Culture medium suitable for cell population containing mesenchymal stem cells

Abstract

The invention belongs to the field of cell preparations, and relates to a culture medium suitable for a cell population containing mesenchymal stem cells, the culture medium comprises a component 1 MEM-alpha, and 5% of a serum substitute is added; component 2: MEM-alpha added with a human bFGF, a human EGF and a human VEGF, wherein the final concentration of the human bFGF, the final concentration of the human EGF and the final concentration of the human VEGF are all 10 ng / ml; wherein the addition amount of the component 2 in the component 1 is less than or equal to 10%; wherein the component 1 is a culture medium adopted during subculture of MSC (mesenchymal stem cells); and the component 2 is an endothelial progenitor cell culture medium. In the culture process of the MSC cells, introduction of animal-derived components is avoided; meanwhile, the content of endothelial progenitor cells carried by the umbilical cord in the process of obtaining the MSC is regulated and controlled, and the optimal performance of the MSC is kept.

Description

Technical Field

[0001] The present invention belongs to the field of cell preparations, and relates to a culture medium suitable for cell populations containing mesenchymal stem cells. Background Art

[0002] MSC cells (MSC, mesenchymal stem cells) are important members of the stem cell family, originating from the mesoderm in the early stage of development, and belong to pluripotent stem cells.

[0003] Under specific induction conditions in vivo or in vitro, MSC cells can differentiate into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, myocardium, and endothelium. They still have multi-directional differentiation potential after continuous passage culture and cryopreservation, and can be used as ideal seed cells for repairing tissue and organ damage caused by aging and diseases.

[0004] A large number of studies have shown that MSC plays an important role in the treatment of cardiovascular diseases, liver cirrhosis, bone and muscle degenerative diseases, brain and spinal cord nerve injuries, Alzheimer's disease, and autoimmune diseases such as lupus erythematosus and scleroderma; and has great development prospects in beauty, health care, anti-aging, etc. Such as Chinese patents CN104136034B Mesenchymal stromal cells and related uses, CN102008507B Human umbilical cord MSC cell anti-hepatic fibrosis injection and its preparation method...

[0005] However, after the MSC preparations summarized in the prior art are administered for treatment, there will be adverse reactions, such as fever, and the highest incidence rate in the literature is about 39%; the main reason for the analysis is that the exact characteristics of human MSC (hMSC) may vary greatly due to various parameters (including tissue source, isolation method, and culture medium components) [Literature 1].

[0006] Human umbilical vein endothelial progenitor cells are isolated from umbilical cord tissue; it is one of the important structural component cells of the umbilical vein and plays an important role in the normal physiological process of the body.

[0007] Literature 1: Isolation, cultivation, and characterization of human mesenchymal stem cells. Mushahary D, Spittler A, Kasper C, Weber V, Charwat V. Cytometry A. 2018 Jan; 93(1): 19 - 31. Summary of the Invention

[0008] The present application provides a culture medium suitable for cell populations containing mesenchymal stem cells (MSCs) based on the current situation. During the culturing process of MSCs, the introduction of animal-derived components is avoided. At the same time, the content of endothelial progenitor cells carried during the acquisition of MSCs from the umbilical cord is regulated to maintain the optimal performance of MSCs. Finally, the preparation containing MSCs obtained in the present application has simple and clear components, no toxic or side effects, and good stability.

[0009] To achieve the above technical objectives, the technical solution adopted in the present application is a culture medium suitable for cell populations containing mesenchymal stem cells, including

[0010] Component 1: MEM-α, with 5% serum replacement added;

[0011] Component 2: MEM-α supplemented with human bFGF, human EGF, and human VEGF, with the final concentrations of human bFGF, human EGF, and human VEGF all being 10 ng / ml;

[0012] Among them, the addition amount of Component 2 in Component 1 is ≤10%;

[0013] Among them, Component 1 is the culture medium used for the passage culture of MSCs; Component 2 is the endothelial progenitor cell culture medium.

[0014] As an improved technical solution of the present application, the content of MSCs in the cell population containing mesenchymal stem cells is ≥90%, and the content of endothelial progenitor cells is ≤10%.

[0015] As an improved technical solution of the present application, the serum replacement includes one of EliteGRO-Adv, UltraGRO-Advanced, KnockOut Serum Replacement, CTS KnockOut SR, and XenoFree Kit.

[0016] Beneficial effects

[0017] Both the MSCs and endothelial progenitor cells in the present application are derived from the umbilical cord, that is, they have the advantages of rich sources, non-invasive sampling, low immunogenicity, and avoiding ethical disputes. The preparation containing MSCs in the present application has good short-term stability at low temperature and good long-term stability under cryopreservation conditions.

[0018] The preparation method of the preparation containing MSCs provided in the embodiments of the present invention has simple steps, high repeatability, and is suitable for large-scale production.

[0019] The preparation containing MSC cells of the present application can be widely applied to various diseases and has a good improvement effect on various diseases; and it has been verified by animal model experiments that it is non-toxic; in particular, it can be adapted to the diseases applicable to MSC cells verified by the prior art.

[0020] It should be understood that all combinations of the foregoing concepts and additional concepts described in more detail below can be regarded as part of the inventive subject matter of the present disclosure as long as such concepts do not contradict each other. Brief Description of the Drawings

[0021] Figure 1 Graph of the body weights of the sham operation group and the model establishment group continuously increasing from 0 to 12 weeks after castration surgery;

[0022] Figure 2 From 0 to 12 weeks after castration surgery, the relative body weight growth rate of the model establishment group was significantly higher than that of the sham operation group (***, P < 0.01);

[0023] Figure 3 During the drug administration period, the body weights of the animals in the sham operation group, the model control group, and the MSC treatment group all increased;

[0024] Figure 4 Comparison of the trabecular bone mineral density at the distal femur of the animals in the sham operation group and the model establishment group 12 weeks after castration surgery: Compared with the sham operation group, the trabecular bone mineral density (Trabeculae Mean BMD) of the animals in the model establishment group had a decreasing trend, but there was no significant difference;

[0025] Figure 5 Comparison of the trabecular bone number of the animals in the sham operation group and the model establishment group 12 weeks after castration surgery: Compared with the sham operation group, the trabecular bone number (Tb.N) of the animals in the model establishment group was significantly decreased (***p < 0.05);

[0026] Figure 6 Comparison of the trabecular bone connectivity density of the animals in the sham operation group and the model establishment group 12 weeks after castration surgery: Compared with the sham operation group, the trabecular bone connectivity density (Conn.D) of the animals in the model establishment group was significantly decreased (**p < 0.05);

[0027] Figure 7 Comparison of the BMD change% of the femoral shaft of the animals in the sham operation group, the model control group, and the MSC treatment group at the experimental end point: The BMD change% of the femoral shaft bone mineral density of the animals in the model control group was significantly lower than that of the sham operation group (*P < 0.05), and the BMD change% of the femoral shaft bone mineral density of the MSC treatment group was significantly higher than that of the model control group (##P < 0.01).

[0028] Figure 8Comparison of the percentage change in BMD of the femoral neck among animals in the sham operation group, model control group, and MSC treatment group at the experimental endpoint showed that the percentage change in BMD of the femoral neck in the MSC treatment group was significantly higher than that in the model control group (#P < 0.05). Detailed implementation manners

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the described embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein shall have the ordinary meanings understood by those of ordinary skill in the art in the field to which the present invention belongs.

[0030] Definition:

[0031] Stability, stability of the final product before injection: The final product of MSCs needs to be uniformly mixed with the injection solution before intravenous injection can be used for administration; during the time from the mixing of MSCs with the injection solution until the completion of MSC injection administration, the cell number and viability of the MSC product must meet the quality standard requirements to ensure safety and effectiveness.

[0032] Umbilical cord MSCs have high differentiation potential and can differentiate in multiple directions. It has broad clinical application prospects in tissue engineering such as bone, cartilage, muscle, tendon, ligament, nerve, liver, endothelium, and myocardium. It has been reported that MSCs are isolated from human umbilical cords, and the cell content and proliferation ability are better than those of bone marrow MSCs, and the immunogenicity is lower than that of bone marrow MSCs. Therefore, the MSCs in this application are derived from umbilical cord tissue.

[0033] Injection solution: A sterile solution for injection into the body, in which MSCs derived from umbilical cords and endothelial progenitor cells derived from umbilical cords are suspended. To ensure the drug effect, when used for intravenous injection, the injection solution includes normal saline, compound electrolyte injection solution, or dextran 40 glucose injection solution. When used for local joint cavity injection, the injection solution includes normal saline, compound electrolyte injection solution, or hyaluronic acid injection solution.

[0034] Sodium chloride injection solution used herein: 0.9%, manufacturer: Sichuan Kelun Pharmaceutical Co., Ltd., batch number: M19051004-2; Bleomycin hydrochloride for injection (bleomycin), production unit: Nippon Kayaku Co., Ltd., batch number: 790670.

[0035] CD34 is a single-chain transmembrane glycoprotein with a relative molecular mass of 110,000 encoded by a gene located at 1q32, and can be expressed in normal endothelial progenitor cells, splenic marginal zone cells, and dendritic interstitial cells surrounding blood vessels, nerves, muscle bundles, skin appendages, and mammary lobule stroma. However, the cells closest to MSC cells after digestion and separation of umbilical cord-derived cells are endothelial progenitor cells.

[0036] This application mainly isolates and obtains MSC and endothelial progenitor cells from the umbilical cord simultaneously, and conducts conditional amplification culture to study the interaction between umbilical cord-derived MSC and endothelial progenitor cells during culture and medicinal use.

[0037] The technical solution of this application will be clearly and completely described below in combination with specific embodiments.

[0038] I. Acquisition methods of umbilical cord-derived MSC cells and umbilical cord-derived endothelial progenitor cells in preparations containing MSC cells

[0039] A) Primary cell acquisition

[0040] In order to improve the purity of cells and avoid the introduction of animal-derived components, this application mainly uses the tissue block adherence method to obtain primary cells (P0 cells, including MSC cells and endothelial progenitor cells), and physically removes amniotic membranes, blood vessels, etc. It can obtain high-purity cells without using materials such as trypsin, and can better maintain the self-activity of cells, which is more suitable for clinical applications than the enzyme digestion method.

[0041] B) Subculture of primary cells

[0042] Since the ultimate use of the cell preparation in this technical solution is for medicinal purposes, it is necessary to avoid using animal-derived reagents during digestion, subculture, cryopreservation, etc.

[0043] Therefore, this technical solution uses a recombinant cell dissociation enzyme without animal origin: such as TrypLE products. TrypLE can be used to dissociate cells under serum replacement and serum-free conditions. It is stable at room temperature, and there is no need to use a specific protease inhibitor to inactivate after digestion; another example is the HyrTryp cell separation reagent product, which meets standards such as cGMP, ISO 9001:2015, and ISO13485:2016, and is a better choice from the perspectives of regulatory safety and downstream purification difficulty.

[0044] In the embodiments of the present application, TrypLE is mainly selected (in actual applications, HyrTryp is also verified, which has the same effect as TrypLE. For the principle of text saving, the verification and description of verification conclusions are not repeated herein). Selection of neutralization solution: To ensure that no animal-derived components are introduced into the process, any neutralization solution containing serum or animal protein is abandoned, and DPBS with clear components is selected in this technical solution.

[0045] Selection of passage medium: The medium used for primary cell passage culture is MEM-α, and 5% serum substitute is added. In this application, the serum substitute includes one of EliteGRO-Adv, UltraGRO-Advanced (HELIOS), KnockOutSerum Replacement (Gibco), CTS KnockOut SR, and XenoFree Kit (Gibco).

[0046] To ensure that a certain proportion of endothelial progenitor cells can be obtained simultaneously, ECM medium (endothelial progenitor cell medium) is also added to the passage medium. The proportion of ECM can be 0-10%, approximately corresponding to the content ratio of endothelial progenitor cells. In this application, ≤10% of endothelial progenitor cell medium is also added to the medium. The ECM is MEM-α supplemented with human bFGF, human EGF, and human VEGF. The ECM is endothelial progenitor cell medium, and the final concentrations of human bFGF, human EGF, and human VEGF are all 10 ng / ml.

[0047] Medium verification:

[0048] The seeding density is 6.0×10 3 / cm 2 , and the culture time is 120 h for medium verification; during cell culture, the medium is MEM-α, and 5% EliteGRO-Adv is added (other types of serum substitutes have similar effects during experimental verification and are not repeated herein). The effects of changes in the content of endothelial progenitor cell medium on the amounts of endothelial progenitor cells and MSC cells are explored. See Table 1. The results show that when the content of endothelial progenitor cell medium is ≤10%, the content ratio of the two types of cells meets the requirements.

[0049] Table 1 Effects of medium design on cell amounts

[0050]

[0051]

[0052] In subsequent experiments, MEM-α medium was selected and 5% EliteGRO-Adv was added, and the medium with 3% endothelial progenitor cell medium content was used for cell passage and cell proliferation.

[0053] After the P0 cells (primary cells) were incubated with TrypLE for 5 minutes and neutralized with DPBS, most cells detached from the culture dish; the P0 cells were centrifuged at 300 g for 10 minutes and then resuspended in MSCM, and the viability was calculated. The results showed that the vast majority of cells were successfully centrifuged, and the cell viability was not less than 80%, meeting the expectations.

[0054] The P0 cells were inoculated in 100 mm culture dishes at different seeding densities for amplification, and the amplification effects are shown in Table 2.

[0055] Table 2 Average total number of cells after amplification at different seeding densities of primary cells

[0056]

[0057] After incubation with TrypLE for 5 minutes and neutralization with DPBS, most cells detached from the culture dish. After centrifugation at 300 g for 5 minutes at room temperature, they were resuspended in MSCM and the viability was calculated. The results showed that the cell viability was not less than 90%.

[0058] This example verified that when the seeding density was 0.5×10 3 / cm 2 -6×10 3 / cm 2 and the culture time was 96 h - 144 h, the cells had good fusion rate and cell proliferation multiple, and the expected total number of cells was obtained. More preferably, when the seeding density was 1.0×10 3 / cm 2 -6.0×10 3 / cm 2 and the culture time was 120 h - 144 h, the cell yield could be maximized, while ensuring the high viability of the cells and avoiding overly frequent digestion and passage, representing the appropriate seeding density and culture duration.

[0059] C) Cell proliferation and passage

[0060] Based on the foregoing experiments, after culturing the cells for 120 h, digestion and passage were performed, and the cells were counted. Inoculate at a density of 6x10 3 / cm 2 and passage after culturing for 120 hours. When culturing for 120 h and performing digestion and passage, if the cell number increased less than 10-fold compared to the inoculation, or obvious changes occurred in the cell morphology, or the positive rate of any one MSC positive marker was lower than 80%, the experiment was terminated.

[0061] Among them, the positive markers of MSC cells are CD90, CD73, and CD105; the positive marker of endothelial progenitor cells is CD34.

[0062] Table 3 Cell Maximum Passage Test

[0063]

[0064] The test results showed that after 9 passages, the proliferation multiple, cell morphology, and the expression of positive markers of the cells all met the requirements. After 10 passages, the cell volume increased significantly, but the proliferation multiple and the expression of positive markers still met the requirements.

[0065] According to the results, it was determined that under the inoculation amount of 0.32x10 6 and the passage interval culture time of 120 hours, the maximum passage number of the cells was 9 times. When the cells were passaged 1 - 8 times, the cells proliferated 16.7 times on average per passage. To ensure the maximum efficiency utilization and the maintenance of the maximum activity of the cell preparation, the MSC cell preparation of this application selected cells passaged 6 - 8 times.

[0066] To simplify the text description, the following experiments were all carried out using: the number of cells at inoculation (100 mm culture dish) 0.32x10 6 and the number of cells 4.1x10 6 after 8 passages to conduct cryopreservation, cytotoxicity, the stability of the preparation containing MSC cells, and verification of animal models. Generally speaking, when the cells passaged 8 times are non-toxic and have therapeutic uses, the cells passaged 3, 4, 5, 6, and 7 times can also meet the relevant requirements.

[0067] D) Cryopreservation of Cells

[0068] Cryopreservation solution test and confirmation of cryopreservation steps: At 1.0x10 7 / ml, 1.3x10 7 / ml, 2x10 7 / ml, 2.5x10 7 / ml, 3.3x10 7 / ml, and 4x10 7Resuspend the cells at a density of / ml in three cryopreservation solutions: MSCM (7.5% DMSO), PRIME-XVF reezIS, and PRIME-XVM SCF reezIS DMSO-Free. After perfusion into cryotubes, place them in a programmable cryobox, store at -80°C for 16 hours, and then transfer them to a liquid nitrogen tank. After 48 hours of storage in liquid nitrogen, thaw the cells in a 37°C water bath. Slowly add the thawed cell suspension to a 15-ml centrifuge tube containing 4 ml of MSCM and centrifuge at 300 g for 5 minutes. Then, resuspend the cells in MSCM to 1 ml and perform tests according to the quality standards. The specific data are shown in Table 4.

[0069] Table 4 Viability of cells cryopreserved with different cryopreservation solutions and thawed

[0070]

[0071] The results showed that the quality of the cells cryopreserved with the three cryopreservation solutions was qualified after thawing. However, at a cryopreservation density of 1.3x10 7 / ml - 3.3x10 7 / ml, the cells cryopreserved in MSCM (7.5% DMSO) had the highest viability.

[0072] IV) Short-term stability verification

[0073] Short-term stability: After thawing the working bank cells in a 37°C water bath, mix them evenly with the injection solution and place them at 2 - 8°C. Detect the cell viability at 0, 1, 2, 4, 8, 12, 16, 24, and 32 hours after thawing to provide a basis for the stable storage time of the MSC final product (the preparation containing MSC cells) at 2 - 8°C. The expected viability is not less than 95%.

[0074] Detection method: The cryopreservation densities of the cells are 1.3x10 7 / ml (Sample 1), 2.0x10 7 / ml (Sample 2), 2.5x10 7 / ml (Sample 3), 3.3x10 7 / ml (Sample 4), 1x10 7 / ml (Sample 5), and 4.3x10 7 / ml (Sample 6). Detect the cell viability at 0, 1, 2, 4, 8, 12, 16, 24, and 32 hours after thawing in a 2 - 8°C environment. The results showed that the viabilities of Samples 1 to 4 could meet the usage requirements even within 32 hours or longer. The results showed that after 16 hours of placement, the cell quantity and viability of Samples 5 to 6 still met the quality requirements. The results are shown in Table 5.

[0075] Table 5 Short-term stability of the preparation containing MSC cells

[0076] Time point 0h 1h 2h 4h 8h 12h 16h 24h 32h Survival rate of sample one (%) 99.9 99.7 99.0 99.0 98.5 98.0 97.0 96.0 95.0 Survival rate of sample two (%) 99.9 99.0 98.7 98.3 98.1 97.8 97.3 96.4 95.3 Survival rate of sample three (%) 99.9 99.1 98.9 98.5 98.0 97.6 97.0 96.0 95.5 Survival rate of sample four (%) 99.9 99.3 98.7 98 97.3 97.2 96.8 96.1 95.7 Survival rate of sample five (%) 99.8 99.1 98.0 95.0 93.0 90.0 84.0 80.0 70.0 Survival rate of sample six (%) 99.9 99.0 97.0 96.0 95.0 93.0 89.0 86.0 80.0

[0077] The unpublicized embodiments of this application are to detect the cell viability at 0, 1, 2, 4, 8, 12, 16, and 24 hours after resuscitation in environments of 2°C, 3°C, 5°C, 6°C, 7°C, and 8°C, with an error of only ±0.4% compared to detecting the cell viability in a 4°C environment.

[0078] V) Long-term stability verification

[0079] Long-term stability: Confirm the longest stable time (product validity period) of cells (MSC cells and endothelial progenitor cells) under cryopreservation to determine the shelf life of the product.

[0080] After storing at -80°C for 16 h, transfer to a liquid nitrogen tank for storage for 3 years, and then resuscitate and detect the cell performance.

[0081] The cryopreservation density used in this experiment is 1.3×10 7 / ml.

[0082] Resuscitation method: Resuscitate the cells in a 37°C water bath, slowly add the resuscitated cell suspension to a 15-ml centrifuge tube containing 4 ml of MSCM, centrifuge at 300 g for 5 minutes, then resuspend the cells to 1 ml with MSCM and detect according to the quality standards.

[0083] Table 6 Long-term stability of the preparation product containing MSC cells

[0084]

[0085]

[0086] Of course, the applicant also verified cryopreservation densities of 2.3×10 7 / ml and 3.3×10 7 / ml, and there is basically no difference in their long-term stability effects compared to a cryopreservation density of 1.3×10 7 / ml, so this is not described separately in this article to save text.

[0087] VI) Cytotoxicity experiment

[0088] In this experiment, a control group and a test group were designed, with 20 NCG mice in each group, half male and half female. The NCG mice were administered once every 2 days for 1 week (4 times in total) by intravenous injection of hMSC100 and observed for 4 weeks to observe the nature, degree, dose-effect and time-effect relationships, and reversibility of the possible toxic reactions caused by the test article.

[0089] hMSC100 was prepared by inoculating 0.32x10 6, the cell suspension obtained by suspending the cells after 8 passages with a cell count of 4.1x10 6 in physiological saline.

[0090] Table 7 Toxicity Test Dose Design Table

[0091]

[0092] Toxicity detection indicators:

[0093] Blood biochemistry:

[0094] At the end of the observation period, no significant abnormal changes were observed in the blood biochemical indicators such as ALB, TP, A / G, AST, ALT, TBIL, CK, CHOL, TG, Crea, Urea, GLU, GGT, ALP, LDH, Na+, K+, and Cl- in male and female mice in each group of hMSC100, indicating normal conditions.

[0095] Gross anatomical observation:

[0096] At the end of the observation period, the main organs such as the brain, heart, liver, spleen, kidney, gastrointestinal tract, and reproductive system were observed macroscopically in male and female mice in each group. No significant abnormal changes were observed in their morphology, color, texture, etc., indicating normal conditions.

[0097] In summary, under the conditions of this experiment, NCG mice were intravenously injected with 3×10 6 and 1×10 7 hMSC100 cells / kg once every 2 days for 1 week (4 times in total), and observed for 4 weeks. No significant abnormal changes were observed in the general observation, body weight, food intake, hematology, blood biochemistry, and gross anatomical observation of each group of mice.

[0098] VII) Pharmacodynamic Study of MSC Cell Preparation on Bleomycin-induced Pulmonary Fibrosis Model Rats

[0099] Experimental grouping:

[0100] A total of 55 male SD rats were used in this experiment: initially, they were randomly divided into a normal control group (10 rats) and a model group (45 rats); after the establishment of the pulmonary fibrosis model, they were randomly divided again into a low-dose test article group (2×10 6 cells / rat / time, 1.5 mL / rat, 11 rats), a high-dose test article group (6×10 6 cells / rat / time, 1.5 mL / rat, 11 rats), a commercially available control group (pirfenidone capsules, batch number: 191006, 240 mg / kg, 5 mL / kg, 11 rats), a model control group (sodium chloride injection, 12 rats), and a normal control group (sodium chloride injection, 10 rats).

[0101] In the D1 and D5 model groups, animals were given bleomycin (5 mg / kg, 1 mL / kg) by aerosol inhalation into the airway to establish the model, and animals in the normal control group were given sodium chloride injection (1 mL / kg) by aerosol inhalation into the airway. Among them, hMSC100 was a preparation obtained by suspending cells with a cell count of 0.32x10 6 at the time of inoculation (100 mm culture dish) and a cell count of 4.1x10 6 after 8 passages in physiological saline.

[0102] Animals in the normal control group, model control group, low-dose and high-dose test article groups were each given the corresponding drug intravenously once on D6, D8, and D10, for a total of 3 administrations; animals in the commercially available control group were given the drug by gavage once a day for a total of 23 times (D6 - D28).

[0103] On D28, animals in each group were subjected to pulmonary function tests, weighed for lung weight, and the lung weight index was calculated; the left lung was excised for determination of hydroxyproline content, and the remaining right lung was perfused with 10% neutral buffered formalin solution and then fixed in 10% neutral buffered formalin solution, paraffin-embedded, sectioned, prepared, stained with HE for evaluation of the degree of inflammatory cell infiltration in lung tissue, and stained with Masson for evaluation of the degree of pulmonary fibrosis.

[0104] Results: In terms of model establishment, during the experiment, animals were induced with pulmonary fibrosis by aerosol inhalation of bleomycin into the airway on D1 and D5 respectively. Under the microscope, pathological changes such as varying degrees of inflammatory cell infiltration and fibrosis were mainly visible in the lungs, indicating successful establishment of the pulmonary fibrosis model.

[0105] Survival rate: One animal died in the model control group on D6 and D11 respectively, and one animal died in the low-dose test article group on D14. The survival rates of animals in the normal control group, model control group, low-dose hMSC100 group, high-dose hMSC100 group, and commercially available control group were 100%, 83%, 91%, 100%, and 100% respectively. The survival rates of animals in each test article group and the commercially available control group were higher than that of the model control group.

[0106] Lung weight index: Compared with animals in the model control group, the mean lung weight index of animals in the normal control group, low-dose test article group, high-dose test article group, and commercially available control group decreased, and there was a statistical difference in the normal control group; the low-dose and high-dose test article groups showed a dose-dependent decrease. Lung

[0107] Hydroxyproline content: Compared with animals in the model control group, the mean hydroxyproline content of animals in the normal control group, low-dose test article group, and high-dose test article group decreased, and there were statistical differences in the low-dose and high-dose test article groups, and there was a dose-dependent decrease.

[0108] (VIII) Pharmacodynamic evaluation of hMSC100 in the treatment of osteoporosis in ovariectomized rats

[0109] hMSC100 was seeded with a cell number of 0.32x10 6 After 8 passages, the number of cells was 4.1x10 6 The cells were suspended in physiological saline to obtain the preparation. Sham group: Sham group, model control group: Model group, MSC treatment group: MSC group.

[0110] Table 8 Animal grouping information

[0111]

[0112] Detection indicators:

[0113] During the experiment, the behavioral state and food intake of animals in each group were observed once a day; the body weight of animals was measured once a week; 3 months after castration and 1 week after the last administration, the animals were sampled and the femoral bone density and bone microstructure of the animals were analyzed by micro-CT.

[0114] Test results

[0115] 1. General observation and body weight

[0116] All animals showed no abnormal behavior during the experiment.

[0117] Three months after castration, the weight of animals in the sham operation and modeling groups continued to increase. The relative weight growth rate of animals in the sham operation group was 20.42%, and the relative weight growth rate of animals in the modeling group was 35.44%. The animal model meets the law of weight growth after castration. For specific weight change trends, see Figure 1 , 2 .

[0118] During the drug administration period, the weight of animals in the sham operation group increased slightly, while the weight of animals in the model control group increased significantly, and the weight of animals in the MSC treatment group did not increase significantly. Figure 3 .

[0119] 2. Bone density and bone microstructure

[0120] The femoral bone density and bone microstructure of the animals were analyzed by micro-CT.

[0121] Three months after castration, compared with the sham operation group, the trabecular bone density (Trabeculae Mean BMD) of the distal femur of the model group animals showed a downward trend, the number of trabeculae (Tb.N) was significantly reduced (p<0.05), and the trabecular connection density (Conn.D) was significantly reduced (p<0.05). The results showed that the model group was in line with the trend of osteoporosis. For specific data, see Figure 4 ,5 , 6. Trabecular bone has the functions of supporting hematopoietic tissue and increasing bone strength. The number, quality, direction, thickness, etc. of trabecular bone will have a great impact on bone strength.

[0122] After the treatment period, the percentage of the difference between the bone mineral density (BMD) values of each group of individuals and the average BMD of the sham operation group was analyzed (BMD change% = (BMD value of each group of individuals - average BMD of the sham operation group)%; change, change rate). The BMD change% of the femoral shaft bone density in the model control group was significantly lower than that in the sham operation group (P < 0.05), and the BMD change% of the femoral shaft bone density in the MSC treatment group was significantly higher than that in the model control group (P < 0.01). The BMD change% of the femoral neck bone density in the MSC treatment group was significantly higher than that in the model control group (P < 0.05). The specific data are shown in Figure 7 , 8 , and the results showed that the bone mineral density of the femoral shaft and femoral neck in ovariectomized SD rats induced with osteoporosis was significantly increased after 4 weeks of treatment with hMSC100, suggesting that MSC has a good therapeutic effect on osteoporosis.

[0123] IX) Pharmacodynamic evaluation of hMSC100 with different components in the treatment of osteoporosis in ovariectomized rats

[0124] Different from the "pharmacodynamic evaluation of hMSC100 in the treatment of osteoporosis in ovariectomized rats", this example verified the influence of the components and content of hMSC100 on the change rate of femoral shaft bone mineral density in animals. The cell number in hMSC100 with different components was 4.1x10 6 , and the preparations used were all obtained by suspending cells in physiological saline. The administration concentration was 0.6×10 6 cells / mL, and the administration volume was 5 mL / kg. The data showed the change rate of femoral shaft bone mineral density in the experimental group (MSC treatment group) relative to the model group (untreated group) (Table 8).

[0125] Table 9 Influence of components and content of hMSC100 on femoral shaft bone mineral density in animals

[0126]

[0127] Experimental verification showed that when the content of MSC cells in the preparation was ≥90% and the content of endothelial progenitor cells was ≤10%, it had a good effect on the treatment of osteoporosis.

[0128] In actual application, the MSC preparation of the present application is also used for the treatment of multiple sclerosis, osteoporosis, systemic scleroderma, hematological malignancies, myocardial infarction, organ transplant rejection, chronic allograft nephropathy, sclerosis, liver failure, heart failure, GvHD, tibial fracture, left ventricular dysfunction, leukemia, myelodysplastic syndrome, Crohn's disease, diabetes, chronic obstructive pulmonary disease, osteogenesis imperfecta, homozygous familial hypercholesterolemia, treatment after meniscectomy, adult periodontitis, angiogenesis in patients with severe myocardial ischemia, spinal cord injury, osteodysplasia, critical limb ischemia, diabetic foot disease, primary Sjogren's syndrome, osteoarthritis, cartilage defect, laminitis, multiple system atrophy, amyotrophic lateral sclerosis, cardiac surgery, systemic lupus erythematosus, living kidney allotransplantation, non-malignant red blood cell disorders, thermal burns, radiation burns, Parkinson's disease, microfracture, epidermolysis bullosa, severe coronary ischemia, idiopathic dilated cardiomyopathy, osteonecrosis of the femoral head, lupus nephritis, bone defect, ischemic stroke, post-stroke, acute radiation syndrome, lung disease, arthritis, bone regeneration, uveitis or a combination thereof. For the principle of text simplicity, only some of the efficacy evaluations are listed in the technical text of the present application.

[0129] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Those of ordinary skill in the art to which the present invention pertains can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to that defined by the claims.

Claims

1. A culture medium suitable for a cell population containing mesenchymal stem cells, characterized in that, Comprising Component 1: MEM-α, with 5% serum replacement added; Component 2: MEM-α supplemented with human bFGF, human EGF, and human VEGF, with the final concentrations of human bFGF, human EGF, and human VEGF all being 10 ng / ml; Among them, the addition amount of Component 2 in Component 1 is ≤10%; Among them, Component 1 is the culture medium adopted when MSC cells are subcultured; Component 2 is the endothelial progenitor cell culture medium.

2. A culture medium suitable for a cell population containing mesenchymal stem cells according to claim 1, characterized in that, The content of MSC cells in the cell population containing mesenchymal stem cells is ≥90%, and the content of endothelial progenitor cells is ≤10%.

3. A culture medium for a cell population containing mesenchymal stem cells according to claim 1, characterized in that, The serum replacement includes one of EliteGRO-Adv, UltraGRO-Advanced, KnockOutSerum Replacement, CTS KnockOut SR, XenoFree Kit.

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