Cell freezing medium for preparation containing MSC (mesenchymal stem cells)

Abstract

The invention belongs to the field of cell preparations, and relates to a cell cryopreservation solution for a preparation containing MSC (mesenchymal stem cells), the cryopreservation solution is selected as follows: a basic culture medium is MSCM, and an additive is DMSO (dimethylsulfoxide) with the volume concentration of 7.5%; the MSCM is an MEM-alpha culture medium added with 5% of a serum substitute, and the serum substitute is one of EliteGRO-Adv, UltraGRO-Advanced, KnockOutSerum Repeller, KnockOutSR (KnockOutSR), and XenoFree Kit, and the MSCM is an MEM-alpha culture medium added with 5% of the serum substitute, and the serum substitute is one of EliteGRO-Adv, UltraGRO-Advanced, KnockOutSR, and XenoFree Kit. The preparation containing the MSC cells has good short-term stability at low temperature and has good long-term stability under a cryopreservation condition.

Description

Technical Field

[0001] The invention belongs to the field of cell preparations and relates to a cell freezing solution for preparations containing MSC cells. Background Art

[0002] MSC cells (MSC, mesenchymal stem cells) are an important member of the stem cell family. They are derived from the mesoderm in the early development and are pluripotent stem cells.

[0003] Under specific induction conditions in vivo or in vitro, MSC cells can differentiate into a variety of tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, myocardium, endothelium, etc. They still have multidirectional differentiation potential after continuous subculture and cryopreservation, and can be used as ideal seed cells for repairing tissue and organ damage caused by aging and pathology.

[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 degeneration diseases, brain and bone marrow nerve damage, Alzheimer's disease, and autoimmune diseases such as lupus erythematosus and scleroderma; and has great development prospects in beauty, health care, anti-aging, etc. For example, Chinese patent CN104136034B mesenchymal stromal cells and related uses, CN102008507B human umbilical cord MSC cell anti-liver fibrosis injection and its preparation method...

[0005] However, the existing technology summarizes that MSC preparations will have adverse reactions after infusion therapy, such as fever, with the highest incidence in the literature being approximately 39%; the main reason for this is that the exact characteristics of human MSCs (hMSCs) may vary greatly depending on a variety of parameters, including tissue source, isolation method, and culture medium composition [Reference 1].

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

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

[0008] Based on the current situation, the present application provides a cell freezing solution for preparations containing MSC cells, which avoids the introduction of animal-derived components during the culture process of MSC cells; at the same time, it regulates the content of endothelial progenitor cells carried in the process of obtaining MSCs from the umbilical cord to maintain the optimal performance of MSC cells; finally, the preparation containing MSC cells obtained in the present application has simple and clear ingredients, no toxic side effects, and good stability.

[0009] To achieve the above technical objectives, the technical solution adopted in the present application is a cell freezing solution for a preparation containing MSC cells, wherein the freezing solution is selected as follows: the basal culture medium is MSCM, and the additive is DMSO with a volume concentration of 7.5%; the MSCM is a MEM-α culture medium supplemented with 5% serum substitute, and the serum substitute includes one of EliteGRO-Adv, UltraGRO-Advanced, KnockOut Serum Replacement, CTS KnockOut SR, and XenoFree Kit.

[0010] As an improved technical solution of this application, the freezing density is 1.3×10 7 / ml-3.3×10 7 / ml.

[0011] As an improved technical solution of the present application, the freezing process conditions are: after being stored at -80°C for 16 hours, transferred to a liquid nitrogen tank.

[0012] As an improved technical solution of the present application, the preparation includes umbilical cord-derived MSC cells and umbilical cord-derived endothelial progenitor cells; wherein the concentration of MSC cells in the preparation is 0.1×10 6 -12×10 6 cells / ml; the content of MSC cells in the preparation is ≥90%, and the content of endothelial progenitor cells is ≤10%.

[0013] As an improved technical solution of the present application, the concentration of the MSC cells is 0.6×10 6 -4×10 6 cells / ml.

[0014] As an improved technical solution of the present application, the preparation also includes an injection solution, and the umbilical cord-derived MSC cells and the umbilical cord-derived endothelial progenitor cells are both suspended in the injection solution.

[0015] As an improved technical solution of the present application, when used for intravenous injection, the injection includes physiological saline, compound electrolyte injection or dextran 40 glucose injection.

[0016] As an improved technical solution of the present application, when used for local joint cavity injection, the injection solution includes physiological saline, compound electrolyte injection solution or hyaluronic acid injection solution.

[0017] Beneficial Effects

[0018] The MSC cells and endothelial progenitor cells of the present application are both derived from the umbilical cord, which has the advantages of abundant sources, non-invasive collection, low immunogenicity and avoidance of ethical disputes. The preparation containing MSC cells of the present application has good short-term stability at low temperatures and good long-term stability under cryopreservation conditions.

[0019] The method for preparing a preparation containing MSC cells provided in the embodiment of the present invention has simple steps and a high repetition rate, and is suitable for large-scale production.

[0020] The preparation containing MSC cells of the present application can be widely applied to many types of diseases and has a good improvement effect on many types of diseases; and it has been verified by animal model experiments that it is non-toxic; in particular, it can be adapted to the diseases that the MSC cells have been verified to be applicable to by the prior art.

[0021] It should be appreciated that all combinations of the foregoing concepts, as well as additional concepts described in greater detail below, may be considered to be part of the inventive subject matter of the present disclosure, provided such concepts are not mutually inconsistent. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The body weight of the sham operation and modeling groups continued to increase from 0 to 12 weeks after castration surgery;

[0023] Figure 2 The relative weight growth rate of the modeling group was significantly higher than that of the sham operation group 0-12 weeks after castration (***, P<0.01);

[0024] Figure 3 During the drug administration period, the weight of animals in the sham operation group, model control group, and MSC treatment group increased;

[0025] Figure 4 Comparison of trabecular bone density of distal femur between sham-operated and model-created animals 12 weeks after castration: Compared with the sham-operated group, the trabecular bone density (Trabeculae Mean BMD) of distal femur of the model-created animals tended to decrease, but there was no significant difference;

[0026] Figure 5 Comparison of femoral trabecular numbers between sham-operated and model-created animals 12 weeks after castration: Compared with the sham-operated group, the trabecular number (Tb.N) of the model-created animals was significantly reduced (***p<0.05);

[0027] Figure 6Comparison of femoral trabecular connection density between sham-operated and model-created animals 12 weeks after castration: Compared with the sham-operated group, the trabecular connection density (Conn.D) of the model-created animals was significantly reduced (**p<0.05);

[0028] Figure 7 Comparison of femoral BMD change% among the sham operation group, model control group and MSC treatment group at the experimental endpoint: the femoral BMD change% of the model control group was significantly lower than that of the sham operation group (*P<0.05), and the femoral BMD change% of the MSC treatment group was significantly higher than that of the model control group (##P<0.01).

[0029] Figure 8 At the end of the experiment, the femoral neck BMD change% of the animals in the sham operation group, model control group and MSC treatment group was compared. The femoral neck BMD change% in the MSC treatment group was significantly higher than that in the model control group (#P<0.05). DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings of the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein should be the common meanings understood by people with ordinary skills in the field to which the present invention belongs.

[0031] definition:

[0032] Stability, stability of the final product before injection: The MSC final product needs to be evenly mixed with the injection solution before it can be administered by intravenous injection; from the time when MSC is mixed with the injection solution until the MSC injection is completed, the cell number and viability of the MSC product must meet the quality standards to ensure safety and effectiveness.

[0033] Umbilical cord MSC cells (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 were isolated from human umbilical cord, and the cell content and proliferation ability are better than bone marrow MSCs, and the immunogenicity is lower than that of bone marrow MSCs. Therefore, the MSC of this application is derived from umbilical cord tissue.

[0034] Injection: A sterile solution for injection into the body, used to suspend umbilical cord-derived MSC cells and umbilical cord-derived endothelial progenitor cells. To ensure the efficacy, when used for intravenous injection, the injection includes physiological saline, compound electrolyte injection or dextran 40 glucose injection. When used for local joint cavity injection, the injection includes physiological saline, compound electrolyte injection or hyaluronic acid injection.

[0035] The sodium chloride injection used in this article: 0.9%, manufacturer: Sichuan Kelun Pharmaceutical Co., Ltd., batch number: M19051004-2; bleomycin hydrochloride for injection (bleomycin), manufacturer: Nippon Kayaku Co., Ltd., batch number: 790670.

[0036] CD34 is a single-chain transmembrane glycoprotein with a relative molecular mass of 110,000 encoded by a gene located at 1q32, which can express normal endothelial progenitor cells, spleen marginal zone cells, and dendritic interstitial cells surrounding blood vessels, nerves, muscle bundles, skin appendages, and mammary lobular matrix. However, after digestion and separation, the cells derived from the umbilical cord are closest to MSC cells, which are endothelial progenitor cells.

[0037] The present application mainly separates and simultaneously obtains MSCs and endothelial progenitor cells from the umbilical cord, and performs conditional amplification culture to study the relationship between umbilical cord-derived MSCs and endothelial progenitor cells during the culture and medicinal use process.

[0038] The technical solution of this application will be clearly and completely described below in conjunction with specific implementation methods.

[0039] 1. Methods of obtaining umbilical cord-derived MSC cells and umbilical cord-derived endothelial progenitor cells in preparations containing MSC cells

[0040] A) Primary cell acquisition

[0041] In order to improve the purity of cells and avoid the introduction of animal-derived components, the present application mainly adopts the tissue block adhesion method when obtaining primary cells (P0 cells, including MSC cells and endothelial progenitor cells), and removes the amniotic membrane, blood vessels, etc. by physical means. It can obtain high-purity cells without using materials such as pancreatic enzymes, and better maintain the activity of the cells themselves. Compared with the enzymatic digestion method, it is more suitable for clinical applications.

[0042] B) Subculture of primary cells

[0043] Since the ultimate use of the cell preparation of this technical solution is for medicinal purposes, the use of animal-derived reagents must be avoided during the processes of digestion, passage, and cryopreservation.

[0044] Therefore, this technical solution uses animal-free recombinant cell dissociation enzymes: such as the TrypLE product. TrypLE can be used to dissociate cells under serum replacement and serum-free conditions. It is stable at room temperature and does not require the use of specific protease inhibitors for inactivation after digestion. Another example is the HyrTryp cell separation reagent product, which complies with cGMP, ISO 9001:2015, ISO13485:2016 and other standards. It is a better choice both from the perspective of regulatory safety and downstream purification difficulty.

[0045] The examples of this application mainly use TrypLE (HyrTryp is also verified in actual applications, and it has the same effect as TrypLE. For the principle of text saving, this article will not repeat the description of the verification and the verification conclusion). Selection of neutralizing solution: In order to ensure that no animal-derived components are introduced into the process, the use of any neutralizing solution containing serum or animal protein is abandoned. This technical solution selects DPBS with clear components.

[0046] Selection of subculture medium: The culture medium used for subculture of primary cells is MEM-α, and 5% serum substitute is added. The serum substitute in this application includes one of EliteGRO-Adv, UltraGRO-Advanced (HELIOS), KnockOut Serum Replacement (Gibco), CTS KnockOut SR, and XenoFree Kit (Gibco).

[0047] To ensure that a certain proportion of endothelial progenitor cells can be obtained at the same time, ECM culture medium (endothelial progenitor cell culture medium) is also added to the subculture culture medium, and the proportion of ECM can be 0-10%, which corresponds approximately to the content ratio of endothelial progenitor cells. The present application also adds ≤10% endothelial progenitor cell culture medium to the culture medium, wherein the ECM is MEM-α with human bFGF, human EGF and human VEGF added, and the ECM is endothelial progenitor cell culture medium, and the final concentrations of human bFGF, human EGF and human VEGF are all 10ng / ml.

[0048] Culture medium validation:

[0049] The seeding density was 6.0×10 3 / cm 2, the culture time was 120h for culture medium verification; during the cell culture process, the culture medium was MEM-α, and 5% EliteGRO-Adv was added (other types of serum substitutes had similar effects during the experimental verification process, and this article will not repeat the description), to explore the effect of changes in the content of endothelial progenitor cell culture medium on the amount of endothelial progenitor cells and MSC cells. See Table 1, the results show that when the endothelial progenitor cell culture medium is ≤10%, the content ratio of the two cells meets the requirements.

[0050] Table 1 Effect of culture medium design on cell mass

[0051]

[0052]

[0053] In subsequent experiments, the culture medium selected was MEM-α, supplemented with 5% EliteGRO-Adv, and the endothelial progenitor cell culture medium with 3% content was used for cell passage and cell proliferation.

[0054] After incubating P0 cells (primary cells) with TrypLE for 5 minutes and neutralizing with DPBS, most of the cells fell off the culture dish; the P0 cells were centrifuged at 300g / 10 minutes and resuspended in MSCM and the viability was calculated. The results showed that most of the cells were successfully centrifuged, and the cell viability was not less than 80%, which was in line with expectations.

[0055] P0 cells were inoculated in 100 mm culture dishes at different inoculation densities for expansion. The expansion effects are shown in Table 2.

[0056] Table 2 Average total number of cells after expansion at different seeding densities of primary cells

[0057]

[0058] After incubation with TrypLE for 5 minutes and neutralization with DPBS, most of the cells fell off the culture dish, were centrifuged at 300g / 5 minutes at room temperature, and then resuspended in MSCM and the viability was calculated. The results showed that the cell viability was not less than 90%.

[0059] This example verifies that at a seeding density of 0.5×10 3 / cm 2 -6×10 3 / cm 2 When the culture time is 96h-144h, the cells have a good fusion rate and cell proliferation multiple, and the expected total number of cells is obtained. More preferably, the inoculation density is 1.0×10 3 / cm 2 -6.0×10 3 / cm 2When the culture time is 120h-144h, the cell yield can be maximized while ensuring high cell viability and avoiding too frequent digestion and subculture, which represents the appropriate inoculation density and culture time.

[0060] C) Cell proliferation and subculture

[0061] Based on the above experiments, the cells were cultured for 120 hours before digestion and cell counting. 3 / cm 2 The cells were inoculated at the same density and subcultured after 120 hours of culture. When the cell number increased less than 10 times compared with that at the time of inoculation after 120 hours of culture and digestion and subculture, or the cell morphology changed significantly, or the positive rate of any MSC positive marker was less than 80%, the experiment was terminated.

[0062] Among them, the positive markers of MSC cells are CD90, CD73, and CD105; the positive marker of endothelial progenitor cells is CD34.

[0063] Table 3 Cell limit passage test

[0064]

[0065] The test results showed that after 9 passages, the proliferation times, cell morphology and expression of positive markers all met the requirements. After 10 passages, the cell volume increased significantly, but the proliferation times and expression of positive markers all met the requirements.

[0066] According to the results, it was determined that the cells were 0.32x10 6 With an inoculation volume of 100 μg / cm2 and a culture time of 120 hours between passages, the maximum number of passages was 9. The cells were passaged 1-8 times, and the average cell proliferation was 16.7 times per passage. In order to ensure the maximum efficiency of the cell preparation and the maintenance of the maximum activity, the MSC cell preparation of this application uses cells of passage 6-8.

[0067] To simplify the text description, the following experiments were performed using the following cell number at inoculation (100 mm culture dish): 0.32 x 10 6 After 8 passages, the number of cells was 4.1x10 6 The cells were tested for cryopreservation, cytotoxicity, stability of preparations containing MSC cells, and animal model validation. Generally speaking, when cells that have been passaged 8 times are non-toxic and have therapeutic uses, cells that have been passaged 3, 4, 5, 6, and 7 times can also meet the relevant requirements.

[0068] D) Cryopreservation of cells

[0069] Cryopreservation solution testing and cryopreservation step confirmation: 1.0x10 7 / ml, 1.3x10 7 / ml, 2x10 7 / ml, 2.5x10 7 / ml, 3.3x10 7 / ml and 4x10 7 / ml density, the cells were resuspended in three kinds of freezing solutions: MSCM (7.5% DMSO), PRIME-XVF reezIS, and PRIME-XVM SCF reezIS DMSO-Free; the cryopreservation tubes were perfused and placed in a program cooling box, stored at -80°C for 16 hours and then transferred to a liquid nitrogen tank; after the cells were stored in liquid nitrogen for 48 hours, they were revived in a 37°C water bath, the revived cell suspension was slowly added to a 15ml centrifuge tube containing 4ml MSCM, and then centrifuged at 300g / 5 minutes, and the cells were resuspended in MSCM to 1ml and tested according to the quality standards. Specific data are shown in Table 4.

[0070] Table 4 Cell viability after cryopreservation and thawing in different cryopreservation solutions

[0071]

[0072] The results showed that the quality of cells frozen in the three freezing solutions was acceptable after thawing, but the 7 / ml-3.3x10 7 / ml freezing density, and the cell viability was the highest when frozen in MSCM (7.5% DMSO).

[0073] 4) Short-term stability verification

[0074] 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 stability time of the MSC final product (preparation containing MSC cells) at 2-8°C. The expected viability is not less than 95%.

[0075] Detection method: The freezing density of cells is 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). Under 2-8℃ environment, the cell viability was tested at 0, 1, 2, 4, 8, 12, 16, 24, and 32 hours after recovery. The results showed that the viability of samples 1 to 4 could meet the use requirements within 32 hours or even longer. The results showed that the cell number and viability of samples 5 and 6 still met the quality requirements after being placed for 16 hours. The results are shown in Table 5.

[0076] Table 5 Short-term stability of preparations containing MSC cells

[0077] Time 0h 1h 2h 4h 8h 12h 16h 24h 32h Sample survival rate (%) 99.9 99.7 99.0 99.0 98.5 98.0 97.0 96.0 95.0 Sample survival rate (%) 99.9 99.0 98.7 98.3 98.1 97.8 97.3 96.4 95.3 Sample three-life rate (%) 99.9 99.1 98.9 98.5 98.0 97.6 97.0 96.0 95.5 Sample survival rate (%) 99.9 99.3 98.7 98 97.3 97.2 96.8 96.1 95.7 Sample survival rate (%) 99.8 99.1 98.0 95.0 93.0 90.0 84.0 80.0 70.0 Sample six-way survival rate (%) 99.9 99.0 97.0 96.0 95.0 93.0 89.0 86.0 80.0

[0078] The undisclosed embodiments of the present application are to detect cell viability at 0, 1, 2, 4, 8, 12, 16, and 24 hours after resuscitation at 2°C, 3°C, 5°C, 6°C, 7°C, and 8°C. Compared with the cell viability detected at 4°C, the error is only ±0.4%. For the sake of text saving, it will not be described in detail here. The applicant can supplement it if necessary.

[0079] 5) Long-term stability verification

[0080] Long-term stability: Confirm the longest stability time (product shelf life) of cells (MSC cells and endothelial progenitor cells) under low-temperature freezing to determine the shelf life of the product.

[0081] After being stored at -80°C for 16 h, the cells were transferred to a liquid nitrogen tank for storage for 3 years and then revived to test cell performance.

[0082] The freezing density used in this experiment was 1.3×10 7 / ml.

[0083] Recovery method: The cells were revived in a 37°C water bath. The revived cell suspension was slowly added to a 15ml centrifuge tube containing 4ml MSCM and centrifuged at 300g / 5 minutes. The cells were then resuspended in MSCM to 1ml and tested according to the quality standards.

[0084] Table 6 Long-term stability of preparations containing MSC cells

[0085]

[0086] Of course, the applicant also has a cryopreservation density of 2.3×10 7 / ml, the freezing density is 3.3×10 7 / ml was used to verify its long-term stability and the freezing density was 1.3×10 7 / ml basically has no difference, so this article will not elaborate on it in order to save text.

[0087] VI) Cytotoxicity assay

[0088] This experiment was designed with a control group and a test group, each group had 20 NCG mice, half male and half female. NCG mice were intravenously injected with hMSC100 once every 2 days for 1 week (4 times in total) and observed for 4 weeks to observe the nature, degree, dose-effect and time-effect relationship and reversibility of the toxic reactions that the test product may cause.

[0089] 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 saline to obtain the preparation.

[0090] Table 7 Toxicity test dosage design table

[0091]

[0092]

[0093] Toxicity detection indicators:

[0094] Blood biochemistry:

[0095] At the end of the observation period, no obvious abnormal changes were found in the blood biochemical indices such as ALB, TP, A / G, AST, ALT, TBIL, CK, CHOL, TG, Crea, Urea, GLU, GGT, ALP, LDH, Na+, K+, and Cl- of female and male mice in each hMSC100 group, indicating normal conditions.

[0096] Gross anatomical observations:

[0097] At the end of the observation period, the female and male mice in each group were grossly dissected and the major organs such as the brain, heart, liver, spleen, kidney, gastrointestinal tract, and reproductive system were observed with the naked eye. No obvious abnormal changes were found in their morphology, color, and texture, indicating that they were normal.

[0098] In summary, under the conditions of this experiment, NCG mice were intravenously injected with 3×10 6 , 1×10 7 The mice in each group were treated with hMSC100 cells / kg and observed for 4 weeks. No obvious abnormal changes were found in general observation, body weight, food intake, hematology, blood biochemistry, and gross anatomical observation.

[0099] 7) Study on the efficacy of MSC cell preparations on bleomycin-induced pulmonary fibrosis model rats

[0100] Experimental groups:

[0101] A total of 55 male SD rats were randomly divided into a normal control group (10 rats) and a model group (45 rats) for the first time; after the pulmonary fibrosis model was established, they were randomly divided into a low-dose test article group (2×10 6 cells / cell / time, 1.5mL / cell, 11 cells), high-dose group (6×10 6 cells / animal / time, 1.5mL / animal, 11 animals), commercial control group (pirfenidone capsules, batch number: 191006, 240mg / kg, 5mL / kg, 11 animals), model control group (sodium chloride injection, 12 animals) and normal control group (sodium chloride injection, 10 animals).

[0102] The animals in the D1 and D5 model groups were given bleomycin (5 mg / kg, 1 mL / kg) by intratracheal aerosolization to construct the model, and the animals in the normal control group were given sodium chloride injection (1 mL / kg) by intratracheal aerosolization. 6 After 8 passages, the number of cells was 4.1x10 6 The cells were suspended in saline to obtain the preparation.

[0103] The animals in the normal control group, model control group, low-dose test article group and high-dose test article group were given the corresponding drugs by intravenous injection once on D6, D8 and D10, for a total of 3 times; the commercially available control group was given the drug by oral gavage once a day, for a total of 23 times (D6-D28).

[0104] On D28, all animals in each group underwent lung function tests, lung weight was weighed, and lung weight index was calculated; the left lung was cut for hydroxyproline content determination, and the remaining right lung was perfused with 10% neutral buffered formalin solution and then placed in 10% neutral buffered formalin solution for fixation, paraffin embedding, sectioning, preparation, HE staining for evaluation of lung tissue inflammatory cell infiltration, and Masson staining for evaluation of lung tissue fibrosis.

[0105] Results: Modeling: During the experiment, animals were treated with intra-airway aerosolized bleomycin on D1 and D5 to induce pulmonary fibrosis. Under the microscope, pathological changes such as inflammatory cell infiltration and fibrosis of varying degrees were observed in the lungs, indicating that the pulmonary fibrosis model was successfully established.

[0106] Survival rate: One animal died in the model control group on D6 and D11, and one animal died in the test sample low-dose group on D14. The survival rates of animals in the normal control group, model control group, hMSC100 low-dose group, hMSC100 high-dose group and commercial control group were 100%, 83%, 91%, 100% and 100%, respectively. The survival rates of animals in the test sample groups and the commercial control group were higher than those in the model control group.

[0107] Lung weight index: Compared with the model control group, the lung weight index of the animals in the normal control group, the test article low-dose group, the test article high-dose group and the commercial control group was reduced, and the normal control group had a statistically significant difference; the test article low-dose group and the high-dose group had a dose-dependent decrease.

[0108] Hydroxyproline content: Compared with the animals in the model control group, the mean hydroxyproline content of the animals in the normal control group, the low-dose test article group, and the high-dose test article group was reduced, and there was a statistical difference between the low-dose test article group and the high-dose test article group, and the reduction was dose-dependent.

[0109] 8) Evaluation of the efficacy of hMSC100 in the treatment of osteoporosis in ovariectomized rats

[0110] 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.

[0111] Table 8 Animal grouping information

[0112]

[0113]

[0114] Detection indicators:

[0115] 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.

[0116] Test results

[0117] 1. General observation and body weight

[0118] All animals showed no abnormal behavior during the experiment.

[0119] 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 .

[0120] 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 .

[0121] 2. Bone density and bone microstructure

[0122] The femoral bone density and bone microstructure of the animals were analyzed by micro-CT.

[0123] 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. Trabeculae support hematopoietic tissue and increase bone strength. The number, quality, direction and thickness of trabeculae have a great impact on bone strength.

[0124] After the treatment period, the percentage of the difference between the individual BMD values ​​of each group and the average BMD value of the sham operation group was analyzed (BMD change% = (individual BMD value of each group - average BMD value of the sham operation group)%; change, change rate). The BMD change% of the femoral shaft bone density of 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 density of the MSC treatment group was significantly higher than that of 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 of the model control group (P < 0.05). For specific data, see Figure 7 , 8 The results showed that the bone density of the femoral shaft and femoral neck of SD rats with osteoporosis induced by castration surgery was significantly improved after 4 weeks of hMSC100 treatment, indicating that MSC has a good therapeutic effect on osteoporosis.

[0125] IX) Evaluation of the efficacy of hMSC100 with different components in the treatment of osteoporosis in ovariectomized rats

[0126] Different from the “Evaluation of the efficacy of hMSC100 in treating osteoporosis in ovariectomized rats”, this example verifies the effect of hMSC100 composition and content on the change rate of femoral bone density in animals. The number of cells in hMSC100 with different compositions is 4.1x10 6 The preparations used were obtained by suspending cells in physiological saline, and the administration concentration was 0.6×106 cells / mL, and the administration volume was 5mL / kg. The data show the change rate of femoral bone density in the experimental group (MSC treatment group) relative to the modeling group (untreated group) (Table 8).

[0127] Table 9 Effects of hMSC100 components and content on femoral bone density in animals

[0128]

[0129] Experimental verification shows that the content of MSC cells in the preparation is ≥90%, and the content of endothelial progenitor cells is ≤10%, and the preparation has a good effect on the treatment of osteoporosis.

[0130] In practical application, the MSC preparation of the present application is also useful for multiple sclerosis, osteoporosis, systemic scleroderma, hematological malignancies, myocardial infarction, organ transplant rejection, chronic allograft nephropathy, cirrhosis, 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, bone dysplasia, Treatment of critical limb ischemia, diabetic foot disease, primary Sjögren's syndrome, osteoarthritis, cartilage defect, laminitis, multiple system atrophy, amyotrophic lateral sclerosis, cardiac surgery, systemic lupus erythematosus, living kidney allograft, non-malignant erythrocyte disease, thermal burns, radiation burns, Parkinson's disease, microfracture, epidermolysis bullosa, severe coronary ischemia, idiopathic dilated cardiomyopathy, femoral head necrosis, lupus nephritis, bone defect, ischemic stroke, post-stroke, acute radiation syndrome, lung disease, arthritis, bone regeneration, uveitis or its combination. The technical text of this application is based on the principle of text simplicity, and only part of the drug efficacy evaluation is listed.

[0131] Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. A person with ordinary knowledge in the technical field to which the present invention belongs may 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 determined by the definition of the claims.

Claims

1. Cell cryopreservation medium for preparations containing MSC cells, Features: The freezing solution is selected as follows: the basic culture medium is MSCM, and the additive is DMSO with a volume concentration of 7.5%; the MSCM is MEM-α culture medium supplemented with 5% serum substitute, and the serum substitute includes one of EliteGRO-Adv, UltraGRO-Advanced, KnockOut Serum Replacement, CTS KnockOutSR, and XenoFree Kit.

2. The cell freezing solution for a preparation containing MSC cells according to claim 1, Features: The cryopreservation density is 1.3×10 7 / ml-3.3×10 7 / ml.

3. The cell freezing solution for a preparation containing MSC cells according to claim 1, Features: The freezing process conditions are: after storing at -80℃ for 16h, transfer to a liquid nitrogen tank.

4. The cell freezing solution for a preparation containing MSC cells according to claim 1, Features: The preparation includes umbilical cord-derived MSC cells and umbilical cord-derived endothelial progenitor cells; wherein the concentration of MSC cells in the preparation is 0.1×10 6 -12×10 6 cells / ml; the content of MSC cells in the preparation is ≥90%, and the content of endothelial progenitor cells is ≤10%.

5. The cell freezing solution for a preparation containing MSC cells according to claim 4, It is characterized in that The concentration of the MSC cells was 0.6×10 6 -4×10 6 cells / ml.

6. The cell freezing solution for a preparation containing MSC cells according to claim 4, It is characterized in that The preparation also includes an injection solution, in which the umbilical cord-derived MSC cells and the umbilical cord-derived endothelial progenitor cells are both suspended.

7. The cell freezing solution for a preparation containing MSC cells according to claim 6, It is characterized in that When used for intravenous injection, the injection includes physiological saline, compound electrolyte injection or dextran 40 glucose injection.

8. The cell freezing solution for a preparation containing MSC cells according to claim 6, It is characterized in that When used for local joint cavity injection, the injection solution includes physiological saline, compound electrolyte injection solution or hyaluronic acid injection solution.

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