Method for culturing and preparing umbilical cord mesenchymal stem cells and preparation

Abstract

The invention belongs to the technical field of biology, particularly relates to a culture and preparation method of umbilical cord mesenchymal stem cells and a preparation thereof, and provides a culture and preparation method of the umbilical cord mesenchymal stem cells and the preparation thereof which are simple, low in cost and easy to operate and conform to clinical grade. The umbilical cord mesenchymal stem cells are subjected to in-vitro culture and amplification by applying a multi-layer plate culture system and a serum-free and animal-source-component-free culture system with high safety, culture is only needed to be performed for about 4 weeks, the number of cells in a working cell bank capable of being used by the mesenchymal stem cells can reach trillion, the number level of clinical application is met, and the clinical application cost is reduced. The cell viability of the cultured and amplified umbilical cord mesenchymal stem cells is 90% or above, the purity is high, morphology, karyotype analysis, cell surface markers and the like all meet identification standards, and biological safety, general biological characteristics, biological effectiveness and microbial safety all meet clinical application standards; a powerful theoretical basis is provided for large-scale amplification of the mesenchymal stem cells.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for culturing and preparing umbilical cord mesenchymal stem cells and preparations thereof. Background Art

[0002] Mesenchymal stem cells (MSCs), as a type of adult stem cells with multidirectional differentiation potential, have been a research hotspot in the field of regenerative medicine and cell therapy since they were first discovered by Freidenstein et al. in 1976. MSCs were initially isolated from bone marrow and subsequently found in a variety of tissues, including fat, umbilical cord, and placenta. Among them, umbilical cord, as an important source of MSCs, has gradually become the focus of researchers and clinicians due to its advantages such as high cell content, strong proliferation ability, low immunogenicity, easy collection, and no ethical controversy.

[0003] Umbilical cord mesenchymal stem cells (hUC-MSCs) not only have the basic characteristics of MSCs, such as adherent growth, expression of specific surface markers (such as CD105, CD73 and CD90, no expression of CD45, CD34, etc.), and differentiation into osteoblasts, adipocytes and chondrocytes in vitro, but also have good application prospects in the treatment of systemic lupus erythematosus, diabetes, rheumatoid arthritis and other diseases due to their unique advantages in inflammation and immune regulation. In addition, hUC-MSCs also have broad clinical application potential in tissue engineering such as bone, cartilage, muscle, tendon, ligament, nerve, liver, endothelium and myocardium.

[0004] However, the number of hUC-MSCs obtained in primary culture is limited and cannot meet the needs of clinical treatment, so they must be expanded in vitro. Traditional expansion methods mainly rely on single-layer dishes or culture flasks. Although simple, low-cost and easy to operate, it is difficult to meet the needs of large-scale expansion. For this reason, the multi-layer dish culture system (also known as the "cell factory") came into being, providing strong support for the large-scale expansion of hUC-MSCs.

[0005] In the culture system of stem cells, culture medium containing animal serum (such as fetal bovine serum) is traditionally used. However, this culture environment may bring the risk of contamination by pathogens such as viruses, as well as allergic reactions caused by xenogeneic serum. In order to avoid these problems, researchers began to explore serum-free and animal-free culture systems to culture and expand hUC-MSCs in vitro, and explore the feasibility of its clinical application. Summary of the invention

[0006] The purpose of the present invention is to provide a method for culturing and preparing umbilical cord mesenchymal stem cells and preparations, which optimizes the serum-free culture system of umbilical cord mesenchymal stem cells as a whole, provides a strong theoretical basis for the establishment of an umbilical cord mesenchymal stem cell bank and clinical application, and establishes a culture and preparation process that can be used for clinical-grade stem cell preparations.

[0007] The objective of the present invention is achieved through the following technical solutions: The present invention provides a method for culturing umbilical cord mesenchymal stem cells, comprising the following steps: (1) Donor screening and collection of umbilical cord tissue; (2) After disinfection and cleaning, the donor umbilical cord tissue is separated from Wharton's jelly and centrifuged, then resuspended in complete culture medium and inoculated into a T175 culture flask for culture. After the culture is completed, the P0 cells are collected; (3) Inoculate the P0 cells at a cell density of 1*10^6 / T175, resuspend the cell suspension, add complete culture medium for culture to obtain P1 cells, and collect and freeze for later use; (4) Collect or resuscitate the P1 cells and inoculate them at a cell density of 1*10^6 / T175, resuspend the cell suspension, add complete culture medium for culture to obtain P2 cells, and collect them for later use. Then, culture them according to the same operation to obtain P3 cells and collect and freeze them for later use.

[0008] (5) Collect or revive the P3 cells and inoculate them at a cell density of 5*10^7 / cell factory. Resuspend the cell suspension, add 1L of complete culture medium to each cell factory, and then add the cell suspension. After culturing, obtain P4 cells, and then culture according to the same operation to obtain P5 cells.

[0009] (6) The cells prepared in steps (1) to (5) are revived, washed by centrifugation, resuspended, and then human albumin is added to obtain a cultured umbilical cord mesenchymal stem cell preparation.

[0010] Furthermore, in step (2), the disinfection and cleaning are to wash the umbilical cord twice with physiological saline containing triple antibodies, add 75% volume concentration alcohol filtered through a 0.22 μm microporous filter membrane to completely immerse the umbilical cord, soak and disinfect for 2 minutes, then take out the umbilical cord and place it in a sterile culture dish, and wash it 2-3 times with physiological saline to clean the residual umbilical cord blood.

[0011] Furthermore, in step (2), the culturing step is to inoculate the resuspended cells into T175 at 1 g per bottle and culture them in the serum-free medium, perform a half-volume medium replacement on the 5th day after inoculation, and perform a full medium replacement on the 10th day, and digest and collect the cells when the confluence reaches 60%-70%, thereby obtaining primary umbilical cord mesenchymal stem cells.

[0012] Furthermore, in step (3), the specific conditions of the culture are 5% CO 2 , and cultured at 37°C for 3 days. The collection was performed by washing the cells with physiological saline and then digesting them with 5ml / T175 of animal-free trypsin TrypLE Express (1X) for 3min, then washing them with physiological saline and centrifuging them at 500g for 5min to discard the supernatant. The freezing was performed by resuspending the cells in freezing solution at 17.5 million / ml, placing the cells in a -80 refrigerator overnight, and transferring them to a liquid nitrogen tank the next day.

[0013] Furthermore, in step (4), the specific conditions of the culture are to add 21 ml of complete culture medium / T175, and then add 1 ml of cell suspension. After labeling, mix well and place in a carbon dioxide incubator at 5% CO 2 , and cultured at 37°C for 3 days. The collection was performed by washing the cells with physiological saline and then digesting them with 5ml / T175 of animal-free trypsin TrypLE Express (1X) for 3min, then washing them with physiological saline and centrifuging them at 500g for 5min to discard the supernatant. The freezing was performed by resuspending the cells in freezing solution at 17.5 million / ml, placing the cells in a -80 refrigerator overnight, and transferring them to a liquid nitrogen tank the next day.

[0014] Furthermore, in step (5), the specific conditions of the culture are as follows: 1 L of complete medium is added to each cell factory, and then the cell suspension is added, and after labeling, the mixture is mixed and placed in a carbon dioxide incubator for culture. The culture conditions are: 5% CO 2 , 37°C, after the cells have been cultured for about 3-4 days, the cells reach 90% confluence and are then collected; the collection comprises washing the cells with physiological saline and then digesting them with 180 ml / cell factory animal-free trypsin TrypLE Express (1X) for 3 min; after washing with physiological saline, centrifuging at 500 g for 5 min and discarding the supernatant; the freezing comprises resuspending the cells in freezing solution at 17.5 million / ml, placing the cells in a -80 refrigerator overnight, and transferring them to a liquid nitrogen tank the next day.

[0015] Furthermore, in step (6), the specific conditions for the recovery are as follows: after taking out the qualified frozen umbilical cord mesenchymal stem cells from liquid nitrogen, quickly placing them in a preheated 37°C water bath, gently shaking them, and melting them within 2 minutes. When the ice crystals in the cell suspension are about to completely disappear by naked eye observation, the cells are taken out and quickly added to sodium chloride injection incubated at room temperature; the centrifugal washing is centrifugation at 500g for 5 min, pouring off the supernatant, adding sodium chloride injection to resuspend, passing through a 100μm cell sieve, mixing and counting, centrifuging at 500g for 5 min, and pouring off the supernatant; the amount of human serum albumin added is 5ml, and the number of cells in the umbilical cord mesenchymal stem cell preparation is 7*10^7 / 100ml / bag.

[0016] Furthermore, the method also includes the step of performing mycoplasma, endotoxin and sterility tests on the supernatant during the culture process.

[0017] Furthermore, the complete culture medium is a complete culture medium prepared by 500 ml of serum-free culture medium base + 5 ml of additive 1.

[0018] The present invention also provides an umbilical cord mesenchymal stem cell preparation prepared by the culture method.

[0019] Beneficial effects: After the mesenchymal stem cells cultured with (A) Youkang medium were used to build a bank, P3, P5, cell preparations, and P10 umbilical cord mesenchymal stem cells were sent to a third-party testing agency with CMA and CNAS for testing: including cell identification inspection, intracellular and extracellular viral factor inspection, infectious source microbial inspection, microbial, mycoplasma, endotoxin inspection, cell activity, induction differentiation ability, immunological response, and tumorigenicity inspection, and the test results all met clinical grade standards. For example, the preparation cell identification and cell surface markers all meet the identification standards of umbilical cord mesenchymal stem cells; mycoplasma, endotoxin, and sterility tests are all negative, which meets biological safety; the preparation has no DMSO residues, the appearance of the preparation has no visible foreign matter, and the pH, osmotic pressure, and filling volume all meet the preparation standards. Therefore, the culture process is a culture and preparation method that meets the clinical grade umbilical cord mesenchymal stem cells and their preparations. It only takes about 4 weeks of culture, and the number of cells in the working cell bank that can be used for mesenchymal stem cells can reach trillions, which meets the number level of clinical application. The cell viability of the cultured and expanded umbilical cord mesenchymal stem cells is above 90%, with high purity, morphology, karyotype analysis, cell surface markers, etc., all meet the identification standards, and biological safety, general biological characteristics, biological effectiveness, and microbial safety all meet the clinical application standards. A simple, low-cost, and easy-to-operate process method is provided. In order to scale up the expansion of mesenchymal stem cells, a multi-layer plate culture system and a highly safe serum-free and animal-derived component-free culture system are also used to culture and expand umbilical cord mesenchymal stem cells in vitro. The in vitro expansion and subculture of umbilical cord mesenchymal stem cells is established, and their three-lineage mesoderm differentiation potential is maintained, the proliferation ability is strong, the doubling time is short, and the umbilical cord mesenchymal stem cells that meet the GMP standard are more suitable for clinical research. It provides a strong theoretical basis for the establishment of an umbilical cord mesenchymal stem cell bank and clinical application, and a culture and preparation process that can be used for clinical-grade stem cell preparations is established. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1The figure shows the number of cells at P0 after primary cell isolation and culture in different culture systems, among which A is the treatment group using 500ml serum-free medium base + 5ml additive 1 purchased from Youkang Biotechnology Co., Ltd., B is the treatment group using 500ml Zhongjia serum-free medium + 33.3ml additives purchased from Zhongjia Biomedical Technology Co., Ltd.; C is the treatment group using 490ml M200 serum-free medium (type B) + 10ml F20 PlusMSC additives purchased from Dakoway Biotechnology Co., Ltd.; Figure 2 These are microscope images of primary cell separation and culture in different culture systems. The meanings of AC in the figure are the same as Figure 1 , the magnification is 100 times; Figure 3 The results of the proliferation of umbilical cord mesenchymal stem cells in different culture systems are shown in Figure 1. Figure 1 ; Figure 4 Figure 2 is a diagram for verifying the biological effectiveness of umbilical cord mesenchymal stem cells in different culture systems. Figure 1 ; Figure 5 Figure 2 shows the cell cycle detection diagram of umbilical cord mesenchymal stem cells in different culture systems. Figure 1 , P5 means passaged to the fifth generation; Figure 6 The results of SA-β-Gal activity detection of umbilical cord mesenchymal stem cells in different culture systems are shown in Figure 1. Figure 1 , P3, P5, and P10 represent the third, fifth, and tenth generations, respectively; Figure 7 The figure is a specific process flow chart of the umbilical cord mesenchymal stem cell culture process of the present invention. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] The test methods used in the following implementations, unless otherwise specified, are all commonly used experimental methods in this field; Unless otherwise specified, the experimental materials used in the following examples are all commonly used experimental materials in this field.

[0028] The specific preparation process of umbilical cord mesenchymal stem cells in the present invention comprises the following steps: (1) Collection of umbilical cord tissue as donor material: 1.1 Donor screening criteria: maternal information, name, age 20-36 years old. Maternal screening items: blood routine and alanine aminotransferase (ALT) indicators are normal, RH blood type test is positive (results within 6 months); infectious disease examination: hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), syphilis spirochete (TP), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human T-lymphotropic virus (HTLV)) screening is no infection (results within 6 months); other examination items: no family genetic diseases, such as thalassemia or glucose-6-phosphate dehydrogenase (G-6-PD) abnormalities, etc.; no history of drug abuse; no blood transfusion or use of blood products within 1 year; single birth, gestational age greater than 34 weeks, and maternal delivery method is caesarean section. The mother shall donate voluntarily and sign the informed consent form.

[0029] 1.2 Newborn screening: no deformity, weight greater than 2500g, newborn score ≥8 points.

[0030] 1.3 Umbilical cord collection: After the fetus is delivered, the umbilical cord is routinely cut; After the umbilical cord is cut, the umbilical cord is disinfected: Use iodine gauze to quickly wipe from the umbilical cord end to the placental end, disinfect twice, and the disinfection range is 10-15cm from the umbilical cord end to remove blood, amniotic fluid, meconium, etc.; After the disinfection is completed, use this gauze to support the umbilical cord end and expose the filled part of the umbilical vein for puncture; Umbilical vein puncture and blood collection: Aseptically remove the blood collection needle cap, puncture the umbilical vein with the needle beveled downward or at a small angle to the side 3-5cm away from the umbilical cord end to collect 4-6ml of blood; then drain the umbilical cord blood from the cut end or squeeze and wipe it with sterile gauze to remove the umbilical cord blood; Clamp the umbilical cord with hemostatic forceps 2-3cm near the placental end, and then tie it with silk thread near the hemostatic forceps, and connect the ligature and the hemostatic forceps Use surgical scissors to cut the umbilical cord; tie the other end of the umbilical cord with a thin thread, and the length of the umbilical cord between the two ligature points must be greater than 20cm; clean the umbilical cord sample after collection: after the umbilical cord is collected, use sterile gauze to wipe off the vernix caseosa, amniotic fluid and other dirt on the surface of the umbilical cord (serosa); hold the umbilical cord with sterile forceps, first rinse it thoroughly with 75% alcohol, and then rinse it with 500ml of saline; if the umbilical cord sample tissue is seriously contaminated, it can be disinfected with iodine before rinsing with alcohol; place the umbilical cord vertically in the umbilical cord storage and transportation bottle (containing 100ml of preservation solution: 1% of the three antibodies), and minimize contact with the bottle mouth and the outside; mark the key information such as the mother's name, hospital number, collection time, collection hospital, and collector on the umbilical cord storage and transportation bottle.

[0031] 1.4 Transportation and handover of umbilical cord: Place the collected maternal peripheral blood, umbilical cord blood, and umbilical cord storage and transportation bottles into a medical vaccine box with an ice box. The umbilical cord storage and transportation bottles can also be temporarily stored at 4-10℃, but avoid direct contact with the ice box. Transport to the laboratory by car.

[0032] 1.5 Verification and receipt of umbilical cord: Umbilical cord blood and peripheral blood were sent for testing of HIV, HBV, HCV, HTLV, EBV, CMV, TP: negative; Umbilical cord tissue: Mycoplasma, endotoxin, sterility test: negative, no deformity, no edema, the umbilical cord was ligated at both ends, no contamination, and the information was complete.

[0033] (2) Isolation and culture of primary cells: 1. Use a 60ml syringe to extract 50ml of 75% alcohol, filter it through a 0.22μm needle filter into a centrifuge tube for later use. Use 100× of the third antibody to prepare sodium chloride washing solution.

[0034] Disinfection and cleaning of the umbilical cord: Aspirate the umbilical cord preservation solution in the umbilical cord preservation bottle, wash twice with saline containing three antibodies, add medical disinfectant alcohol (75%) filtered with a 0.22μm microporous filter membrane to completely immerse the umbilical cord, and soak for disinfection for 2 minutes. Take out the umbilical cord and place it in a sterile culture dish, wash it with saline 2-3 times to clean the remaining umbilical cord blood. Cut the umbilical cord into small pieces of about 2-3 cm, and wash it with saline again 2-3 times to clean the remaining umbilical cord blood.

[0035] Separation of umbilical cord Wharton's jelly: Cut the umbilical cord along the vein and remove the vein wall. After the vein wall is completely removed, the umbilical cord will be fully unfolded, and then the two arteries are removed. After the veins and arteries are completely removed, carefully separate the Wharton's jelly, taking care to avoid the epidermis. Transfer the separated Wharton's jelly into a 50 mL centrifuge tube for centrifugation (keep 20 ml of the supernatant for bacterial inspection, mycoplasma detection, etc.), then add 3-5 drops of physiological saline to keep it moist, use elbow surgical scissors to cut the Wharton's jelly into 1-4 mm3 pieces, and then weigh it.

[0036] Tissue block inoculation: Cut the Wharton's jelly into pieces and resuspend them in complete culture medium. Inoculate them into T175 culture flasks, with 1g of Wharton's jelly and 10ml of complete culture medium per flask, and culture them in a carbon dioxide incubator.

[0037] Medium change: The first half-volume medium change was on the 5th day after inoculation. Cells began to crawl out of the tissue block. The culture bottle was tilted 30 degrees upright to allow the tissue block to naturally settle to one corner of the culture bottle. Half of the supernatant was aspirated and discarded. 10 ml of fresh rewarmed complete medium was slowly added. Mix gently and return to the incubator for continued culture. The second full-volume medium change was on the 10th day after inoculation. The crawling cells were in good condition and began to grow in stacks. The culture bottle was tilted 30 degrees upright to allow the tissue block to naturally settle to one corner of the culture bottle. The supernatant was aspirated and discarded. 15 ml of fresh rewarmed complete medium was slowly added. Mix gently and return to the carbon dioxide incubator for continued culture.

[0038] Collect P0 cells of MSC: Gently shake the flask back and forth, and keep it flat on the table. Most of the tissue will fall off. If there are still tissue pieces that have not fallen off, poke or suck them away with a pipette or gun tip; suck off the culture supernatant and tissue pieces (leave 20 ml for bacterial inspection, mycoplasma, and endotoxin testing), add physiological saline to wash once, and discard. Add 5 ml of rewarmed digestion solution to digest for 3 minutes, then add an equal volume of complete culture medium to terminate digestion, collect cells and centrifuge at 500×g for 5 min, discard the supernatant, and resuspend the cell pellet in complete culture medium for counting.

[0039] (3) Expansion and culture of P1 primary cell bank cells 1. P0 to P1: After counting the cells, inoculate at a cell density of 1*10^6 / T175, resuspend the cell suspension, add 21ml of complete medium / T175, and then add 1ml of cell suspension. After labeling, mix well and culture in a carbon dioxide incubator. The culture conditions are: 5% CO2, 37℃. After about 3 days of cell culture, the cells reach 90% confluence and are collected.

[0040] 2. Collection and cryopreservation of P1 cells: The cell supernatant was tested for mycoplasma, endotoxin, and sterility. The cells were washed with saline and then digested with 5 ml / T175 of animal-free trypsin TrypLE Express (1X) for 3 min. After washing with saline, the cells were centrifuged at 500 g for 5 min and the supernatant was discarded. The cell pellet was resuspended in saline and counted. After counting, the cells were centrifuged at 500 g for 5 min and the supernatant was discarded. The cells were resuspended in cryopreservation solution at 17.5 million / ml for cell cryopreservation. The cells were placed in a -80 refrigerator overnight and transferred to a liquid nitrogen tank the next day.

[0041] 3. Testing and storage of P1 generation original cell bank: On the third day, randomly sample a cell for resuscitation, place in a 37℃ water bath for 2 minutes, quickly transfer to physiological saline, wash and centrifuge at 500g for 5 minutes, discard the supernatant, add physiological saline for resuspending, and perform mycoplasma, endotoxin and sterility tests on the cell suspension. After the test is negative, transfer the cells to a meteorological liquid nitrogen tank and mark them as original cell banks. At the same time, the original cells are sampled for testing including cell identification, cell counting, flow cytometry, etc.

[0042] (4) Expansion and culture of P3 master cell bank cells 1. P1 to P2: Collect P1 cells and directly pass them to P2, or resuscitate P1 cells and pass them to P2. After counting the cells, inoculate them at a cell density of 1*10^6 / T175, resuspend the cell suspension, add 21ml of complete medium / T175, and then add 1ml of cell suspension. After labeling, mix well and culture in a carbon dioxide incubator. The culture conditions are: 5% CO2, 37℃. After about 3 days of cell culture, the cells reach 90% confluence and are collected.

[0043] 2. Collection of P2 cells: The cell supernatant was tested for mycoplasma, endotoxin and sterility. The cells were washed with saline and then digested with 5ml / T175 animal-free trypsin TrypLE Express (1X) for 3min. After washing with saline, the cells were centrifuged at 500g for 5min and the supernatant was discarded. The cell pellet was resuspended in saline and counted. After counting, the cells were centrifuged at 500g for 5min and the supernatant was discarded. The cell pellet was resuspended in complete medium and counted.

[0044] 3. P2 passage P3: After counting the P2 cells, inoculate them at a cell density of 1*10^6 / T175, resuspend the cell suspension, add 21ml of complete medium / T175, and then add 1ml of cell suspension. After labeling, mix well and culture in a carbon dioxide incubator. The culture conditions are: 5% CO2, 37℃. After about 3 days of cell culture, the cells reach 90% confluence and are collected.

[0045] 4. Collection and cryopreservation of P3 cells: The cell supernatant was tested for mycoplasma, endotoxin and sterility. The cells were washed with saline and then digested with 5ml / T175 animal-free trypsin TrypLE Express (1X) for 3min. After washing with saline, centrifuged at 500g for 5min and the supernatant was discarded. The cell pellet was resuspended in saline and counted. After counting, centrifuged at 500g for 5min and the supernatant was discarded. The cells were resuspended in cryopreservation solution at 17.5 million / ml for cell cryopreservation. The cells were placed in a -80 refrigerator overnight and transferred to a liquid nitrogen tank the next day.

[0046] 5. Testing and storage of the P3 generation master cell bank: On the third day, randomly sample a cell for resuscitation, bathe at 37℃ for 2 minutes, quickly transfer to physiological saline, wash and centrifuge at 500g for 5 minutes, discard the supernatant, add physiological saline for resuspending, and perform mycoplasma, endotoxin and sterility tests on the cell suspension. After the test is negative and qualified, the cells are transferred to the meteorological liquid nitrogen tank, marked, and used as the master cell bank. At the same time, the master cells are sampled for testing including cell identification, cell counting, flow phenotype, microbial examination, cell activity, population doubling time, induced differentiation ability, immunological response, etc.

[0047] (5) Expansion and culture of P5 working cell bank cells 1. P3 to P4: Collect P3 cells and pass them to P4 directly, or resuscitate P3 cells and pass them to P4. After counting the cells, inoculate them at a cell density of 5*10^7 / cell factory, resuspend the cell suspension, add 1L of complete medium to each cell factory, and then add the cell suspension. After labeling, mix well and culture in a carbon dioxide incubator. The culture conditions are: 5% CO2, 37℃. After about 3-4 days of cell culture, the cells reach 90% confluence and are collected.

[0048] 2. Collection of P4 cells: The cell supernatant was tested for mycoplasma, endotoxin and sterility. The cells were washed with saline and then digested with 180 ml / cell factory animal-free trypsin TrypLE Express (1X) for 3 min. After washing with saline, centrifuged at 500 g for 5 min and the supernatant was discarded. The cell pellet was resuspended in saline and counted. After counting, centrifuged at 500 g for 5 min and the supernatant was discarded. The cell pellet was resuspended in complete medium and counted.

[0049] 3. P4 to P5: After counting the cells, inoculate at a cell density of 5*10^7 / cell factory, resuspend the cell suspension, add 1L of complete medium to each cell factory, and then add the cell suspension. After labeling, mix well and culture in a carbon dioxide incubator. The culture conditions are: 5% CO2, 37℃. After about 3-4 days of cell culture, the cells reach 90% confluence and are collected.

[0050] 4. Collection and cryopreservation of P5 cells: The cell supernatant was tested for mycoplasma, endotoxin, and sterility. The cells were washed with saline and then digested with 180 ml / cell factory animal-free trypsin TrypLE Express (1X) for 3 min. After washing with saline, centrifuged at 500 g for 5 min and the supernatant was discarded. The cell pellet was resuspended in saline and counted. After counting, centrifuged at 500 g for 5 min and the supernatant was discarded. The cells were resuspended in cryopreservation solution at 17.5 million / ml for cell cryopreservation. The cells were placed in a -80 refrigerator overnight and transferred to a liquid nitrogen tank the next day.

[0051] 5. Testing and storage of P5 generation working cell bank: On the third day, randomly sample a cell for resuscitation, bathe at 37℃ for 2 minutes, quickly transfer to physiological saline, wash and centrifuge at 500g for 5 minutes, discard the supernatant, add physiological saline for resuspending, and perform mycoplasma, endotoxin and sterility tests on the cell suspension. After the test is negative and qualified, the cells are transferred to the meteorological liquid nitrogen tank and marked as the working cell bank. At the same time, the working cells are sampled for testing, including cell identification, cell counting, cell activity, flow phenotype, microbial examination, cell morphology, cytopathic effect, exogenous viral factors in cells, induction differentiation ability, immunological response, etc.

[0052] (6) Preparation of umbilical cord mesenchymal stem cell preparations 1. Cell recovery: After taking out the qualified frozen umbilical cord mesenchymal stem cells from liquid nitrogen, quickly put them into a preheated 37°C water bath and gently shake them to melt within 2 minutes. When the ice crystals in the cell suspension are about to disappear completely (remaining mung bean-sized ice crystals), take them out and quickly add the cells to the sodium chloride injection solution incubated at room temperature.

[0053] 2. Centrifugal washing: Centrifuge at 500g for 5 min, discard the supernatant, add sodium chloride injection solution to resuspend, pass through a cell sieve (100μm), mix and count, centrifuge at 500g for 5 min, and discard the supernatant.

[0054] 3. Preparation packaging: resuspend the cells with sodium chloride injection, add 5ml human albumin, so that the number of cells is 7*10^7 / 100ml / bag, and pack them in the reinfusion bag. Before packing, keep 2ml of cell suspension and keep it at 4℃ for 2 months. Heat seal the reinfusion bag with a heat sealer and then check the preparation.

[0055] 4. Verify the preparation: the results of microbial mycoplasma, endotoxin and sterility tests are all negative, the color is uniform and free of foreign matter, the preparation is sealed and leak-proof, and the information is complete and accurate.

[0056] 5. Transportation of preparations: Refrigerate the umbilical cord mesenchymal stem cell preparations at 5±3℃ for temporary storage. If they are to be transported to other places, place them in a medical vaccine box with an ice box, but add a spacer to avoid direct contact with the ice box, and place a real-time temperature monitor before transporting them to the destination.

[0057] Example 1 Optimization of serum-free culture system for umbilical cord mesenchymal stem cells 1. Experimental Materials 500ml serum-free medium base + 5ml additive 1 were purchased from Youkang Biotechnology Co., Ltd.; 490ml M200 serum-free medium (type B) + 10ml F20 Plus MSC supplement were purchased from Dakoway Biotechnology Co., Ltd.; 500ml Zhongjia serum-free medium + 33.3ml supplement were purchased from Zhongjia Biomedical Technology Co., Ltd.; non-animal-derived recombinant enzymes were purchased from Thermo Fisher, USA; cell freezing solution was purchased from Zhongjia Biomedical Technology Co., Ltd.; 0.9% sodium chloride injection was purchased from Sichuan Kelun Pharmaceutical Co., Ltd.; cycle kits, antibodies, osteogenic / adipogenic induction kits and AOPI fluorescent dyes were purchased from Shanghai Ruiyu Biotechnology Co., Ltd.; mycoplasma detection kits were purchased from Junyan Biotechnology Co., Ltd.; endotoxin detection kits were purchased from Zhanjiang Andus Biological Co., Ltd.; ethanol was purchased from Guilin Lifeng Medical Supplies Co., Ltd.; cryopreservation tubes were purchased from Thermo Fisher; culture dishes were purchased from Wuxi Nice Life Science Co., Ltd.; 50ml centrifuge tubes were purchased from Wuxi Nice Life Science Co., Ltd.; T75 and T175 culture flasks were purchased from Corning, USA; 5ml, 10ml, and 25ml pipettes were purchased from Wuxi Nice Life Science Co., Ltd.; 0.22μm microporous filter membranes were purchased from Pall, USA; Pasteur pipettes were purchased from Hefei White Shark Biotechnology Co., Ltd.; gun tips were purchased from Eppendorf, USA; syringes were purchased from China Shuangge Group Co., Ltd.; sealing films were purchased from Bemis, USA; umbilical cord collection sterile bottles were purchased from Costar, USA; tertiary antibodies (100×) were purchased from Gibco, USA; 10-layer cell factories were purchased from Wuxi Nice Life Science Co., Ltd.; and cell senescence β-galactosidase staining kit was purchased from Shanghai Bio-Tech Co., Ltd.

[0058] 2. Experimental Methods 2.1 Primary Isolation and Extraction of Umbilical Cord Mesenchymal Stem Cells (1) Collection: After the umbilical cord is collected, it is placed in umbilical cord preservation solution (ncMission Basal Medium), transported at 4°C, and processed within 24 hours.

[0059] (2) Disinfection and cleaning of the umbilical cord: Aspirate the umbilical cord preservation liquid in the umbilical cord preservation bottle, wash twice with saline, add medical disinfectant alcohol (75%) filtered with a 0.22μm microporous filter membrane to completely immerse the umbilical cord, and soak for disinfection for 2 minutes. Take out the umbilical cord and place it in a sterile culture dish. Wash it 2-3 times with saline to clean the remaining umbilical cord blood. Cut the umbilical cord into small pieces of about 2-3 cm, and wash it again with saline 2-3 times to clean the remaining umbilical cord blood.

[0060] (3) Separation of Wharton's jelly from the umbilical cord: Cut the umbilical cord along the vein and remove the venous wall. After the venous wall is completely removed, the umbilical cord will unfold completely. Then remove the two arteries. After the veins and arteries are completely removed, carefully separate the Wharton's jelly, taking care to avoid the epidermis. Transfer the separated Wharton's jelly into a 50ml centrifuge tube and centrifuge (keep 20ml of the supernatant for bacterial inspection, mycoplasma detection, etc.), then add 3-5 drops of physiological saline to keep it moist, use elbow surgical scissors to cut the Wharton's jelly into pieces of 1-4mm3, and then weigh it.

[0061] (4) Tissue block inoculation: Cut the Wharton's jelly into pieces and resuspend them in complete culture medium. Inoculate them into T175 culture flasks, with 1 g of Wharton's jelly and 10 ml of complete culture medium per flask, and place them in an incubator for culture.

[0062] (5) Medium change: The first half-volume medium change was performed on the 5th day after inoculation. Cells began to crawl out of the tissue blocks. The culture flask was tilted 30 degrees to allow the tissue blocks to naturally settle to one corner of the culture flask. Half of the supernatant was aspirated and slowly added 10 ml of fresh rewarmed complete medium. Mix gently and return to the incubator for continued culture. The second full-volume medium change was performed on the 10th day after inoculation. The crawled cells were in good condition and began to grow in stacks. The culture flask was tilted 30 degrees to allow the tissue blocks to naturally settle to one corner of the culture flask. The supernatant was aspirated and slowly added 15 ml of fresh rewarmed complete medium. Mix gently and return to the incubator for continued culture.

[0063] (6) Subculture digestion: Gently shake the flask back and forth, and keep it flat on the table. Most of the tissue will fall off. If there are still tissue pieces that have not fallen off, poke or suck them away with a pipette or gun tip; aspirate the culture supernatant and tissue pieces (leave 20ml for bacterial inspection, mycoplasma detection, etc.), add physiological saline to wash once, and aspirate and discard. Add 5ml of rewarmed digestion solution and digest for 3 minutes, then add an equal volume of complete culture medium to terminate digestion, collect cells and centrifuge (500×g, 5min).

[0064] 2.2 Cell recovery (1) After removing the frozen MSCs from liquid nitrogen, quickly place them in a preheated 37°C water bath and gently shake them to thaw within 2 minutes. When the ice crystals in the cell suspension are about to disappear completely (remaining mung bean-sized ice crystals), remove the cells and quickly add them to complete culture medium.

[0065] (2) Centrifuge at 500g for 5 min, discard the supernatant, add complete culture medium to resuspend and mix, count, then add the cells to 21 ml of T175 culture medium, blow gently with a pipette to mix, and gently place in a carbon dioxide incubator for culture.

[0066] 2.3 Cell passaging (1) Incubate trypsin (non-animal recombinant enzyme), 0.9% NaCl injection solution, and complete culture medium (to be used within 2 weeks) at room temperature for 30 min in advance. After taking the cells out of the incubator, discard the supernatant with a pipette, wash once with about 20 ml of 0.9% NaCl injection solution, and discard the liquid.

[0067] (2) Add 5 ml of trypsin / T175 and spread it evenly to the bottom of the culture flask. Digest the cells for 2-3 minutes. Then add an equal amount of cell culture medium supernatant diluent to blow off the cells. Transfer the cell fluid to a centrifuge tube and wash the culture flask once with about 20 ml of 0.9% NaCl injection solution. Add the liquid to the centrifuge tube as well.

[0068] (3) Centrifuge at 500g for 5 min, discard the supernatant, repeat the washing and centrifugation once, add complete culture medium to resuspend evenly, take a sample and count, take 1*10^6 cells into a culture bottle with 21ml culture medium, mix well and place in a carbon dioxide incubator for culture.

[0069] 2.4 Cell cryopreservation (1) Incubate pancreatic enzyme (non-animal recombinant enzyme), 0.9% NaCl injection solution, and complete culture medium (to be used within 2 weeks) at room temperature for 30 min in advance. Place the programmed cooling box in a 4°C refrigerator. After taking the cells out of the incubator, discard the supernatant with a pipette, wash once with about 20 ml of 0.9% NaCl injection solution, and discard the liquid.

[0070] (2) Add 5 ml of trypsin / T175 and spread it evenly to the bottom of the culture flask. Digest the cells for 2-3 minutes. Then add an equal amount of cell culture medium supernatant diluent to blow off the cells. Transfer the cell fluid to a centrifuge tube and wash the culture flask once with about 20 ml of 0.9% NaCl injection solution. Add the liquid to the centrifuge tube as well.

[0071] (3) Centrifuge at 500g for 5 minutes, discard the supernatant, resuspend in 0.9% NaCl injection solution and count evenly, centrifuge at 500g for 5 minutes, discard the supernatant, add a certain amount of freezing solution (so that the freezing density is 1.05*10^7 / tube), mix well and add to the freezing tube and place in a program cooling box. Place in a -80℃ ultra-low temperature refrigerator overnight, transfer to liquid nitrogen the next day, and transfer to a gas phase liquid nitrogen tank for storage after the test meets the requirements.

[0072] 2.5 Cell counting and viability detection (1) Prepare the samples to be tested, Countstar counting plate, pipette and pipette tip.

[0073] (2) Counting: After mixing the cell suspension, take 20 μl of sample and 20 μl of AOPI dye and mix thoroughly, then add them into the Countstar counting plate at a uniform speed. Insert the counting plate into the Countstar Rigel S2 counting instrument to count the cells and obtain the cell number, viability and diameter.

[0074] 2.6 Cell expansion multiples and doubling time (1) Cells of generations P0-P4 were inoculated into T175 culture flasks at a cell number of 6000 / cm2, and 21 ml of complete culture medium of four different systems was added for culture. Each group was replicated with 3 flasks.

[0075] (2) After 3-4 days, when the cells have grown to 90% confluence, they are digested with trypsin and collected. The doubling time and expansion multiple of each generation are counted (calculation formula DT = t*[lg2 / (lgNt-lgN0)]), where t is the culture time, N0 is the number of cells recorded in the first flask, and Nt is the number of cells after t time.

[0076] 2.7 Cell sterility test (1) Prepare the cell sample to be tested (20 ml), aerobic / anaerobic culture bottle (colorimetric method), and syringe.

[0077] (2) Use a syringe to draw up the sample liquid to be tested and inject it into aerobic / anaerobic culture bottles respectively (air cannot be injected), and place them into the Meihua BC64 blood culture instrument. The results will be available after 5 days of culture.

[0078] 2.8 Mycoplasma Detection (1) Extraction of mycoplasma from test samples: Place the sample to be tested in a 1.5 ml centrifuge tube and centrifuge at 500 g for 5 min to precipitate the cells; place the supernatant in a new 1.5 ml centrifuge tube and centrifuge at 20,000 g for 10 min to enrich the supernatant; carefully remove the supernatant (leave 20-30 μl of supernatant in the centrifuge tube), and mix the remaining cell pellet by blowing to form a suspension. Add the remaining suspension to the remaining cell pellet, make up to 100 μl with DNA diluent, and vortex to mix for later use. Add 10 μl of 5 M NaCl, 100 μl of lysis buffer, and 10 μl of proteinase K to each 100 μl sample, vortex for 30 seconds, centrifuge for a short time, and incubate at 65°C for 1 hour. After incubation, centrifuge the centrifuge tube for a short time, then add 209.2 μl of working binding solution, 250 μl of isopropanol, and 20 μl of magnetic beads in sequence, vortex for 10 minutes, and centrifuge quickly for 10 seconds. Place the centrifuge tube on the magnetic rack and gently rotate it left and right. After the magnetic beads gather on the tube wall close to the magnetic rack, keep the centrifuge tube fixed on the magnetic rack and use a pipette to remove the supernatant. Be careful not to touch the magnetic beads. Add 500μl of washing solution and vortex for 30s to ensure that the magnetic beads are dispersed and there are no aggregated magnetic beads on the wall of the centrifuge tube. After rapid centrifugation for 10s, place the centrifuge tube back on the magnetic rack and gently rotate it left and right. After the magnetic beads gather on the tube wall close to the magnetic rack, let it stand for 1min and carefully remove the supernatant with a pipette. Add 500μl of freshly prepared 80% ethanol and vortex for 30s to ensure that the magnetic beads are dispersed and there are no aggregated magnetic beads on the wall of the centrifuge tube. After rapid centrifugation for 10s, place the centrifuge tube back on the magnetic rack and gently rotate it left and right. After the magnetic beads gather on the tube wall close to the magnetic rack, let it stand for 1min and carefully remove the supernatant with a pipette. To ensure that the residual ethanol is sucked out as much as possible, the centrifuge tube can be quickly centrifuged for 10 seconds and then placed on a magnetic rack and aspirated with a 10μl pipette. Open the tube cover and dry at room temperature for 3-5 minutes. Remove the centrifuge tube from the magnetic rack, add 100μl of elution solution to each tube, vortex for 1 minute, incubate at 70℃ for 7 minutes, and vortex every 2-3 minutes. After the incubation is completed, centrifuge the centrifuge tube at high speed for 1 minute, then place it on the magnetic rack. After the magnetic beads are separated, use a pipette to carefully transfer the supernatant to a new 1.5ml centrifuge tube.

[0079] (2) QPCR sample preparation and loading: Set the sample wells according to the experimental group samples, mark them and configure QPCRMix according to the instructions. The specific components of QPCRMix are shown in Table 1.

[0080] Table 1 Specific components of QPCRMix Components Single reaction volume 2×qPCRReactionMix 12.5μl Mycoplasma Primer & Probe Mix 2.1μl InternalControl 0.4μl Total volume 15μl Slightly shake the prepared QPCRMix and centrifuge it instantaneously. Add 15μl / well to the eight-well tube. Add 10μl / sample / PC positive control / test sample to the corresponding eight-well tube. Centrifuge it instantaneously. The total volume of the sample is 25μl. Set up 3 sets of replicate wells for each group of test samples. Run the program set in the instructions.

[0081] (3) Result analysis and judgment The final result judgment method is shown in Table 2 2.9 Endotoxin detection (gel method) (1) Take one bottle of horseshoe crab reagent and add the indicated amount of test water to dissolve it, then shake gently.

[0082] (2) Take 5 10×75 mm test tubes, 2 of which are used as test tubes, 1 as a negative control tube, 1 as a positive control tube, and 1 as a test sample positive control tube.

[0083] (3) Add 0.1 ml of dissolved horseshoe crab reagent to each test tube.

[0084] (4) Add 0.1 ml of test sample to each test sample tube; add 0.1 ml of test water to the negative control tube; add 0.1 ml of endotoxin solution with a concentration of 0.5 Eu / ml to the positive control tube; and add 0.1 ml of test sample containing 0.5 Eu endotoxin to the positive control tube.

[0085] (5) Seal the tube, shake gently, and place vertically in a thermostat at 37±1℃ for 60±2 minutes, then take out and observe the results. Avoid any vibration during the incubation period.

[0086] (6) Result judgment: Gently remove the test tube from the thermostat and slowly invert it 180°. If the contents of the tube are in the form of a solid gel that does not deform or slide off the tube wall, the result is recorded as positive (+). If the contents do not gel or if a gel is formed but cannot remain intact and slides off the tube wall, the result is recorded as negative (-).

[0087] 2.10 Cell cycle (1) Collect P3 cells in the logarithmic growth phase: discard the culture medium, wash with 0.9% NaCl injection PBS, digest with trypsin, dilute with complete culture medium and collect the cells in the corresponding labeled centrifuge tube, centrifuge at 500g for 5 minutes at room temperature, and discard the supernatant. Resuspend the cells with 3ml of pre-cooled PBS, centrifuge at 500g for 5 minutes at room temperature, and discard the supernatant.

[0088] (2) Fixing cells: Resuspend the cells in 0.5 ml of pre-cooled PBS solution, add pre-cooled 70% ethanol solution drop by drop and mix gently. Fix the cells in the dark at 4°C for 18 h.

[0089] (3) Propidium iodide (PI) staining cells: collect cells and centrifuge at 1000 rpm for 5 min, wash once with PBS, treat cells with staining working solution, add 500uL of PI / RNaseA staining working solution (RnaseA:PI working solution is prepared in a volume ratio of 1:9), and incubate at room temperature at 4°C in the dark for 30 min.

[0090] (4) Flow cytometry: Detection by flow cytometry within 1 hour. Filter the cell fluid with a 200-mesh cell sieve before loading the flow cytometer. Record the red fluorescence at an excitation wavelength of 488 nm and analyze the histogram of PI fluorescence.

[0091] 2.11 Flow cytometry (1) Collect P1, P3, and P5 cells in the logarithmic growth phase: discard the culture medium, wash with 0.9% NaCl injection PBS, digest with trypsin, dilute with complete culture medium and collect the cells in the corresponding labeled centrifuge tubes, centrifuge at 500g for 5 min at room temperature, and discard the supernatant.

[0092] (2) Resuspend the cells to 2*10^6 / ml in 0.9% NaCl injection solution, filter through a 100μm filter, take 100μl of cells and add the corresponding antibodies to incubate for 15 minutes, detect the cell phenotype and analyze it on the machine.

[0093] 2.12 Adipogenic differentiation (1) Collect P3 cells in the logarithmic growth phase, inoculate them into a six-well plate at 1×10^5 / well, add 2.5 ml of the corresponding culture medium for culture, replace the medium after 24 hours, and add adipogenic culture medium for culture.

[0094] (2) The medium was changed every three days, and adipogenic differentiation was detected by Oil Red O staining after 14 days.

[0095] 2.13 Osteogenic differentiation (1) Collect P3 cells in the logarithmic growth phase, inoculate them into a six-well plate at 5×10^4 / well, add 2.5 ml of the corresponding culture medium, and change to osteogenic culture medium after 24 hours.

[0096] (2) The medium was changed every three days, and osteogenic differentiation was detected by Alizarin red staining after 21 days.

[0097] 2.14 Chondrogenic Differentiation (1) Collect P3 cells in the logarithmic growth phase and transfer them to a 15 ml centrifuge tube at a rate of 3 × 10^5 / tube. Wash them by centrifugation twice. Resuspend the precipitated cells in 0.5 ml of chondrogenic culture medium and centrifuge them. Loosen the tube cap and culture them. Keep the tube in place for 24 hours. When the cell clusters begin to aggregate (usually after 24 or 48 hours, depending on the actual cell growth), gently tap the bottom of the centrifuge tube to detach the chondrogenic balls from the bottom of the tube and suspend them in the liquid.

[0098] (2) The medium was changed every three days. After 21-28 days of induction, the chondrocytes were fixed with formalin, embedded in paraffin and sectioned. Finally, Alecian blue staining was performed to detect the cartilage differentiation.

[0099] 2.15 Analysis of immune regulation ability The immunoregulatory ability of umbilical cord mesenchymal stem cells was analyzed.

[0100] 2.16 β-galactosidase SA-β-Gal detection (1) Resuscitation of P3, P5, and P10 cells: After centrifugation and discarding the supernatant, the cells were inoculated at 10^5 / well in a 6-well plate. After culturing for 3 days until the confluence reached 90%, the cell culture medium was aspirated, the cells were washed once with PBS, and 1 ml of β-galactosidase staining fixative was added. The cells were fixed at room temperature for 15 minutes.

[0101] (2) Aspirate the cell fixative solution, wash with PBS three times, 3 min each time, and aspirate the PBS.

[0102] (3) Add 1 ml of staining solution to each well, seal the plate with sealing film, and incubate at 37°C overnight. Count and photograph the cells under a microscope the next day.

[0103] 2.17 Experimental data processing and statistics All data were expressed as mean ± standard error (mean ± SEM), and GraphPad prism 8.0.2 software was used for statistical T test and one-way analysis of variance. When p < 0.05, the results were considered statistically significant.

[0104] Three different serum-free culture media on the market were used to separate, culture and amplify umbilical cord mesenchymal stem cells from the same umbilical cord tissue according to the above process, and relevant indicators were tested. The final experimental results are as follows: 3. Experimental results 3.1 Isolation and culture of primary cells All three serum-free culture mediums had spindle-shaped cells migrate out in about 5-7 days. The morphology met the identification criteria for umbilical cord mesenchymal stem cells. Clones were formed in about 10 days, and dense clones were formed in about 15 days. The crawling rate and cell harvest rate were high after digestion and collection of P0. The number of primary cells crawling out for the first time reached tens of millions (10 cm umbilical cord tissue). In addition, the yield of primary cells cultured in medium A and medium B was better, and they were more suitable for the culture of primary cells in this culture process. For details, see Figure 1-2 .

[0105] 3.2 Proliferation of umbilical cord mesenchymal stem cells The average doubling time of umbilical cord mesenchymal stem cells cultured in the three serum-free culture systems is within 40 hours at P5 and before. Normal culture is generally passaged once every 4 days, and it takes about 32 days from primary cell isolation to reaching the storage quantity. The cell viability of umbilical cord mesenchymal stem cells cultured in the three serum-free culture systems is above 90% throughout the culture process to P10. The cumulative population doubling number is about 30 times, and it can reach about 1×1012 (30cm long umbilical cord tissue) at P5 of the working bank cells, which can meet the quantity requirements of umbilical cord mesenchymal stem cells for clinical use. For details, see Figure 3 .

[0106] 3.3 Microbial safety of umbilical cord mesenchymal stem cells The umbilical cord mesenchymal stem cells cultured in the three serum-free culture systems were tested for sterility, endotoxin and mycoplasma from P0 to P10 and were all negative, meeting the quality control standards.

[0107] 3.4 Flow cytometry analysis of umbilical cord mesenchymal stem cells Flow cytometry index CD34 of umbilical cord mesenchymal stem cells cultured in three serum-free culture systems + 、CD45 + 、CD19 + 、CD11b + , HLA-DR + The proportion of cells is less than 2%, CD105 + 、CD73 + 、CD90 + The cell proportions were all higher than 95%, meeting the quality standards of mesenchymal stem cells. The specific results are shown in Table 3.

[0108] Table 3 Flow cytometry results of umbilical cord mesenchymal stem cells cultured in three serum-free culture systems Cell surface marker expression% CD105 CD90 CD73 CD45 CD34 CD19 CD11b HLA-DR A-P1 99.8 99.9 99.9 0.0 0.4 0.0 0.0 0.0 B-P1 99.5 99.5 99.5 0.0 0.0 0.0 0.0 0.0 C-P1 99.2 99.8 99.7 0.0 0.3 0.0 0.0 0.0 A-P3 99.3 99.4 99.4 0.0 0.0 0.1 0.0 0.0 B-P3 99.9 99.9 99.9 0.0 0.3 0.0 0.0 0.0 C-P3 97.3 99.4 98.0 0.0 1.8 0.2 0.0 0.0 A-P5 97.5 99.2 99.3 0.0 0.1 0.6 0.0 0.0 B-P5 96.1 99.5 98.8 0.0 1.7 0.2 0.0 0.0 C-P5 97.1 99.2 98.3 0.0 1.9 0.0 0.0 0.0 3.5 Biological effectiveness of umbilical cord mesenchymal stem cells After adipogenic induction, the umbilical cord mesenchymal stem cells cultured in the three serum-free culture systems contained red lipid droplets inside the cells stained with Oil Red O (100×). In the osteogenic differentiation experiment, alizarin red staining can produce orange-red calcium nodules (100×), and no such structure appeared in the negative control group. The umbilical cord mesenchymal stem cells cultured in the three serum-free culture systems had an inhibition rate of 81.2%, 75.5%, and 58.7% on the proliferation of lymphocytes, respectively. That is, they have the ability of immune regulation and stem cell differentiation, and are biologically effective. For details, see Figure 4 .

[0109] 3.6 Analysis of aging of umbilical cord mesenchymal stem cells during passage (cycle, β-galactosidase) The results of cell cycle detection are shown in Figure 5 , indicating that compared with P3, the cell cycle of umbilical cord mesenchymal stem cells cultured in the three serum-free culture systems showed S phase cell cycle arrest in P5, indicating that the cells were in a state of increased proliferation; while compared with P5, P10 showed G0 / G1 phase cell arrest, that is, senescent cells were arrested in the G1 phase, lost the ability to respond to mitogens and synthesize DNA, and could not enter the S phase, indicating that the cells have gradually aged.

[0110] The results of SA-β-Gal activity detection are shown in Figure 6 , showing that the number of β-galactosidase-positive cells in three generations of umbilical cord mesenchymal stem cells cultured in three serum-free culture systems is A<C<D, and the SA-β-gal activity level of P3 and P5 umbilical cord mesenchymal stem cells does not change much. However, after long-term in vitro subculture, the number of β-galactosidase-positive cells in P10 umbilical cord mesenchymal stem cells increases, and like normal somatic cells, they gradually enter the senescence stage, which is manifested by flattening of cell morphology and significantly enhanced SA-β-gal activity, without the characteristics of unlimited proliferation.

[0111] 4. Experimental conclusion Various experiments have proved that the umbilical cord mesenchymal stem cells cultured in the three serum-free systems according to this culture process can meet the clinical-grade umbilical cord mesenchymal stem cell culture requirements, and various safety and effectiveness test indicators meet the requirements; the P3 generation umbilical cord mesenchymal stem cell bank can be used as the main cell bank, and the P5 generation umbilical cord mesenchymal stem cell bank can be used as the working cell bank.

[0112] (1) In primary culture using the tissue block direct adherence method, cells crawl out and form dense clones in about 7-10 days; (2) The cells can proliferate quickly after passaging, with a doubling time of less than 30 hours. They can be passaged every 3-4 days and reach the cell number required for storage or clinical use in about 4 weeks; (3) The relevant endotoxin and bacterial tests of the cells cultured by this process before storage were all negative, meeting the storage requirements; at the same time, all flow cytometry detection indicators met and exceeded the requirements of the International Association of Cell Therapy for the identification of mesenchymal stem cells, proving that the cultured mesenchymal stem cells are of high purity; (4) The adipogenic and osteogenic differentiation of umbilical cord mesenchymal stem cells cultured by this process also achieved the expected results, proving that the umbilical cord mesenchymal stem cells cultured by this system have stemness and the ability to differentiate into adipogenic and osteogenic cells. In addition, umbilical cord mesenchymal stem cells have immunomodulatory functions and can inhibit the proliferation of lymphocytes.

[0113] Considering that the cost of the culture medium for the three serum-free systems is 1120 yuan / 500ml, 1060 yuan / 500ml, and 1300 yuan / 500ml respectively, the cost of culture reagent A is slightly higher than that of B, but at the same time, the doubling number of umbilical cord mesenchymal stem cells cultured in group A is slightly higher than that in group B. Therefore, under the same conditions of other cryopreservation reagents and consumables, the cost of umbilical cord mesenchymal stem cells cultured in serum-free culture systems A and B is similar. Therefore, we prefer culture medium A as the culture and expansion medium for umbilical cord mesenchymal stem cells used in the final process.

[0114] After the mesenchymal stem cells cultured with (A) Youkang medium were used to build a bank, P3, P5, cell preparations, and P10 umbilical cord mesenchymal stem cells were sent to a third-party testing agency with CMA and CNAS for testing: including cell identification inspection, intracellular and extracellular viral factor inspection, infectious source microbial inspection, microbial, mycoplasma, endotoxin inspection, cell activity, induction differentiation ability, immunological response, and tumorigenicity inspection. The test results all met clinical grade standards. For example, the preparation cell identification and cell surface markers all met the identification standards of umbilical cord mesenchymal stem cells; mycoplasma, endotoxin, and sterility tests were all negative, which met the biological safety; the preparation had no DMSO residues, no visible foreign matter on the appearance of the preparation, and the pH, osmotic pressure, and filling volume all met the preparation standards.

[0115] Therefore, this culture process is a culture and preparation method for clinical-grade umbilical cord mesenchymal stem cells and their preparations. It only takes about 4 weeks of culture, and the number of mesenchymal stem cells that can be used in the working cell bank can reach trillions, which meets the number level of clinical application. In addition, the cell viability of the cultured and expanded umbilical cord mesenchymal stem cells is above 90%, with high purity, morphology, karyotype analysis, cell surface markers, etc., all meet the identification standards, and biological safety, general biological characteristics, biological effectiveness, and microbial safety all meet clinical application standards. A simple, low-cost, and easy-to-operate process method is provided. In order to scale up the expansion of mesenchymal stem cells, a multi-layer plate culture system and a highly safe serum-free, animal-derived component-free culture system are also used to culture and expand umbilical cord mesenchymal stem cells in vitro. The in vitro expansion and subculture of umbilical cord mesenchymal stem cells was established, and their tri-lineage mesoderm differentiation potential was maintained. The proliferation ability was strong and the doubling time was short. The umbilical cord mesenchymal stem cells that meet the GMP standards are more suitable for clinical research, providing a strong theoretical basis for the establishment of umbilical cord mesenchymal stem cell banks and clinical applications. A culture process that can be used for clinical-grade stem cell manufacturing was established. The specific process flow chart of the culture process finally obtained is shown in the figure below. Figure 7 shown.

[0116] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for culturing umbilical cord mesenchymal stem cells, characterized in that: The following steps are involved: (1) Collecting umbilical cord tissue from donors; (2) After disinfection and cleaning, the donor umbilical cord tissue is separated from Wharton's jelly and centrifuged, then resuspended in complete culture medium and inoculated into a T175 culture flask for culture. After the culture is completed, the P0 cells are collected; (3) Inoculate the P0 cells at a cell density of 1*10^6 / T175, resuspend the cell suspension, add complete culture medium for culture to obtain P1 cells, and collect and freeze for later use; (4) Collect or resuscitate the P1 cells and inoculate them at a cell density of 1*10^6 / T175, resuspend the cell suspension, add complete medium for culture to obtain P2 cells, and collect them for use. Then, culture them according to the same operation to obtain P3 cells and collect and freeze them for use; (5) Collect or resuscitate P3 cells and inoculate them at a cell density of 5*10^7 / cell factory, resuspend the cell suspension, add 1L of complete culture medium to each cell factory, then add the cell suspension, culture to obtain P4 generation cells, and then culture according to the same operation to obtain P5 generation cells; (6) The cells prepared in steps (1) to (5) are revived, washed by centrifugation, resuspended, and then human albumin is added to obtain a cultured umbilical cord mesenchymal stem cell preparation.

2. The culture method according to claim 1, characterized in that In step (2), the disinfection and cleaning are to wash the umbilical cord twice with physiological saline containing triple antibodies, add 75% volume concentration alcohol filtered through a 0.22 μm microporous filter membrane to completely immerse the umbilical cord, soak and disinfect for 2 minutes, then take out the umbilical cord and place it in a sterile culture dish, and wash it 2-3 times with physiological saline to clean the residual umbilical cord blood.

3. The culture method according to claim 1, characterized in that In step (2), the culturing step is to inoculate the resuspended cells into T175 at 1 g per bottle and culture them in the serum-free medium, replace half of the medium on the 5th day after inoculation, and replace the full medium on the 10th day. When the confluence reaches 60%-70%, the cells are digested and collected to obtain primary umbilical cord mesenchymal stem cells.

4. The culture method according to claim 1, characterized in that In step (3), the specific conditions of the culture are culturing for 3 days under 5% CO2 and 37°C. The collection is washing the cells with physiological saline and then digesting them with 5ml / T175 of animal-free trypsin TrypLE Express (1X) for 3 minutes, then washing with physiological saline and centrifuging at 500g for 5 minutes to discard the supernatant. The freezing is resuspending the cells in freezing solution at 17.5 million / ml, placing the cells in a -80 refrigerator overnight, and transferring them to a liquid nitrogen tank the next day.

5. The culture method according to claim 1, characterized in that In step (4), the specific conditions of the culture are to add 21 ml / T175 of complete medium and then add 1 ml of cell suspension. After labeling, the mixture is placed in a carbon dioxide incubator and cultured for 3 days at 5% CO2 and 37°C. The collection is to wash the cells with saline and then digest them with 5 ml / T175 of animal-free trypsin TrypLE Express (1X) for 3 minutes, then wash with saline and centrifuge at 500g for 5 minutes to discard the supernatant; the freezing is to resuspend the cells in freezing solution at 17.5 million / ml, place the cells in a -80 refrigerator overnight, and transfer them to a liquid nitrogen tank the next day.

6. The culture method according to claim 1, characterized in that In step (5), the specific conditions of the culture are to add 1L of complete culture medium to each cell factory, then add the cell suspension, mark and mix well, and then culture in a carbon dioxide incubator. The culture conditions are: 5% CO2, 37°C. After the cells are cultured for about 3-4 days, the cells reach 90% confluence and are collected; the collection is to wash the cells with saline and then digest them with 180ml / cell factory of animal-free trypsin TrypLE Express (1X) for 3min; after washing with saline, centrifuge at 500g for 5min and discard the supernatant; the freezing is to resuspend the cells in freezing solution at 17.5 million / ml, place the cells in a -80 refrigerator overnight, and transfer them to a liquid nitrogen tank the next day.

7. The culture method according to claim 1, characterized in that In step (6), the specific conditions for the recovery are as follows: after taking out the qualified frozen umbilical cord mesenchymal stem cells from liquid nitrogen, quickly placing them in a preheated 37°C water bath, gently shaking them, and melting them within 2 minutes. When the ice crystals in the cell suspension are about to completely disappear by naked eye observation, the cells are taken out and quickly added to sodium chloride injection incubated at room temperature; the centrifugal washing is centrifuged at 500g for 5 min, the supernatant is discarded, sodium chloride injection is added to resuspend, the cells are passed through a 100μm cell sieve, mixed and counted, centrifuged at 500g for 5 min, and the supernatant is discarded; the amount of human serum albumin added is 5ml, and the number of cells in the umbilical cord mesenchymal stem cell preparation is 7*10^7 / 100ml / bag.

8. The culture method according to claim 1, characterized in that The method also includes the steps of performing mycoplasma, endotoxin and sterility tests on the supernatant during the culture process.

9. The culture method according to claim 1, wherein The complete culture medium is a complete culture medium prepared by 500 ml serum-free culture medium base + 5 ml additive 1.

10. An umbilical cord mesenchymal stem cell preparation obtained according to the culture method according to any one of claims 1 to 9.

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