sandy state under a microscope, and human umbilical cord mesenchymal stem cell serum-free culture medium is added to terminate the digestion, the cell suspension is collected and centrifuged at 1300 rpm for 5 minutes, and the supernatant is discarded, and the volume is fixed with human umbilical cord mesenchymal stem cell serum-free culture medium, and then samples are counted, and the cell suspension is divided into several culture bottles, mixed and placed in an incubator for culture, and the master cell bank cells are obtained after repeated digestion and subculture; In S7, the cell cryopreservation specification of the master cell bank is 2.0×10^ 6 cells / tube; When the cells in S8 are resuscitated and cultured, the cells are taken out from the main cell bank and quickly transferred to a 37°C water bath for cell resuscitation. When the cells are completely dissolved, they are transferred to a biosafety cabinet, washed with PBS, centrifuged, fixed to volume with human umbilical cord mesenchymal stem cell serum-free culture medium, and then sampled and counted. The cell suspension is divided into several culture bottles, mixed, and then cultured in an incubator. After repeated digestion and subculture, the working cell bank cells are obtained; In S8, the cryopreservation specification of the working cell bank is 2.5×10^ 7 cells / tube or 5.0×10^ 6 cells / tube; The cell culture flasks used in S7 and S8 were T175 cell culture flasks. During subculturing, the cell seeding density was 0.9-1.2×10^ 6 cells.

7. The method for isolating and preparing clinical-grade human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: At each stage of cell culture, including the tissue preservation solution used to collect human umbilical cord tissue, the full medium change on the 7th day of umbilical cord tissue culture, the harvesting of cells from the master cell bank, and the harvesting of cells from the working cell bank, bacteria, fungi, mycoplasma, and endotoxin testing are required. If the test results are negative, continue to culture or freeze for later use.

8. The method for isolating and preparing clinical-grade human umbilical cord mesenchymal stem cells according to any one of claims 1 to 7, characterized in that: The method further comprises the following steps: S7', when the master cell bank is frozen in S7, the cells are resuspended in a cell freezing solution, and then the cell freezing suspension is dispensed into a plurality of cryopreservation tubes according to the freezing specifications, 20-30 μL of the cell freezing suspension is retained and inoculated into a T25 cell culture flask, and the cell growth state is further cultured and observed to indirectly determine the quality of the frozen cells; S8', when the working cell bank is frozen in S8, the cells are resuspended in a cell freezing solution, and then the cell freezing suspension is dispensed into a plurality of cryopreservation tubes according to the freezing specifications, 20-30 μL of the cell freezing suspension is retained and inoculated into a T25 cell culture flask, and the cell growth state is further cultured and observed to indirectly determine the quality of the frozen cells.

9. The method for isolating and preparing clinical-grade human umbilical cord mesenchymal stem cells according to claim 8, characterized in that: In step S7', the quality of the frozen cells is determined when the cells in the master cell bank are harvested and frozen. In addition to the cell morphology and viability meeting the quality standards, an appropriate amount of cells need to be taken for phenotypic testing. The phenotypic test results meet the basic standards set by the International Society for Cellular Therapy (ISCT). In step S8', the quality of the frozen cells is determined when the working cell bank is harvested and frozen. In addition to the cell morphology and viability reaching the quality standards, an appropriate amount of cells need to be taken for phenotypic detection, multidirectional differentiation ability, and immunomodulatory ability detection. The results of the phenotypic detection and multidirectional differentiation ability detection meet the basic standards set by the International Society for Cellular Therapy (ISCT), and the immunomodulatory ability meets the quality standards.

10. The method for isolating and preparing clinical-grade human umbilical cord mesenchymal stem cells according to claim 9, characterized in that: In step S7' or S8', the standard for determining the quality of the frozen cells is that the cell morphology is a long spindle-shaped cell that grows adherently and has a uniform morphology; and the positive expression rate of CD73, CD90 and CD105 is greater than or equal to 95%, and the positive expression rate of CD11b, CD19, CD34, CD45 or HLA-Ⅱ molecules should not exceed 2%; under in vitro induced differentiation conditions, it has the ability to differentiate into osteoblasts, chondrocytes and adipocytes; the infectious disease and pathogenic microorganism test results are all negative, and the endotoxin result is less than 0.25EU / mL; in step S8', the quality standard for determining the immune regulation ability of the frozen cells is that the inhibition rate of the pro-inflammatory lymphocyte Th1 of the cells is greater than 50%, the inhibition rate of Th17 is greater than 40%, and the promotion rate of the anti-inflammatory lymphocyte Treg is greater than 40%; the PBMC inhibition rate is greater than 50%.

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