CN115400149A - A preparation containing MSC cells - Google Patents

Abstract

This application belongs to the field of cell preparations, and relates to a preparation containing MSC cells, including MSC cells derived from umbilical cord and endothelial progenitor cells derived from umbilical cord; wherein, the concentration of MSC cells is 0.1×10 ⁶ -12×10 ⁶ cells/ ml, and the content of the MSC cells is ≥90%, and the content of the endothelial progenitor cells is ≤10%. The technical solution is to realize the maintenance of the optimal performance of the MSC cells from the culture process of the MSC cells, and then realize that the obtained MSC cell preparation has good stable performance.

Description

A preparation containing MSC cells

technical field

The invention belongs to the field of cell preparations and relates to a preparation containing MSC cells.

Background technique

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

Under specific induction conditions in vivo or in vitro, MSC cells can differentiate into fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, myocardium, endothelial cells, etc. It can be used as an ideal seed cell for the repair of tissue and organ damage caused by aging and disease.

A large number of studies have shown that MSC has an important role in the treatment of cardiovascular diseases, liver cirrhosis, bone and muscle degenerative diseases, brain and bone marrow nerve damage, senile dementia, and autoimmune diseases such as lupus erythematosus and scleroderma; There are huge development prospects in beauty, health care, anti-aging and other aspects. For example, Chinese patent CN104136034B mesenchymal stromal cells and related applications, CN102008507B human umbilical cord MSC cell anti-hepatic fibrosis injection and its preparation method...

However, the MSC preparations summarized in the prior art will have adverse reactions after infusion therapy, 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 MSCs (hMSCs) may vary due to various parameters (including tissue source, isolation method, and medium composition) and there are great differences [Document 1].

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

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.

Contents of the invention

Based on the current situation, this application provides a preparation containing MSC cells, which avoids the introduction of animal-derived components during the culture process of MSC cells; at the same time, regulates the content of endothelial progenitor cells in the process of obtaining MSCs from the umbilical cord, and realizes the optimal preparation of MSC cells. Performance maintenance; finally, the composition of the preparation containing MSC cells obtained in the present application is simple and clear, has no toxic and side effects, and has good stability.

In order to achieve the above-mentioned technical purpose, the technical solution adopted by the present application is a preparation containing MSC cells, the preparation includes MSC cells derived from umbilical cord and endothelial progenitor cells derived from umbilical cord; wherein, the MSC cells in the preparation The concentration is 0.1×10 ⁶ -12×10 ⁶ cells/ml; the content of the MSC cells is ≥90%, and the content of the endothelial progenitor cells is ≤10%.

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.

As an improved technical solution of the present application, the content of the MSC cells is ≥95%, and the content of the endothelial progenitor cells is ≤5%.

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

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.

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

As an improved technical solution of the present application, the cells in the preparation containing MSC cells are obtained by separating the umbilical cord tissue using the tissue block attachment method, digesting it with animal-derived recombinant cell dissociating enzyme, and neutralizing it with DPBS. Obtained by subculture.

As an improved technical solution of the present application, the MSC cells are MSC cells extracted from umbilical cord and passaged for 6-8 passages; the endothelial progenitor cells are endothelial progenitor cells extracted from umbilical cord and passaged for 6-8 passages.

As an improved technical solution of the present application, the seeding density of the cells in the preparation containing MSC cells during subculture is 0.5×10 ³ /cm ² -7×10 ³ /cm ² .

As an improved technical solution of the present application, the duration of cell passage in the preparation containing MSC cells is set to 96-144 hours.

As an improved technical solution of the present application, the centrifugation speed of the collected cells during the subculture of the MSC cells is not greater than 300g/10min.

As an improved technical solution of the present application, the medium used for the subculture of the MSC cells is MEM-α, and 5% serum replacement is added.

As an improved technical solution of the present application, the serum substitute includes one of EliteGRO-Adv, UltraGRO-Advanced, KnockOutSerum Replacement, CTS KnockOut SR, and XenoFree Kit.

As an improved technical solution of the present application, the culture medium taken by the MSC cells during subculture also contains ≤10% endothelial progenitor cell culture medium, and the ECM is MEM-α supplemented with human bFGF, human EGF and human VEGF , ECM is endothelial progenitor cell culture medium, and the final concentrations of human bFGF, human EGF and human VEGF are all 10ng/ml.

As an improved technical solution of the present application, the cell cryopreservation method in the preparation containing MSC cells is selected as follows:

Design the cryopreservation density of cells in preparations containing MSC cells to be 1.3×10 ⁷ /ml-3.3×10 ⁷ /ml;

The cryopreservation solution is selected as follows: the basic medium is MSCM, and the additive is DMSO with a volume concentration of 7.5%; the MSCM is the MEM-α medium that adds 5% serum substitute, and the serum substitute includes EliteGRO-Adv, One of UltraGRO-Advanced, KnockOutSerum Replacement, CTS KnockOut SR, XenoFree Kit;

The cryopreservation process conditions are as follows: after storing at -80°C for 16 hours, transfer to a liquid nitrogen tank.

As an improved technical solution of the present application, the MSC cell preparation can be used for 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 mellitus, chronic obstructive pulmonary disease, osteogenesis imperfecta, homozygous familial hypercholesterolemia disease, post-meniscectomy treatment, periodontitis in adults, angiogenesis in patients with severe myocardial ischemia, spinal cord injury, bone dysplasia, critical limb ischemia, diabetic foot disease, primary Sjögren syndrome, Osteoarthritis, Cartilage Defects, Laminitis, Multiple System Atrophy, Amyotrophic Lateral Sclerosis, Cardiac Surgery, Systemic Lupus Erythematosus, Living Donor Kidney Allograft, Nonmalignant Red Blood Cell Disorders, 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, pulmonary disease, arthritis, bone regeneration, uveitis, or combinations thereof.

Beneficial effect

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

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

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 does not have toxicity; especially it can be adapted to the MSC cells that have been verified in the prior art. Applicable conditions.

It should be understood that all combinations of the foregoing concepts, as well as additional concepts described in more detail below, may be considered part of the inventive subject matter of the present disclosure, provided such concepts are not mutually inconsistent.

Description of drawings

Fig. 1 The body weight of both the sham operation group and the model group continued to increase at 0-12 weeks after castration operation;

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

During the administration of Figure 3, the body weight of the animals in the sham operation group, the model control group, and the MSC treatment group all increased;

Figure 4 Comparison of trabecular bone density of the distal femur between the sham operation group and the model group at 12 weeks after castration: Compared with the sham operation group, the trabecular bone density (Trabeculae Mean BMD) of the model group had significantly Decreasing trend, but no significant difference;

Figure 5 Comparison of femoral trabecular bone number between sham operation group and model group at 12 weeks after castration: Compared with sham operation group, the number of trabecular bone (Tb.N) in model group decreased significantly (***p< 0.05);

Fig.6 Comparison of femoral trabecular connection density between sham operation group and model group at 12 weeks after castration: Compared with sham operation group, the trabecular connection density (Conn.D) of model group animals was significantly lower (**p <0.05);

Figure 7. Experimental endpoints. Comparison of femoral shaft BMD change% among animals in the sham operation group, model control group and MSC treatment group: the BMD change% of the femoral shaft in the model control group was significantly lower than the BMD change% in the sham operation group (*P<0.05), 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).

Figure 8 Experimental endpoints Comparing the BMD change% of the femoral neck in the sham operation group, the model control group and the MSC treatment group, 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).

Detailed ways

In order to make the purpose, 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 in conjunction with the drawings of the embodiments of the present invention. Apparently, the described embodiments are some, not all, embodiments of the present invention. Based on the described embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative work shall fall within the protection scope of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein shall have the usual meanings understood by those skilled in the art to which the present invention belongs.

definition:

Stability, the stability of the final product before injection: the MSC final product needs to be uniformly mixed with the injection solution before it can be administered through intravenous injection; during the time when MSC is mixed with the injection solution until the MSC injection is completed, the MSC product The number of cells and viability must meet the quality standards to ensure safety and effectiveness.

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

Injection: a sterile solution for injection into the body, used to suspend MSC cells derived from umbilical cord and endothelial progenitor cells derived from umbilical cord in the injection. In order 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.

Sodium chloride injection used in this article: 0.9%, manufacturer: Sichuan Kelun Pharmaceutical Co., Ltd., batch number: M19051004-2; bleomycin hydrochloride for injection (bleomycin), production unit: Nippon Kayaku Co., Ltd. Club, batch number: 790670.

CD34 is a single-chain transmembrane glycoprotein with a relative molecular mass of 110,000 encoded by a gene located at 1q32, which can be expressed in normal endothelial progenitor cells, spleen marginal zone cells, surrounding blood vessels, nerves, muscle bundles, skin appendages and mammary glands Dendritic stromal cells surrounding the lobular stroma. However, the cells derived from umbilical cords are endothelial progenitor cells which are closest to MSC cells after digestion and separation.

This application mainly separates and obtains MSCs and endothelial progenitor cells from the umbilical cord, and conducts conditional expansion culture to study the relationship between umbilical cord-derived MSCs and endothelial progenitor cells in the process of culture and medicinal use.

The following will give a clear and complete description of the technical solutions of the present application in conjunction with specific implementation methods.

1. The method of obtaining umbilical cord-derived MSC cells and umbilical cord-derived endothelial progenitor cells in preparations containing MSC cells

A) Primary cell acquisition

In order to improve the purity of cells and avoid the introduction of animal-derived components, this application mainly adopts the method of tissue block adhesion when obtaining primary cells (P0 cells, including MSC cells and endothelial progenitor cells), and removes amniotic membrane, blood vessels, etc. by physical means. , which can obtain high-purity cells without using trypsin and other materials, and can better maintain the cell's own activity, which is more suitable for clinical application than enzymatic digestion.

B) Subculture of primary cells

Since the final use of the cell preparation of the technical solution is medicinal, the use of animal-derived reagents must be avoided in the processes of digestion, subculture, and cryopreservation.

Therefore, this technical solution uses animal-derived recombinant cell dissociation enzymes: such as TrypLE products, TrypLE is suitable for serum replacement and dissociation of cells under serum-free conditions. It is stable at room temperature and does not need to be used after digestion. Specific protease inhibitors are inactivated; another example is the HyrTryp cell separation reagent product, which complies with cGMP, ISO 9001:2015, ISO13485:2016 and other standards, and is a better choice in terms of regulatory safety and downstream purification difficulty.

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

Selection of subculture medium: The medium used for subculture of primary cells is MEM-α, and 5% serum substitutes are added. Serum substitutes in this application include EliteGRO-Adv, UltraGRO-Advanced (HELIOS), One of KnockOutSerum Replacement (Gibco), CTS KnockOut SR, XenoFree Kit (Gibco).

In order to ensure that a certain proportion of endothelial cells can be obtained at the same time, ECM medium (endothelial progenitor cell medium) is also added to the subculture medium. The proportion of ECM can be 0-10%, which is approximately corresponding to the proportion of endothelial cells. In the present application, an endothelial progenitor cell medium containing ≤10% is also added to the medium, the ECM is MEM-α added with human bFGF, human EGF and human VEGF, ECM is an endothelial progenitor cell medium, human bFGF, human EGF and human VEGF final concentration are 10ng/ml.

Media Validation:

The inoculation density was 6.0×10 ³ /cm ² , and the culture time was 120 hours to verify the medium; during the cell culture process, the medium was MEM-α, and 5% EliteGRO-Adv was added (other types of serum substitutes were tested in the experiment. It has similar effects in the verification process, and will not be described again in this article), to explore the influence of changes in the content of endothelial 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 kinds of cells meets the requirements.

Table 1 Effect of culture medium design on cell mass

test group Endothelial cell medium content Cell morphology CD90, CD73 and CD105 CD34 1 0 Fibrocytic All≥95% ≤1% 2 1% Fibrocytic All≥95% ≤1.5% 3 3% Fibrocytic All≥95% ≤2% 4 5% Fibrocytic All≥95% ≤5% 5 8% Fibrocytic All≥95% ≤8% 6 10% Fibrocytic All≥90% ≤10% 7 12% Fibrocytic All≥85% ≤15%

In the follow-up experiments, the medium was selected as MEM-α, and 5% EliteGRO-Adv was added, and the medium content of endothelial cell medium was 3% for cell passage and cell proliferation.

After P0 cells (primary cells) were incubated with TrypLE for 5 minutes and neutralized with DPBS, most of the cells fell off from the culture dish; P0 cells were centrifuged at 300g/10 minutes and resuspended in MSCM to calculate the viability. Most of the cells were successfully centrifuged, and the cell viability was not lower than 80%, which was in line with expectations.

P0 cells were inoculated in 100mm culture dishes at different seeding densities for expansion, and the expansion effects are shown in Table 2.

Table 2 The average total number of cells after expansion of primary cells at different seeding densities

Inoculation density 0.1×10³/cm² 0.5×10³/cm² 1×10³/cm² 2×10³/cm² 4×10³/cm² 6×10³/cm² 10×10³/cm² 72h 0.01×10⁶ 0.7×10⁶ 2×10⁶ 3.3×10⁶ 3.8×10⁶ 3.5×10⁶ 3.9×10⁶ 96h 0.04×10⁶ 1.2×10⁶ 2.5×10⁶ 4.1×10⁶ 4.8×10⁶ 5.1×10⁶ 4.1×10⁶ 120h 0.08×10⁶ 2×10⁶ 3.6×10⁶ 4.8×10⁶ 5.1×10⁶ 5.5×10⁶ 4.3×10⁶ 144h 0.1×10⁶ 2.3×10⁶ 3.7×10⁶ 5.1×10⁶ 5.2×10⁶ 5.8×10⁶ 4.9×10⁶ 168h 0.07×10⁶ 1.8×10⁶ 3.3×10⁶ 5.7×10⁶ 5.0×10⁶ 5.3×10⁶ 3.9×10⁶

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

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

C) Cell proliferation and passage

Based on the previous experiments, the cells were digested and passaged after 120 h of culture, and the cells were counted. Inoculate at a density of 6x10 ³ /cm ² and subculture after 120 hours. When cultured for 120 hours and digested and subcultured, the number of cells increases by less than 10 times compared with that of inoculation; or the cell morphology changes significantly; or any one of the MSC positive markers When the positive rate is lower than 80%, the test is terminated.

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

Table 3 Cell limit passage test

Number of passages The number of cells at the time of inoculation (100mm Petri dish) Cell number at passage multiplication factor Cell morphology CD90, CD73 and CD105 CD34 1 0.32x10⁶ 5.3x10⁶ 16.6 Fibrocytic All≥95% ≤2% 2 0.32x10⁶ 6.0x10⁶ 18.8 Fibrocytic All≥95% ≤2% 3 0.32x10⁶ 5.3x10⁶ 16.6 Fibrocytic All≥95% ≤2% 4 0.32x10⁶ 5.4x10⁶ 16.9 Fibrocytic All≥95% ≤2% 5 0.32x10⁶ 7.0x10⁶ 21.8 Fibrocytic All≥95% ≤2% 6 0.32x10⁶ 5x10⁶ 15.6 Fibrocytic All≥95% ≤2% 7 0.32x10⁶ 4.6x10⁶ 14.4 Fibrocytic All≥95% ≤2% 8 0.32x10⁶ 4.1x10⁶ 12.8 Fibrocytic All≥95% ≤2% 9 0.32x10⁶ 3.8x10⁶ 11.9 Fibrocytic All≥95% ≤2% 10 0.32x10⁶ 2.8x10⁶ 8.8 Fibrocytic (increased in size) All≥95% ≤2%

The test results show that after the cells have been passaged for 9 times, the proliferation multiple, cell morphology and the expression of positive markers all meet the requirements. After 10 passages, the cell volume increased significantly, but the proliferation multiples and the expression of positive markers met the requirements.

According to the results, it was determined that the maximum number of passages of the cells was 9 times under the inoculum amount of 0.32x10 ⁶ and the culture time between passages of 120 hours. 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 utilization and the maintenance of the maximum activity of the cell preparation, the MSC cell preparation of this application selects the cells of passage 6-8.

In order to simplify the text description, follow-up experiments are used: cells with a cell number of 0.32x10 ⁶ at the time of inoculation (100mm culture dish) and 4.1x10 ⁶ cells after 8 passages are used for cryopreservation, cytotoxicity, and stability of preparations containing MSC cells and animal model validation. Generally speaking, when the cells passaged 8 times are non-toxic and have therapeutic uses, the cells passed 3 times, 4 times, 5 times, 6 times, and 7 times can also meet the relevant requirements.

D) Cryopreservation of cells

Cryopreservation solution test and cryopreservation procedure confirmation: repopulate cells at densities of 1.0x10 ⁷ /ml, 1.3x10 ⁷ /ml, 2x10 ⁷ /ml, 2.5x10 ⁷ /ml, 3.3x10 ⁷ /ml and 4x10 ⁷ /ml Suspended in MSCM (7.5% DMSO), PRIME-XVF reezIS, PRIME-XVM SCF reezIS DMSO-Free three freezing storage solutions; perfuse the cryopreservation tube and put it into the programmed cooling box, store it at -80°C for 16 hours and then transfer to the liquid In a nitrogen tank; after the cells were stored in liquid nitrogen for 48 hours, they were revived in a 37°C water bath, and the revived cell suspension was slowly added to a 15ml centrifuge tube filled with 4ml of MSCM, then centrifuged at 300g/5 minutes, and then the cells were washed with MSCM Resuspend to 1ml and test according to the quality standard. See Table 4 for specific data.

Table 4 Cell viability after cryopreservation and recovery in different cryopreservation solutions

1.0x10⁷/ml 1.3x10⁷/ml 2x10⁷/ml 2.5x10⁷/ml 3.3x10⁷/ml 4x10⁷/ml MSCM (7.5% DMSO) 88% 98% 99% 98% 97% 93% PRIME-XV FreezIS 90% 93% 92% 94% 93% 88% PRIME-XV MSC FreezIS DMSO-Free 89% 90% 91% 93% 92% 90%

The results showed that the quality of cells cryopreserved in the three cryopreservation solutions was qualified after recovery, but at the cryopreservation density of 1.3x10 ⁷ /ml-3.3x10 ⁷ /ml, the survival rate of cells cryopreserved in MSCM (7.5%DMSO) was the highest .

4) Short-term stability verification

Short-term stability: Resuscitate the working bank cells in a 37°C water bath, mix them evenly with the injection solution and place them at 2-8°C, and detect the cells at 0, 1, 2, 4, 8, 12, 16, 24, and 32 hours after recovery The viability rate provides the basis for the stability time of the final MSC product (preparation containing MSC cells) at 2-8°C, and the expected viability rate is not less than 95%.

Detection method: The cryopreservation 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 five) and 4.3x10 ⁷ /ml (sample six). In an environment of 2-8°C, the cell viability was detected at 0, 1, 2, 4, 8, 12, 16, 24, and 32 hours after recovery, and the results showed the viability of samples 1 to 4 in 32 hours or even longer Can meet the requirements of use. The results showed that the cell quantity and viability of samples 5 to 6 still met the quality requirements after being placed for 16 hours. The results are shown in Table 5.

Table 5 Short-term stability of preparations containing MSC cells

point in time 0h 1h 2 hours 4 hours 8 hours 12 hours 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 secondary survival rate (%) 99.9 99.0 98.7 98.3 98.1 97.8 97.3 96.4 95.3 Three live rate of sample (%) 99.9 99.1 98.9 98.5 98.0 97.6 97.0 96.0 95.5 Sample four 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 survival rate (%) 99.9 99.0 97.0 96.0 95.0 93.0 89.0 86.0 80.0

The undisclosed example of this application is to detect cells at 0, 1, 2, 4, 8, 12, 16, and 24 hours after recovery under the environment of 2°C, 3°C, 5°C, 6°C, 7°C, and 8°C The viability rate is only ±0.4% error compared with the cell viability rate detected at 4°C, and will not be detailed here for the sake of saving text, and the applicant can supplement it if necessary.

5) Long-term stability verification

Long-term stability: Confirm the longest stable time (product expiration date) of cells (MSC cells and endothelial progenitor cells) under cryopreservation to determine the shelf life of the product.

After being stored at -80°C for 16 hours, it was transferred to a liquid nitrogen tank for storage for 3 years, and then recovered to detect the performance of the cells.

The cryopreservation density used in this experiment was 1.3×10 ⁷ /ml.

Resuscitation method: Cells are resuscitated in a 37°C water bath, slowly add the resuscitated cell suspension into a 15ml centrifuge tube containing 4ml of MSCM, centrifuge at 300g/5 minutes, then resuspend the cells to 1ml in MSCM and test according to the quality standards .

Table 6 Long-term stability of preparation products containing MSC cells

Detection Indicator Test items Detection method Test results physical properties Exterior visual inspection MSC cell preparation product stability Cell Differentiation Cell morphology microscope observation Cells grow adherently, fibroblast-like and uniform in size MSC cell purity CD90/CD73/CD105 Flow Cytometry Analysis All surface markers are ≥95% Endothelial progenitor cell purity CD34 Flow Cytometry Analysis ≤2% cell growth activity cell viability Cell Counter with Trypan Blue Exclusion 96% Immunogenicity HLA-DR Flow Cytometry Analysis ≤2% general security bacteria Culture method feminine general security fungus Culture method feminine general security Mycoplasma Culture method feminine general security endotoxin gel method ≤0.5EU/mL

Of course, the applicant also verified the frozen storage density of 2.3× ^{10 7} /ml and the frozen storage density of 3.3×10 ⁷ /ml. In order to save text, this article will not describe it further.

6) Cytotoxicity test

In this experiment, a control group and an experimental group were designed, with 20 NCG mice in each group, half male and half male. NCG mice were dosed once every 2 days, injected intravenously with hMSC100 for 1 week (total 4 times) and observed for 4 weeks to observe the nature, degree, dose-effect and time-effect relationship and reversibility of the possible toxic reactions caused by the test product.

hMSC100 is a preparation obtained by suspending cells with a cell number (100mm culture dish) of 0.32x10 ⁶ at the time of inoculation and 4.1x10 ⁶ cells in physiological saline after 8 passages.

Table 7 Dose Design Table for Toxicity Test

group Route of administration Dosage (cells/Kg) Dosing concentration (cells/mL) Dosing volume (ml/Kg) female animal (only) male animal (only) control group intravenous injection - - 5 10 10 hMSC100 low dose group intravenous injection 3×10⁶ 0.6×10⁶ 5 10 10 hMSC100 high dose group intravenous injection 1×10⁷ 4×10⁶ 5 10 10

Toxicity detection index:

Blood Biochemistry:

At the end of the observation period, ALB, TP, A/G, AST, ALT, TBIL, CK, CHOL, TG, Crea, Urea, GLU, GGT, ALP, LDH, Na+, K+, Cl- No obvious abnormal changes were found in other blood biochemical indicators, which represented normal.

Gross Anatomy Observation:

At the end of the observation period, the female and male mice in each group were grossly dissected to observe the brain, heart, liver, spleen, kidney, gastrointestinal tract, reproductive system and other major organs with the naked eye. There were no obvious abnormal changes in their shape, color, texture, etc., representing normal.

To sum up, under the conditions of this experiment, NCG mice were intravenously injected with 3×10 ⁶ and 1×10 ⁷ cells/kg of hMSC100 once every 2 days for 1 week (4 times in total) and observed for 4 weeks. There were no obvious abnormal changes in the general observation, body weight, food intake, hematology, blood biochemistry, and gross anatomy of the mice in the group.

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

Experimental group:

This experiment shared 55 male SD rats: they were randomly divided into normal control group (10 rats) and model group (45 rats) for the first time; ⁶ cells/time, 1.5mL/time, 11 animals), the high-dose test group (6×10 ⁶ cells/time, 1.5mL/time, 11 animals), the commercially available control group (pirfenib Ketone capsules, batch number: 191006, 240mg/kg, 5mL/kg, 11 rats), model control group (sodium chloride injection, 12 rats) and normal control group (sodium chloride injection, 10 rats).

Animals in D1 and D5 model groups were given intratracheal nebulization of bleomycin (5 mg/kg, 1 mL/kg) for model building, and animals in the normal control group were given intratracheal nebulization of sodium chloride injection (1 mL/kg). Among them, hMSC100 is a preparation obtained by suspending cells with a cell number (100mm culture dish) of 0.32x10 ⁶ at the time of inoculation and 4.1x10 ⁶ cells in saline after 8 passages.

Animals in the normal control group, model control group, low-dose and high-dose groups of the test product were intravenously injected with the corresponding drug once on D6, D8, and D10, and administered for 3 times in total; the commercially available control group was intragastrically administered once a day. times, a total of 23 times (D6-D28).

On D28, the animals in each group were tested for lung function, weighed the lungs, and calculated the lung mass index; the left lung was cut off to measure the content of hydroxyproline, and the remaining right lung was perfused with 10% neutral buffered formalin solution and then placed in Fixation in 10% neutral buffered formalin solution, embedding in paraffin, sectioning, preparation, HE staining to evaluate the degree of inflammatory cell infiltration in lung tissue, and Masson staining to evaluate the degree of lung tissue fibrosis.

Results: During the modeling, during the experiment, the animals were aerosolized with bleomycin in the airway on D1 and D5 respectively to induce pulmonary fibrosis. Under the microscope, pathological changes such as inflammatory cell infiltration and fibrosis of different degrees were mainly seen in the lungs, indicating that The pulmonary fibrosis model was established successfully.

Survival rate: 1 mouse died in the model control group on D6 and D11 respectively, and 1 mouse died in the low-dose group of the test product on D14 day. The survival rates of normal control group, model control group, hMSC100 low-dose group, hMSC100 high-dose group and commercially available control group were 100%, 83%, 91%, 100% and 100%, respectively. The survival rate of the animals in the control group was higher than that in the model control group.

Lung weight index: Compared with the model control group animals, the average value of the lung weight index of the normal control group, the low-dose test product group, the high-dose test product group and the commercially available control group were all reduced, and the normal control group had a statistically significant difference ; Low-dose and high-dose groups of the test article had a dose-dependent decrease. lung

Hydroxyproline content: Compared with the model control group animals, the average value of the hydroxyproline content of the animals in the normal control group, the low-dose group of the test product, and the high-dose group of the test product all decreased, and the low-dose group of the test product and the high-dose group of the test product The dose groups were statistically different and had a dose-dependent reduction.

8) Efficacy evaluation of hMSC100 in the treatment of osteoporosis in ovariectomized rats

hMSC100 is a preparation obtained by suspending cells with a cell number (100mm culture dish) of 0.32x10 ⁶ at the time of inoculation and 4.1x10 ⁶ cells in physiological saline after 8 passages. Sham operation group: Sham group, model control group: Model group, MSC treatment group: MSC group.

Table 8 Animal grouping information

group Dosing preparation Dosecells/kg Dosing concentration cells/mL Dosing volume mL/kg Route of administration Dosing Frequency and Period number of animals mock surgical group Cytolytic / / 5 intravenous injection once a week for four weeks 10 Model control group Cytolytic / / 5 intravenous injection once a week for four weeks 20 MSC treatment group hMSC100 3×10⁶ 0.6×10⁶ 5 intravenous injection once a week for four weeks 20

Detection Indicator:

During the test period, the behavioral state and food intake of animals in each group were observed once a day; the body weight of the animals was measured once a week; 3 months after the castration operation and 1 week after the last administration, the animals were collected, and the femur BMD and BMD of the animals were analyzed by micro-CT. bone microstructure.

test results

1. General observation and body weight

All animals showed no abnormal behavior during the experiment.

Three months after castration, the body weight of the animals in the sham operation and model groups continued to increase. The relative body weight growth rate of animals in the sham operation group was 20.42%, and the relative body weight growth rate of animals in the model group was 35.44%. The animal model satisfies the law of weight gain after castration. See Figures 1 and 2 for specific body weight trends.

During the period of administration, the body weight of the animals in the sham operation group increased slightly, while the body weight of the animals in the model control group increased significantly, and the body weight of the animals in the MSC treatment group did not increase significantly. The specific body weight changes are shown in Figure 3.

2. Bone density and bone microstructure

Animal femur bone mineral density and bone microstructure were analyzed by micro-CT.

Three months after castration, compared with the sham operation group, the Trabeculae Mean BMD of the distal femur in the model group tended to decrease, and the number of trabeculae (Tb.N) decreased significantly (p< 0.05), the trabecular connection density (Conn.D) decreased significantly (p<0.05). The results showed that the model group was in line with the trend of osteoporosis. See Figures 4, 5, and 6 for specific data. Trabecular bone has the function of supporting hematopoietic tissue and increasing the strength of bone. The number, quality, direction and thickness of trabecular bone will greatly affect the strength of bone.

After the treatment period, the percentage of the difference between the individual bone mineral density value of each group and the mean value of bone density of the sham operation group was used for analysis (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 in the model control group was significantly lower than the BMD change% in the sham operation group (P<0.05), and the BMD change% of the femoral shaft in the MSC treatment group was significantly higher than the BMD change% in the model control group ( P<0.01). BMD change% of femoral neck bone mineral density in MSC treatment group was significantly higher than BMD change% in model control group (P<0.05). The specific data are shown in Figures 7 and 8. The results show that the bone mineral density of the femoral shaft and femoral neck in SD rats induced by castration surgery after 4 weeks of treatment with hMSC100 was significantly improved, suggesting that MSC has a good effect on osteoporosis. the therapeutic effect.

9) Efficacy evaluation of hMSC100 with different components in the treatment of osteoporosis in ovariectomized rats

Different from the "drug efficacy evaluation of hMSC100 for treating osteoporosis in ovariectomized rats", this example verifies the effect of the composition and content of hMSC100 on the change rate of animal femoral shaft bone density. The number of cells in hMSC100 of different components was 4.1×10 ⁶ , and the preparations obtained by suspending cells in physiological saline were used, the dosage concentration was 0.6×10 ⁶ cells/mL, and the dosage volume was 5 mL/kg. The data show the change rate of femoral shaft bone mineral density in the experimental group (MSC treatment group) relative to the model group (no treatment group) (Table 8).

Table 9 Effects of hMSC100 composition and content on animal femoral shaft bone mineral density

test group Proportion of MSC cells endothelial podocyte content Fermoral Sharpt BMD change% (experimental group VS modeling group) group one 98% 2% +6% group two 96% 4% +4% group three 95% 5% +3% group four 94% 6% +1.5% group five 90% 10% +1% group six 85% 15% +0.5%

Experiments have verified that the content of MSC cells in the preparation is ≥90%, and the content of endothelial progenitor cells is ≤10%, which has a good effect on the treatment of osteoporosis.

In actual application, the MSC preparation of the present application also has the potential to be used for multiple sclerosis, osteoporosis, systemic scleroderma, hematological malignancies, myocardial infarction, organ transplant rejection, chronic allograft nephropathy, cirrhosis, and liver failure. , Heart Failure, GvHD, Tibia Fracture, Left Ventricular Dysfunction, Leukemia, Myelodysplastic Syndrome, Crohn's Disease, Diabetes, Chronic Obstructive Pulmonary Disease, Osteogenesis Imperfecta, Homozygous Familial Hypercholesterolemia, Treatment after meniscectomy, periodontitis in adults, angiogenesis in patients with severe myocardial ischemia, spinal cord injury, bone dysplasia, critical limb ischemia, diabetic foot disease, primary Sjögren's syndrome, osteoarthritis inflammation, cartilage defects, laminitis, multiple system atrophy, amyotrophic lateral sclerosis, cardiac surgery, systemic lupus erythematosus, living donor kidney allograft, non-malignant red blood cell disorders, 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 a combination thereof. The technical text of this application is based on the principle of text brevity, and only a part of the drug efficacy evaluation is listed.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Those skilled in the art of the present invention can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims (15)

1. A preparation containing MSC cells, comprising MSC cells derived from an umbilical cord and MSC cells derived from an umbilical cordEndothelial progenitor cells; wherein the concentration of MSC cells in said preparation is 0.1X 10 ⁶ -12×10 ⁶ Cells/ml; the content of MSC cells in the preparation is more than or equal to 90 percent, and the content of endothelial progenitor cells is less than or equal to 10 percent.

2. The formulation of claim 1, wherein said concentration of MSC cells is 0.6 x10 ⁶ -4×10 ⁶ Cells/ml.

3. The formulation of claim 1, wherein the content of MSC cells in the formulation is greater than or equal to 95% and the content of endothelial progenitor cells is less than or equal to 5%.

4. The preparation of claim 1, further comprising an injection solution, wherein the MSC cells derived from umbilical cord and the progenitor endothelial cells derived from umbilical cord are suspended in the injection solution.

5. The preparation of claim 4, wherein the injection comprises normal saline, compound electrolyte injection or dextran 40 glucose injection.

6. The preparation of claim 4, wherein the injection comprises normal saline, compound electrolyte injection or hyaluronic acid injection when used for local joint cavity injection.

7. The preparation of claim 1, wherein the cells in the preparation are obtained by separating umbilical cord tissue by tissue block adherence, digesting with animal origin-free recombinant cell dissociation enzyme, neutralizing with DPBS, and subculturing.

8. The preparation of claim 1, wherein the MSC cells are cells obtained by extracting MSC cells from umbilical cord and passaging for 6-8 passages; the endothelial progenitor cells are cells obtained by extracting the endothelial progenitor cells from the umbilical cord and passaging for 6-8 generations.

9. The MSC cell-containing preparation according to claim 7 or 8, wherein the cells in the MSC cell-containing preparation are seeded at a density of 0.5X 10 during passaging ³ /cm ² -7×10 ³ /cm ² 。

10. The MSC-containing preparation according to claim 7 or 8, wherein the cell passage time of the MSC-containing preparation is set to 96-144h.

11. The preparation of claim 7 or 8, wherein said MSC cells are subcultured in MEM- α and supplemented with 5% serum replacement.

12. The MSC cell-containing formulation of claim 11, wherein the serum Replacement comprises one of EliteGRO-Adv, ultraGRO-Advanced, knockOutSerum Replacement, CTS KnockOut SR, xenoFree Kit.

13. The preparation of claim 11, wherein the culture medium adopted by the MSC cells during subculture further comprises 10% or less of an endothelial progenitor cell culture medium, wherein the ECM is MEM- α supplemented with human bFGF, human EGF and human VEGF, and the ECM is the endothelial progenitor cell culture medium, and the final concentrations of the human bFGF, the human EGF and the human VEGF are all 10ng/ml.

14. The MSC-containing preparation according to claim 1, wherein the cells in the MSC-containing preparation are cryopreserved by selecting the following steps:

design of cells containing MSCThe frozen density of the cells in the preparation of (1.3X 10) ⁷ /ml-3.3×10 ⁷ /ml;

The frozen stock solution is selected as follows: the basic culture medium is MSCM, and the additive is DMSO with the volume concentration of 7.5%; the MSCM is MEM-alpha culture medium added with 5% of serum substitute, and the serum substitute comprises one of EliteGRO-Adv, ultraGRO-Advanced, knockOutSerum Replacement, CTS KnockOut SR and XenoFree Kit;

the freezing storage process conditions are as follows: after being stored for 16h at-80 ℃, the mixture is transferred to a liquid nitrogen tank.

15. The MSC cell-containing preparation according to any one of claims 1 to 7, the preparation can be used for treating multiple sclerosis, osteoporosis, systemic scleroderma, hematologic malignancy, 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, post-meniscectomy treatment, adult periodontitis, angiogenesis in patients with severe myocardial ischemia, spinal cord injury, bone dysplasia, severe limb ischemia, chronic renal failure, chronic myelodysplasia, and chronic myelodysplasia diabetic foot disease, primary sjogren's syndrome, osteoarthritis, cartilage defects, laminitis, multiple system atrophy, amyotrophic lateral sclerosis, cardiac surgery, systemic lupus erythematosus, in vivo kidney allograft, non-malignant erythrocytic disease, thermal burns, radiation burns, parkinson's disease, microfracture, epidermolysis bullosa, severe coronary ischemia, idiopathic dilated cardiomyopathy, femoral head necrosis, lupus nephritis, bone defects, ischemic stroke, post stroke, acute radiation syndrome, lung disease, arthritis, bone regeneration, uveitis, or combinations thereof.

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