UMBILICAL CORD MESENCHYMAL STEM CELLS (MSCS) AND CULTURE METHOD AND APPLICATION THEREOF

Abstract

An umbilical cord mesenchymal stern cells (MSCs) and culture method and application thereof, which comprises an MSCs culture step comprising the following substeps: primary culture: culturing Wharton's jelly of an umbilical cord in a serum-free medium under a hypoxic condition; subculture: collecting P1 primary cells to prepare a single cell suspension which is centrifuged to obtain a cell pellet; adding a serum-free medium to the cell pellet, culturing under a hypoxic condition until passage, and continuously culturing up to P2-P3; adding a ligustrazine hydrochloride and a Shenmai injection during each subculture, digesting cells grown to a predetermined fusion degree, and collecting the cells cultured to P6; phenotypic test: testing the phenotype of the collected cells for later use. An MSCs preparation cultured by the method reduces easy aggregation of stem cells, thus avoiding cell adhesion, rouleau formation of red cells and cell cluster embolism after intravenous infusion into human bodies.

Claims

1. A culture method of umbilical cord mesenchymal stem cells (MSCs), wherein comprising an MSCs culture step which comprises the following substeps: primary culture: culturing Wharton's jelly of umbilical cords in a serum-free medium under hypoxic condition until reaches the standard of passage; subculture: collecting P1 primary cells obtained in the primary culture step and removing residual medium to prepare a single cell suspension which is centrifuged to obtain a cell pellet; adding a serum-free medium to the cell pellet, culturing under a hypoxic condition until passage, and continuously culturing up to P2-P3; adding ligustrazine hydrochloride and Shenmai injection during each subsequent subculture, digesting and collecting cells grown to a predetermined fusion degree; phenotypic test: testing the phenotype of the cells collected in said subculture step, and taking cells with CD31-negative, HLA-DR-negative, CD34-negative, CD45-negative, CD44-positive, CD73-positive, CD90-positive and CD105-positive as target MSCs for later use.

2. The culture method of MSCs according to claim 1, wherein the concentration of the ligustrazine hydrochloride added is 40-80 mg/L and the Shenmai injection added is 0.5% by volume in said subculture step.

3. The culture method of MSCs according to claim 1, wherein in the MSCs culture step, the hypoxic condition is as follows: culturing in a carbon dioxide incubator with an oxygen concentration of 3-10%; and the serum-free medium is selected from a mesenchymal stem cell serum-free medium or a medium obtained by adding cytokines to a DMEM, F12, DMEM/F12 or RPMI1640 basal medium.

4. The culture method of MSCs according to claim 1, wherein the standard of passage is that the cells grow to a fusion degree of 80-90% in the primary culture step; in the subculture step, the ligustrazine hydrochloride and the Shenmai injection are added to continue the subculture to P4-P6.

5. The culture method of MSCs according to claim 1, wherein further comprising the following MSCs preparation steps before the MSCs culture step, and the following quality control steps after the MSCs culture step; preparation of MSCs: collecting umbilical cord tissues obtained by caesarean sections, transporting the tissues under refrigeration, making sure that the samples are not exposed to high energy ray irradiation, cleaning and stripping blood vessels in the tissues to obtain Wharton's jelly from the tissues; quality control: identifying the cells and testing the purity, cell growth activity, bacteria and mycoplasma, endotoxin, exogenous pathogenic factors, abnormal immune response, tumorigenicity and/or residual volume of ingredients added of the obtained target MSCs.

6. The culture method of MSCs according to claim 2, wherein further comprising the following MSCs preparation steps before the MSCs culture step, and the following quality control steps after the MSCs culture step; preparation of MSCs: collecting umbilical cord tissues obtained by caesarean sections, transporting the tissues under refrigeration, making sure that the samples are not exposed to high energy ray irradiation, cleaning and stripping blood vessels in the tissues to obtain Wharton's jelly from the tissues; quality control: identifying the cells and testing the purity, cell growth activity, bacteria and mycoplasma, endotoxin, exogenous pathogenic factors, abnormal immune response, tumorigenicity and/or residual volume of ingredients added of the obtained target MSCs.

7. The culture method of MSCs according to claim 3, wherein further comprising the following MSCs preparation steps before the MSCs culture step, and the following quality control steps after the MSCs culture step; preparation of MSCs: collecting umbilical cord tissues obtained by caesarean sections, transporting the tissues under refrigeration, making sure that the samples are not exposed to high energy ray irradiation, cleaning and stripping blood vessels in the tissues to obtain Wharton's jelly from the tissues; quality control: identifying the cells and testing the purity, cell growth activity, bacteria and mycoplasma, endotoxin, exogenous pathogenic factors, abnormal immune response, tumorigenicity and/or residual volume of ingredients added of the obtained target MSCs.

8. The culture method of MSCs according to claim 4, wherein further comprising the following MSCs preparation steps before the MSCs culture step, and the following quality control steps after the MSCs culture step; preparation of MSCs: collecting umbilical cord tissues obtained by caesarean sections, transporting the tissues under refrigeration, making sure that the samples are not exposed to high energy ray irradiation, cleaning and stripping blood vessels in the tissues to obtain Wharton's jelly from the tissues; quality control: identifying the cells and testing the purity, cell growth activity, bacteria and mycoplasma, endotoxin, exogenous pathogenic factors, abnormal immune response, tumorigenicity and/or residual volume of ingredients added of the obtained target MSCs.

9. The culture method of MSCs according to claim 5, wherein further comprising the following donor screening steps before said MSCs preparation step: 1) donors are tested for fulminating infectious diseases, including HIV, hepatitis B virus, hepatitis C virus, treponema pallidum, cytomegalovirus, EB virus and human T-cell lymphotropic virus before sample collection, and donors not infected with the above fulminating infectious diseases may be eligible; 2) donors are subject to a DNA test for genetic disorders, including achondroplasia, congenital deafness, vitamin D-resistant rickets, phenylketonuria, hemophilia, progressive muscular dystrophy and glucose-6-phosphate dehydrogenase deficiency before sample collection, and donors may be eligible if tested negative for all the above infectious diseases; 3) donors have not traveled to or stayed in epidemic areas within 3 months before sample collection; those who meet all the above criteria are eligible donors.

10. The culture method of MSCs according to claim 6, wherein further comprising the following donor screening steps before said MSCs preparation step: 1) donors are tested for fulminating infectious diseases, including HIV, hepatitis B virus, hepatitis C virus, treponema pallidum, cytomegalovirus, EB virus and human T-cell lymphotropic virus before sample collection, and donors not infected with the above fulminating infectious diseases may be eligible; 2) donors are subject to a DNA test for genetic disorders, including achondroplasia, congenital deafness, vitamin D-resistant rickets, phenylketonuria, hemophilia, progressive muscular dystrophy and glucose-6-phosphate dehydrogenase deficiency before sample collection, and donors may be eligible if tested negative for all the above infectious diseases; 3) donors have not traveled to or stayed in epidemic areas within 3 months before sample collection; those who meet all the above criteria are eligible donors.

11. The culture method of MSCs according to claim 7, wherein further comprising the following donor screening steps before said MSCs preparation step: 1) donors are tested for fulminating infectious diseases, including HIV, hepatitis B virus, hepatitis C virus, treponema pallidum, cytomegalovirus, EB virus and human T-cell lymphotropic virus before sample collection, and donors not infected with the above fulminating infectious diseases may be eligible; 2) donors are subject to a DNA test for genetic disorders, including achondroplasia, congenital deafness, vitamin D-resistant rickets, phenylketonuria, hemophilia, progressive muscular dystrophy and glucose-6-phosphate dehydrogenase deficiency before sample collection, and donors may be eligible if tested negative for all the above infectious diseases; 3) donors have not traveled to or stayed in epidemic areas within 3 months before sample collection; those who meet all the above criteria are eligible donors.

12. The culture method of MSCs according to claim 8, wherein further comprising the following donor screening steps before said MSCs preparation step: 1) donors are tested for fulminating infectious diseases, including HIV, hepatitis B virus, hepatitis C virus, treponema pallidum, cytomegalovirus, EB virus a.nd human T-cell lymphotropic virus before sample collection, and donors not infected with the above fulminating infectious diseases may be eligible; 2) donors are subject to a DNA test for genetic disorders, including achondroplasia, congenital deafness, vitamin D-resistant rickets, phenylketonuria, hemophilia, progressive muscular dystrophy and glucose-6-phosphate dehydrogenase deficiency before sample collection, and donors may be eligible if tested negative for all the above infectious diseases; 3) donors have not traveled to or stayed in epidemic areas within 3 months before sample collection; those who meet all the above criteria are eligible donors.

13. A stem cell cultured by the culture method of MSCs according to claim 1.

14. An MSCs injection, wherein comprising the stem cell of claim 13, heparin sodium, a compound amino acid and pharmaceutically acceptable adjuvants.

15. The MSCs injection according to claim 14, wherein the compound amino acid comprises L-isoleucine, L-arginine, L-leucine, L-aspartic acid, L-lysine, L-cysteine, L-glutamic acid, L-methionine, L-histidine, L-phenylalanine, L-proline, L-threonine, L-serine, L-tryptophan, L-tyrosine, L-valine, L-glycine and L-alanine; and the concentration of said compound amino acid is 102 g/100 ml.

16. The MSCs injection according to claim 14, wherein the concentration of the MSCs is (0.5-1.5)10.sup.9/100 ml.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 is a cell graph of P3 cells in Example 1 on Day 1 after a Shenmai injection is added;

[0037] FIG. 2 is a cell graph of P3 cells in Example 1 on Day 1 after a ligustrazine hydrochloride is added;

[0038] FIG. 3 is a cell graph of P3 cells in Example 1 on Day 1 after the ligustrazine hydrochloride and the Shenmai injection are added;

[0039] FIG. 4 is a cell graph of P4 cells in Example 1 on Day 1 after the Shenmai injection is added;

[0040] FIG. 5 is a cell graph of P4 cells in Example 1 on Day 1 after the ligustrazine hydrochloride is added;

[0041] FIG. 6 is a cell graph of P4 cells in Example 1 on Day 1 after the ligustrazine hydrochloride and the Shenmai injection are added;

[0042] FIG. 7 is a cell graph of P5 cells in Example 1 on Day 1 after the Shenmai injection is added;

[0043] FIG. 8 is a cell graph of P5 cells in Example 1 on Day 1 after the ligustrazine hydrochloride is added;

[0044] FIG. 9 is a cell graph of PS cells in Example 1 on Day 1 after the ligustrazine hydrochloride and the Shenmai injection are added;

[0045] FIG. 10 is a cell graph of peripheral blood of the control group in Example 3; and

[0046] FIG. 11 is a cell graph of peripheral blood of the treatment group in Example 3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] The invention is described more fully hereinafter with reference to the accompanying drawings for understanding the invention, in which preferred embodiments of the invention are shown. The invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that the disclosure of the invention will be thorough and complete.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. The terminology used herein in the Specification of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. Also, as used herein, the term and/or includes any and all combinations of one or more associated listed. items.

EXAMPLE 1

[0049] An MSC, which was cultured by the following method:

[0050] I. Donor Screening

[0051] 1. Blood of donors was drawn for testing for fulminating infectious diseases within one month before sample collection, including but not limited to human immunodeficiency virus (HIV), hepatitis B virus (HBW), hepatitis C virus (HCV), treponema pallidum (TP), cytomegalovirus (CMV), EB virus and human T-cell lymphotropic virus (HLTV). Donors carrying pathogen (including those previously infected with treponema pallidum) of any of the seven infectious diseases were excluded.

[0052] 2. Blood of donors was drawn for a DNA test for severe genetic diseases within one month before sample collection, including but not limited to achondroplasia, congenital deafness, vitamin D-resistant rickets, phenylketonuria, hemophilia, progressive muscular dystrophy and. glucose-6-phosphate dehydrogenase (G-6-PD) deficiency (favism). As the DNA test for severe genetic diseases is an important basis for the traceability of important links in cell culture sampling, donors with positive test results were excluded.

[0053] 3. Donors did not travel to or stay in epidemic areas over the past 3 months.

[0054] Those who meet all the criteria are eligible donors.

[0055] II. Preparation of MSCs

[0056] 1. Sample collection

[0057] The donors who passed the screening signed a sample donation agreement. After full-term pregnancy, donors delivered by caesarean sections, placenta and umbilical cord tissues were refrigerated (2-8 C.) within 48 hours and transported to a laboratory in special sterile sampling bags sealed with a proper amount of refrigerated tissue preservation solution. During transport, the sampling bags were kept unbroken without content leakage, and samples were kept unexposed to X ray, ray and other high-energy ray irradiation.

[0058] It is very important to use umbilical cords obtained by caesarean sections as the samples because umbilical cords from natural delivery will produce various microbial contamination when passing through birth canals, affecting the quality of MSCs.

[0059] 2. Sample preparation

[0060] 2.1 The samples transported to the laboratory were unbagged in a sterile biosafety cabinet, and the umbilical cord tissues were cleaned off blood stains on the surfaces with a pre-cold cleaning solution.

[0061] 2.2 The umbilical cords were cut into 2-3 cm segments and washed several times again.

[0062] 2.3 The arteries and veins in the umbilical cord tissues were stripped with tissue forceps, then Wharton's jelly was stripped from the umbilical cords and placed in a precooled tissue cleaning solution, and epidermal tissues could not be mixed in the Wharton's jelly.

[0063] III. MSCs culture

[0064] 1. Primary culture

[0065] 1.1 All the stripped Wharton's jelly was cleaned with a precooled tissue preservation solution several times and then placed in a 50 ml centrifugal tube, and the tissues were cut to a volume of not greater than 333 mm.sup.3.

[0066] 1.2 A proper amount of serum-free medium (containing 100 U/ml of penicillin/streptomycin) (serum-free medium for MSCs of Yocon Biology) was added to the cut tissues to obtain a culture solution, then 10 ml of the culture solution was inoculated in each T75 culture flask on average, and the culture flask was placed in a 37 C. constant temperature carbon dioxide incubator for normal culture under a hypoxic condition with oxygen concentration of 3-10%, preferably 5%.

[0067] 1.3 All medium was changed on Day 5th of the primary culture, and half of medium was changed 3 days later. Under normal circumstances, some cells migrated out of tissue masses on Day 10 and grew to a fusion degree of 90% on Day 15th, which met the standard of passage.

[0068] 2. Subculture

[0069] 2.1 The special medium in the culture flask for passage was collected and centrifuged at 3000 rpm for 10 minutes, and a supernatant was collected for later use.

[0070] 2.2 Primary cells in the culture flask were washed twice with physiological saline to remove residual medium.

[0071] 2.3 Then 3 ml of 0.05% trypsin was added to digest the cells and the culture flask was tapped to allow all adherent cells to fall off, then 5 ml of special medium after centrifugation was added to terminate pancreatic enzymes and the cells were gently blown to obtain single cell suspension.

[0072] 2.4 After the cell suspension was collected, the culture flask was washed twice with physiological saline, then all liquid was collected and filtered through a 100 m filter screen to remove undigested tissue masses.

[0073] 2.5 The filtered single cell suspension was centrifuged at 1300 rpm for 6 min to remove the supernatant.

[0074] 2.6 To the cell pellet, 15 ml of physiological saline was added to resuscitate the cells. After mixing, a small amount of liquid was taken for counting, and the mixture was centrifuged again at 1300 rpm for 6 min to remove the supernatant.

[0075] 2.7 Every 500 ml of a mixture of the cell pellet and a serum-free medium (antibiotic-free) was prepared into a cell suspension. The serum-free medium was a serum-free medium for mesenchymal stem cells of Yocon Biology, or a medium obtained by adding cytokines to a DMEM, F12, DMEM/F12 or RPMI1640 basal medium of GIBCO. According to the cell count result, the cell suspension was inoculated in a T175 culture flask at a density of 10000/cm.sup.2, and the culture flask was placed in a 37 C. constant temperature carbon dioxide incubator for normal culture under a hypoxic condition with oxygen concentration of 3-10%, preferably 5%.

[0076] 2.8 The cells grown to a fusion degree of 90% (about 3 days) were subcultured again to P2.

[0077] 2.9 A ligustrazine hydrochloride (GYZZ H20041175 injection of Harbin Medisan Pharmaceutical Co., Ltd., which follows the Second Supplement to the National Standard and can be directly injected into human bodies) and a Shenmai injection (GYZZ 263021721 of Yunnan Phytopharmaceutical Co., Ltd., which follows the National Drug Standard (Amendment) WS3-B-3428-98-2010 and can be directly injected into human bodies) were added to P3-P5 cells respectively. The concentration of the ligustrazine hydrochloride was 40-80 mg/L and the Shenmai injection was 0.5% by volume. After culture for 24 hours, change the medium and culture for another one week, the cells grown to a fusion degree of 80-90% were collected and digested for tests. The products obtained in the steps are biodetected to eliminate etiological contamination.

[0078] The cells were divided into three groups: a. only the Shenmai injection was added; b. only the ligustrazine hydrochloride was added; and c. both the Shenmai injection and the ligustrazine hydrochloride were added for comparison. The results were shown in FIG. 1 to FIG. 9, it can be seen from the figures that the agglomeration of the cells was improved to some extent when the ligustrazine hydrochloride or the Shenmai injection was added, but the best effect was obtained when both the ligustrazine hydrochloride and the Shenmai injection were added at the same time.

[0079] 3. Phenotypic test

[0080] 3.1 The cells collected were subject to a phenotypic test, and cells with CD31-negative, HLA-DR-negative, CD34-negative, CD45-negative, CD44-positive, CD73-positive, CD90-positive and CD105-positive were taken as qualified target MSCs.

[0081] 3.2 The qualified target MSCs were resuspended in a special freezing medium at a density of 110.sup.7/ml according to the count result. Single cells suspended in the freezing medium were added to freezing tubes (1 ml each) and labeled, with label information, including but not limited cell type, cell number, passage number of frozen cells, cell count of frozen cells in each tube and freezing date. The freezing tubes were sealed with a sealing film, then slowly cooled to 90 C. by a programmed cooling instrument, and directly placed in liquid nitrogen at 205 C. to 185 C. (preferably 196 C.) for long-term storage after program cooling.

[0082] IV. Quality control

[0083] The quality control of the umbilical cord MSCs in the example follows the quality testing standards for stem cell preparations in the Guiding Principles for Quality Control and Preclinical Research of Stem Cell Preparations (Trial) issued by China Food and Drug Administration in 2015.

[0084] 1. Cell identification and purity test: cell morphology, multilineage differentiation potentials and surface markers were used to test and identify whether the cultured cells were MSCs and their purity. The surface markers included positive indicators (>95%): CD44, CD73, CD90, CD105, and negative indicators (<2%): CD34, CD45, HLA-DR, multilineage differentiation potentials.

[0085] 2. Cell growth activity: cell growth activity was tested by CCK8, cell doubling time, cell cycle, clone forming efficiency, telomerase activity and telomere length, and cell senescence was tested by (3 galactosidase.

[0086] 3. Test of bacteria and mycoplasma: bacterial, fungal and mycoplasma contamination of the samples was tested in accordance with procedures for testing sterility and mycoplasma of biological preparations in Chinese Pharmacopoeia (2015 Edition).

[0087] 4. Endotoxin test: endotoxin of the samples was tested in accordance with procedures for testing endotoxin in Chinese Pharmacopoeia (2015 Edition).

[0088] 5. Test of exogenous pathogenic factors: DNA test for pathogens of donor-derived fulminating infectious diseases, including human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), treponema pallidum (TP), cytomegalovirus (CMV), EB virus and human T-cell lymphotropic virus (HLTV); test of bovine-derived specific viruses, including mad cow disease and aftosa; and swine-derived specific viruses, including porcine parvovirus.

[0089] 6. Abnormal immune response: effects of the MSC on the proliferation of human total lymphocytes and specific lymphocyte subsets (including CD4+T cells, CD8+T cells, B cells, NK cells) and the secretion of associated cytokines (including INF-, TNF-, IL-4, IL-6 and IL-10) were tested.

[0090] 7. Tumorigenicity test: the tumorigenicity of cells was tested by implanting high-dose MSCs into immunodeficient animals to observe tumorigenesis.

[0091] 8. Test of residue of added ingredients: the residual volume of ingredients (including bovine serum albumin (BSA), antibiotics, specific cytokines and phenol red) that affect the safety of stem cell preparations during preparation in the final product was tested.

EXAMPLE 2

[0092] An MSCs injection, which was obtained by the following method:

[0093] 1. Preparatory work

[0094] 1.1 Water bath was reheated to stabilize the water temperature at 37 C.

[0095] 1.2 A biosafety cabinet was subject to ultraviolet disinfection and ventilation 30 min in advance.

[0096] 1.3 Physiological saline was provided according to the cell count of thawed cells and added to a centrifuge tube and the volume of the physiological saline was at least 10 times than that of all freezing medium.

[0097] 2. Information checking

[0098] Frozen cells were taken out of a liquid nitrogen tank according to the predetermined demand, and key information such as cell type, cell code, cell count and cell passage number was carefully checked to prevent errors.

[0099] 3. The frozen cells taken out of the liquid nitrogen tank were immediately placed in the reheated water bath for thawing for 2 min until the freezing medium completely thawed, and the frozen cells were constantly shaken while ensuring that the freezing tube mouth did not contact with the water in the water bath.

[0100] 4. Thawed freezing tubes were taken out and transferred to a biosafety cabinet after the tube bodies were wiped with 75% alcohol.

[0101] 5. The cell suspension in the freezing tubes was completely sucked into a centrifuge tube containing physiological saline, and the tubes were washed with physiological saline for 2-3 times, then all the liquid was transferred into the centrifuge tube and mixed.

[0102] 6. The cell suspension was centrifuged at 1300 rpm for 6 min to remove the supernatant, then the cells were resuspended in fresh physiological saline, centrifuged and washed repeatedly for 3 times.

[0103] 7. After washing, 100 ml physiological saline was added to prepare a group of composite preparations.

[0104] The number of cells containing hypoxic MSCs in each group of composite preparations is (0.5-1.5)10.sup.9, the physiological saline in each group of composite preparations contained 10-20Um low molecular weight heparin sodium, and each group of composite preparations contains 18 L-compound amino acids.

[0105] The compound amino acid injection is composed of 18 amino acids, including L-isoleucine, L-arginine, L-leucine, L-aspartic acid, L-lysine, L-cysteine, L-glutamic acid, L-methionine, L-histidine, L-phenylalanine, L-proline, L-threonine, L-serine, L-tryptophan, L-tyrosine, L-valine, L-glycine and L-alanine, and contained 102 g amino acids per 100 ml (said compound amino acid injection was a compound amino acid injection 18AA-III that was commercially available).

[0106] The cells were resuspended and filtered through a 40 m filter screen to remove agglomerated cells to obtain a single cell suspension.

[0107] 8. A small amount of cell suspension was taken to count the cells to obtain the total cell count and cell viability so as to ensure that the cell count and viability were not lower than predetermined requirements.

[0108] 9. The cell suspension was loaded in a special cell reinfusion bags (bottles) and completely sealed. Before the cell suspension was loaded in the reinfusion bags, samples were taken for endotoxin test, with 1 ml of each sample reserved.

[0109] 10. The endotoxin content of the cell suspension to be reinfused was tested according to standard operating procedures of limulus reagent gel method, and the endotoxin content of the reinfused cell suspension was lower than 0.5 EU/ml according to the requirements of Chinese Pharmacopoeia (2015 Edition).

[0110] 11. The products passing the endotoxin test were released, otherwise the products would be destroyed centrally and the causes would be investigated.

[0111] 12. The reserved cell suspension was labeled and stored at 20 C. for at least one year, with label information including but not limited to product number, cell type, cell number, date of manufacture and responsible person.

[0112] 13. Released products were sent out of the laboratory after carefully checking of the product information by a quality inspector, and transported to reinfusion sites within 4 hours under refrigeration (2-8 C.) for reinfusion. During transport, the cell suspension was kept unexposed to X-ray or y-ray and other high energy ray irradiation.

EXAMPLE 3

[0113] An application of MSCs injection in the treatment of myocardial infarction.

[0114] The umbilical cord MSCs preparation of the invention has extensive applications. At present, we have prepared the MSCs of Example 1 into a compound preparation as shown in Example 2 and applied the compound preparation to the treatment of patients with myocardial infarction to observe the efficacy.

[0115] I. The morphology of peripheral blood cells was observed under microscope.

[0116] 1. Method

[0117] The peripheral blood in the control group (MSCs cultured without ligustrazine hydrochloride and Shenmai injection) and in the treatment group (the compound preparation of Example 2) were observed under microscope.

[0118] 2. Results

[0119] The results are shown in FIG. 10 and FIG. 11. FIG. 10 shows the peripheral blood of the subjects in the control group, and FIG. 11 shows the peripheral blood of the subjects in the treatment group. It can be seen from FIG. 10 that cell adhesion, aggregation, rouleau formation of red cells and cell cluster embolism occur in the peripheral blood of the subjects in the control group. However, it is clear from FIG. 11 that, with the MSC preparation cultured by adding the ligustrazine hydrochloride and the Shenmai injection which are traditional Chinese medicine injections for activating blood circulation to remove blood stasis, supplementing qi and nourishing yin, the red blood cell morphology of the subjects is normal without aggregation.

[0120] From the above results, it is clear that red blood cells in the blood of the subjects are normally separated (FIG. 11) in half an hour after injection of the MSCs preparation of the invention, which is consistent with the theory of traditional Chinese medicine that qi commands the blood and the blood carries qi.. According to the traditional Chinese medicine, normal flow of qi ensures normal flow of blood while stagnation of qi causes stagnation of blood. Myocardial infarction with blood stasis and stagnation will hinder the circulation of qi. Therefore, in addition to a hydrochloride of ligustrazine which is an active ingredient of traditional Chinese medicine Ligusticum wallichii with the characteristics of activating blood circulation to remove blood stasis, ginseng for tonifying heart qi and the Radix Ophiopogonis preparation Shenmai injection for nourishing heart and yin blood are added to treat myocardial infarction to keep qi and blood unobstructed, and the effect is better.

[0121] II. Clinical Effect

[0122] The preparation has been applied to dozens of cases with myocardial infarction and severe lesions in three coronary arteries in Foshan. Typical cases have survived since 2001. The cases are as follows:

[0123] The 47-year-old male, Mr. Mai, was admitted to a hospital for chest tightness for seven days. Cardiography showed 80%-90% stenosis in proximal-middle segments of the left anterior descending coronary artery, and approximately 80%-90% stenosis in proximal-middle segments of the right coronary artery. The patient was diagnosed with: latent coronary heart disease with lesions in three coronary arteries, and 2. hyperuricemia. The patient was advised to receive further coronary intervention, but both the patient and his family refused. The patient was later introduced to the applicant office, and given the preparation by intravenous infusion for four times at an interval of 7-15 days. The patient has survived without any symptoms and discomfort. The reexamination in Shunde Hospital of Southern Medical University in August 2018 revealed that the stenosis was improved to 60-70% stenosis in the middle segment of the right coronary artery, approximately 70-85% lumen stenosis in the proximal-middle segments of the left anterior descending coronary artery, and significant improvement in old occlusion of the distal segment of the circumflex artery.

[0124] The technical features of the above examples can be combined in any way. For brevity, not all possible combinations of the technical features of the above examples are described. However, it should be considered that the technical features are included in the scope of the specification provided that there is no contradiction in the combinations thereof.

[0125] The above examples illustrate several embodiments of the invention only, but should not be construed as limiting the scope of the invention despite of specific and detailed description. It should be noted that a person skilled in the art can make various changes and improvements without departing from the concept of the invention, which should be incorporated in the protection scope of the invention. Therefore, the scope of protection of the invention patent shall be subject to appended claims.