Mesenchymal stem cell-containing product, preparation method, quality control method and application of mesenchymal stem cell-containing product in prevention and treatment of digestive system diseases

Abstract

The invention belongs to the technical field of stem cells, and particularly relates to a product containing mesenchymal stem cells, a preparation method, a quality control method and application in prevention and treatment of digestive system diseases. According to the preparation method, 1-3 complete culture media are provided for culturing the mesenchymal stem cells, and the obtained mesenchymal stem cells are good in subpackage form; cD73, CD90 and CD105 are highly expressed, and CD19, CD34, CD45, CD11b and HLA-DR are not expressed; the cell proliferation capacity is strong, the osteogenesis and adipogenic differentiation capacity is strong, and the medicine is safe and free of toxic and side effects. The prepared mesenchymal stem cells are high in quality, have good performance of secreting HGF, KGF and IGF-1, and can effectively treat digestive system diseases.

Description

Technical Field

[0001] The present invention belongs to the field of stem cell technology, and specifically relates to products containing mesenchymal stem cells, preparation methods, quality control methods, and applications in the prevention and treatment of digestive system diseases. Background Art

[0002] Umbilical cord mesenchymal stem cells (MSCs) refer to a type of multifunctional stem cells found in the umbilical cord tissue of newborns. They can differentiate into many types of tissue cells and have broad prospects for clinical application. The secretions of umbilical cord mesenchymal stem cells play an important role in the treatment of diseases. These secretions include a variety of growth factors, cytokines, chemokines, and immunomodulatory molecules, which have therapeutic effects on diseases through different mechanisms.

[0003] The influence of culture methods on umbilical cord mesenchymal stem cells is significant. Different culture methods will directly affect the growth rate and proliferation ability of umbilical cord mesenchymal stem cells. For example, there are differences in cell separation and culture efficiency between the tissue adhesion method and the enzyme digestion method (such as trypsin and collagenase digestion method) in the primary culture method. Studies have shown that the success rate of culturing umbilical cord mesenchymal stem cells by the tissue adhesion method is higher, and the cells can better maintain their basic properties during the culture process). In addition, the formula of the culture medium, the culture conditions (such as temperature, CO concentration, humidity) and the choice of culture vessel will also affect the growth of cells.

[0004] In terms of the culture medium formula, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) can promote stem cell growth, proliferation and maintain stem cell stemness, while stromal cell-derived factor-1α (SDF-1α) has the effect of promoting stem cell homing, chemotaxis and proliferation. Its use in the early stage of mesenchymal stem cell separation and culture can accelerate the speed of stem cell separation from tissues such as the umbilical cord and help maintain stem cell activity; glutathione and α-ketoglutaric acid can provide energy for stem cell growth, reduce oxygen free radical damage and participate in the regulation of stem cell differentiation; while vitamins C and E can regulate stem cell function, maintain cell membrane integrity and delay cell aging. Recent research results show that hydrogen can prolong the in vitro replication life of cells and protect the cell's antioxidant capacity. Hypoxic conditions can promote the proliferation potential of MSCs and maintain the stemness of MSCs well.

[0005] Different culture methods also have a significant effect on the secretion of bioactive molecules such as insulin-like growth factor-1 (IGF-1) and keratinocyte growth factor (KGF) by umbilical cord mesenchymal stem cells. Studies have shown that the introduction of IGF-1 gene into umbilical cord mesenchymal stem cells through gene transfection technology can significantly improve the ability of cells to secrete IGF-1. In addition, optimizing culture conditions (such as adjusting the ratio of nutrients in the culture medium, adding growth factors, etc.) may also have a positive effect on the secretion of IGF-1. The effects of different culture methods on the secretion of KGF by umbilical cord mesenchymal stem cells are equally important. On the one hand, suitable culture conditions can maintain the activity and stability of cells, which is conducive to the secretion of KGF. On the other hand, by constructing a recombinant plasmid expressing KGF and transforming it into umbilical cord mesenchymal stem cells, efficient expression of KGF can be achieved. This method can not only increase the production of KGF, but also further study the effects of KGF on biological processes such as cell proliferation and differentiation. LIF is a multifunctional cytokine with multiple biological activities such as promoting cell growth and inhibiting cell apoptosis. However, there are relatively few studies on the specific effects of different culture methods on the secretion of LIF by umbilical cord mesenchymal stem cells, and the effects may vary due to factors such as experimental conditions and cell sources.

[0006] Gene transfection technology and recombinant plasmids play an important role in biomedical research and treatment, but they do have certain hazards. Gene transfection technology may cause cell damage, gene integration problems (after the foreign gene is introduced into the cell, it may randomly integrate into the host cell genome, resulting in changes in the stability of the genome and genetic characteristics), immune response and ethical issues. Recombinant plasmids may also have toxicity problems, genetic stability problems and horizontal gene transfer.

[0007] Therefore, there is an urgent need to develop a safe and effective culture method to obtain high-quality umbilical cord mesenchymal stem cell secretions and improve the therapeutic effect. Summary of the invention

[0008] In order to solve the above problems, the present invention provides a method for preparing mesenchymal stem cells, which can effectively improve the quality of cells and has significant therapeutic effects on the digestive system and diseases.

[0009] In one aspect, the present invention provides a method for preparing mesenchymal stem cells, the steps of the preparation method are as follows:

[0010] (1) incubating the treated tissue and adding complete culture medium 1 for culturing; culturing until the cell confluence reaches 70-90%, digesting with digestion solution to obtain P0 generation mesenchymal stem cells;

[0011] (2) centrifuging the P0 generation mesenchymal stem cells, discarding the supernatant, and adding complete culture medium 2 for the first subculture to obtain P1 generation mesenchymal stem cells;

[0012] (3) centrifuging the P1 generation mesenchymal stem cells, discarding the supernatant, and adding complete culture medium 3 for a second subculture to obtain P2 generation mesenchymal stem cells;

[0013] (4) Centrifuging the P2 mesenchymal stem cells, discarding the supernatant, harvesting the mesenchymal stem cells, and obtaining seed bank cells.

[0014] (5) The seed bank cells were added to complete culture medium 3 and cultured until P5 to obtain mesenchymal stem cells.

[0015] The complete culture solution 1 is composed of FBS, bFGF, EGF, SDF-1α and basal culture medium;

[0016] The complete culture medium 2 is composed of FBS, bFGF, EGF, SDF-1α glutathione, hydrogen and basal culture medium;

[0017] The complete culture medium 3 consists of FBS, bFGF, EGF, SDF-1α, vitamin C, glutathione and basal culture medium.

[0018] Specifically, the culture conditions in step (1) include 5% CO2, 1%-2% O2;

[0019] The conditions of the first subculture in step (2) include 5% CO2, 5%-10% O2;

[0020] The conditions of the second subculture in step (3) include 5% CO2 and 18%-20% O2;

[0021] The culture conditions in step (5) include 5% CO2.

[0022] Further specifically, in some embodiments, the culture conditions in step (1) include 5% CO2, 1% O2;

[0023] The conditions of the first subculture in step (2) include 5% CO2 and 5% O2;

[0024] The conditions of the second subculture in step (3) include 5% CO2 and 20% O2;

[0025] The culture conditions in step (5) include 5% CO2.

[0026] In other embodiments, the culture conditions in step (1) include 5% CO2, 2% O2;

[0027] The conditions of the first subculture in step (2) include 5% CO2 and 10% O2;

[0028] The conditions of the second subculture in step (3) include 5% CO2 and 18% O2;

[0029] The culture conditions in step (5) include 5% CO2.

[0030] Specifically, the basic culture medium includes one or more of MesenCult-XF Medium, Essential 8 Medium, StemPro hESC SFM, CTS Essential 6 Medium, DMEM / F12 or DMEM.

[0031] Preferably, the basal culture medium is DMEM / F12.

[0032] Specifically, the complete culture solution 1 consists of 10-15% v / v FBS, 5-10 μg / L bFGF, 5-10 μg / L EGF, 100-200 μg / L SDF-1α and DMEM / F12 culture medium;

[0033] The complete culture solution 2 consists of 10-15% v / v FBS, 5-10 μg / L bFGF, 5-10 μg / L EGF, 100-200 μg / L SDF-1α, 1-25 μg / L glutathione, 0.5-1.5 mg / L hydrogen and DMEM / F12 culture medium;

[0034] The complete culture medium 3 consists of 10-15% v / v FBS, 5-10 μg / L bFGF, 5-10 μg / L EGF, 100-200 μg / L SDF-1α, 5-10 mg / L vitamin C, 1-25 μg / L glutathione and DMEM / F12 culture medium.

[0035] Further specifically, the complete culture solution 1 consists of 10% v / v FBS, 5-10 μg / L bFGF, 5-10 μg / L EGF, 100-200 μg / L SDF-1α and DMEM / F12 culture medium;

[0036] The complete culture solution 2 consists of 10% v / v FBS, 5-10 μg / L bFGF, 5-10 μg / L EGF, 100-200 μg / L SDF-1α, 1-25 μg / L glutathione, 0.5-1.5 mg / L hydrogen and DMEM / F12 culture medium;

[0037] The complete culture medium 3 consists of 10% v / v FBS, 5-10 μg / L bFGF, 5-10 μg / L EGF, 100-200 μg / L SDF-1α, 5-10 mg / L vitamin C, 1-25 μg / L glutathione and DMEM / F12 culture medium.

[0038] Preferably, in some embodiments, the complete culture solution 1 consists of 10% v / v FBS, 5 μg / L bFGF, 10 μg / L EGF, 100 μg / L SDF-1α and DMEM / F12 medium;

[0039] The complete culture medium 2 consists of 10% v / v FBS, 5 μg / L bFGF, 10 μg / L EGF, 100 μg / L SDF-1α, 1 μg / L glutathione, 0.5 mg / L hydrogen and DMEM / F12 culture medium;

[0040] The complete culture medium 3 consists of 10% v / v FBS, 5 μg / L bFGF, 10 μg / L EGF, 100 μg / L SDF-1α, 10 mg / L vitamin C, 1 μg / L glutathione and DMEM / F12 culture medium.

[0041] In other embodiments, the complete culture medium 1 consists of 10% v / v FBS, 10 μg / L bFGF, 5 μg / L EGF, 200 μg / L SDF-1α and DMEM / F12 medium;

[0042] The complete culture medium 2 consists of 10% v / v FBS, 10 μg / L bFGF, 5 μg / L EGF, 200 μg / L SDF-1α, 25 μg / L glutathione, 1.5 mg / L hydrogen and DMEM / F12 culture medium;

[0043] The complete culture medium 3 consists of 10% v / v FBS, 10 μg / L bFGF, 5 μg / L EGF, 200 μg / L SDF-1α, 5 mg / L vitamin C, 25 μg / L glutathione and DMEM / F12 culture medium.

[0044] Specifically, the mesenchymal stem cells are derived from bone marrow, adipose tissue, umbilical cord, umbilical cord blood or placental tissue.

[0045] More specifically, the mesenchymal stem cells are derived from umbilical cord.

[0046] In another aspect, the present invention provides mesenchymal stem cells obtained by the aforementioned preparation method.

[0047] Specifically, the concentration of the mesenchymal stem cells is 1×106 -1×10 8 cells / mL, such as 1×10 6 cells / mL, 2×10 6 cells / mL、3×10 6 cells / mL、4×10 6 cells / mL、5×10 6 cells / mL、6×10 6 cells / mL、8×10 6 cells / mL、8×10 6 cells / mL、9×10 6 cells / mL、1×10 7 cells / mL、1×10 8 cells / mL, and any point within the concentration range.

[0048] In yet another aspect, the present invention provides a culture comprising the aforementioned mesenchymal stem cells, wherein the biological efficacy detection indexes thereof include the levels of HGF, IGF-1 and KGF secreted by the mesenchymal stem cells.

[0049] In another aspect, the present invention provides use of the aforementioned mesenchymal stem cells or cultures in the preparation of drugs for treating digestive system diseases.

[0050] Specifically, the digestive system diseases include inflammatory bowel disease, chronic liver fibrosis and acute liver failure.

[0051] Specifically, the drug is in the form of an injection, a gel, a cell membrane, a patch, a microsphere, a microparticle or a composite material.

[0052] More specifically, the composite material comprises mesenchymal stem cells combined with other biological materials, growth factors, drugs, etc. to form a composite material with specific functions.

[0053] In another aspect, the present invention provides the aforementioned quality control method for mesenchymal stem cells, comprising performing one or more of cell morphology, cell flow cytometry phenotype, cell proliferation, osteogenic and adipogenic differentiation ability or biological efficacy detection on the mesenchymal stem cells.

[0054] Specifically, the quality control method includes testing the cell morphology, cell flow cytometry phenotype, cell proliferation, osteogenic and adipogenic differentiation ability and biological efficacy of mesenchymal stem cells.

[0055] Specifically, the cell flow cytometry phenotype includes one or more of CD73, CD90, CD105, CD19, CD34, CD45, CD11b or HLA-DR.

[0056] More specifically, the cell flow cytometry phenotype includes CD73, CD90, CD105, CD19, CD34, CD45, CD11b and HLA-DR.

[0057] Specifically, the biological efficacy assay includes one or more of HGF, KGF or IGF-1.

[0058] More specifically, the biological efficacy assay includes HGF, KGF and IGF-1.

[0059] The technical effects achieved by the present invention are:

[0060] (1) The mesenchymal stem cells prepared by the present invention have good cell morphology.

[0061] (2) The mesenchymal stem cells prepared by the present invention highly express CD73, CD90, and CD105, but do not express CD19, CD34, CD45, CD11b, and HLA-DR.

[0062] (3) The mesenchymal stem cells prepared by the present invention have strong proliferation ability.

[0063] (4) The mesenchymal stem cells prepared by the present invention have strong osteogenic and adipogenic differentiation abilities.

[0064] (5) The mesenchymal stem cells prepared by the present invention are safe, and the results of tests for bacteria, mycoplasma, viruses, etc. are all negative.

[0065] (6) The mesenchymal stem cells prepared by the present invention can effectively treat digestive system diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 Mesenchymal stem cells.

[0067] Figure 2 This is the result of cell proliferation ability test.

[0068] Figure 3 The result is the cell differentiation ability.

[0069] Figure 4 The effect on the survival rate of rats (Mean±SD, n=9-12).

[0070] Figure 5 The effect on the body weight of rats (Mean±SD, g, n=9-12), compared with the sham operation group: *P<0.05; compared with the ethanol control group: # P<0.05, ## P<0.01.

[0071] Figure 6The effect on DAI scores of rats (Mean±SD, n=9-12), compared with the sham operation group: *P<0.05, **P<0.01, ***P<0.001; compared with the ethanol control group: # P<0.05, ### P<0.001; compared with the model group: & P<0.05, && P<0.01.

[0072] Figure 7 The effect on the macroscopic score of rat colon (Mean±SD, n=9-12), compared with the sham operation group: *P<0.05, ***P<0.001; compared with the ethanol control group: ### P<0.001; compared with the model group: &&& P<0.001.

[0073] Figure 8 The effect on the colon weight of rats (Mean±SD, mg, n=9-12), compared with the sham operation group: **P<0.01, ***P<0.001; compared with the ethanol control group: # P<0.05, ### P<0.001; compared with the model group: && P<0.01, &&& P<0.001.

[0074] Fig. 9 The effect on the intestinal coefficient of rats (Mean±SD, n=9-12), compared with the sham operation group: ***P<0.001; compared with the ethanol control group: ### P<0.001; compared with the model group: && P<0.01, &&& P<0.001.

[0075] Fig.10 The weight changes of animals in each experimental group during the experimental period, where A is the weight change curve of animals in each experimental group during the experimental period (D0-D42); B is the weight of animals in each experimental group at the end of the experiment (D42); C is the range of weight change of animals in each experimental group during the experimental period (D0-D42); Compared with the normal control group, the model control group: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0076] Fig.11The effect on the chronic liver injury process of model animals, where A is AST; B is ALT; C is ALP; D is ALB; model control group compared with normal control group: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; hUC-MSC treatment group compared with model control group: # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001.

[0077] Fig.12 The following are the histopathological diagnostic scores of the livers of the animals in each experimental group, where A is the liver fibrosis score; B is the liver confluent necrosis score; C is the liver inflammatory infiltration score; D is the liver HYP; D is the liver CVF; Compared with the normal control group, the model control group: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; 2. Compared with the model control group, the hUC-MSC treatment group: # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001. DETAILED DESCRIPTION

[0078] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0079] The experimental materials used in the present invention are shown in Table 1:

[0080] Table 1 Experimental materials

[0081]

[0082]

[0083] Example 1

[0084] A method for preparing umbilical cord mesenchymal stem cells, the specific steps are as follows:

[0085] (1) Human umbilical cord tissue was derived from healthy fetuses born by caesarean section in a cooperative obstetrics and gynecology hospital. Umbilical cord tissue from healthy donors with a length of more than 15 cm was selected, and the umbilical cord tissue was cut into small pieces with a diameter of 1-1.5 mm without washing in order to preserve the original environment of the tissue; each piece of umbilical cord tissue was then directly inoculated into a T75 culture bottle and placed in a 5% CO2 incubator at a temperature of 38°C for incubation for 24 hours. No cell culture medium was added during this process. After the incubation, the culture bottle containing the umbilical cord tissue was placed in a 5% CO2, 1% O2 incubator at 37°C, 5 mL of complete culture medium 1 was added to the culture bottle, and 10 mL of complete culture medium 1 was added after overnight culture. Thereafter, the culture medium was changed every 3-5 days. After the cell fusion degree reached 80%, the cells were digested with pancreatic enzyme digestion solution to obtain P0 generation human umbilical cord mesenchymal stem cells.

[0086] (2) The P0 generation human umbilical cord mesenchymal stem cells obtained in step (1) were centrifuged at 300-400gx5min, the supernatant was discarded, and complete culture medium 2 with a H2 concentration of 0.5mg / L was added, and the cells were subcultured at 37°C in an incubator with a 5% CO2+5% O2 environment for 48-72 hours, which was recorded as P1 generation. The preparation method of complete culture medium 2 with a H2 concentration of 0.5mg / L is as follows: using a hydrogen generator, the hydrogen generated by the generator is connected to the complete culture medium 2 through a silicone tube, and the hydrogen concentration in the culture medium is detected using a hydrogen electrode.

[0087] (3) The P1 human umbilical cord mesenchymal stem cells obtained in step (2) were centrifuged at 400 g for 5 min, the supernatant was discarded, complete culture medium 3 was added, and the cells were placed in an incubator at 37° C. with a 5% CO 2 environment and 20% O 2 for subculture for 72 hours, which was recorded as the P2 generation.

[0088] (4) The P2 human umbilical cord mesenchymal stem cells obtained in step (3) were centrifuged at 400 g for 5 min, the supernatant was discarded, a portion was tested, and the other portion was frozen, and the cells that passed the test were used as a seed bank.

[0089] (5) Take the seed bank cells described in step (4), continue to culture to P5 generation and freeze, the culture system and environment are: complete culture medium 3, 37°C, 5% CO2.

[0090] (6) The frozen P5 mesenchymal stem cells described in step (5) are tested as a reference seed bank, and cells that pass the test are kept as a working bank, denoted as W1, and unqualified cells are discarded.

[0091] The complete culture medium 1 is: 10% v / v fetal bovine serum (FBS), 5 μg / L basic fibroblast growth factor (bFGF), 10 μg / L epidermal growth factor (EGF), 100 μg / L SDF-1α and DMEM / F12 (DF12) culture medium;

[0092] The complete culture medium 2 is: 10% v / v FBS, 5 μg / L bFGF, 10 μg / L EGF, 100 μg / L SDF-1α, 1 μg / L glutathione, 0.5 mg / L hydrogen and DMEM / F12 (DF12) culture medium;

[0093] The complete culture medium 3 comprises: 10% v / v FBS, 5 μg / L bFGF, 10 μg / L EGF, 100 μg / L SDF-1α, 10 mg / L vitamin C, 1 μg / L glutathione and DMEM / F12 (DF12) culture medium.

[0094] Example 2

[0095] A method for preparing umbilical cord mesenchymal stem cells, the specific steps are as follows:

[0096] (1) Human umbilical cord tissue was derived from healthy fetuses born by caesarean section in a cooperative obstetrics and gynecology hospital. Umbilical cord tissue from healthy donors with a length of more than 15 cm was selected, and the umbilical cord tissue was cut into small pieces with a diameter of 1-1.5 mm without washing in order to preserve the original environment of the tissue; each piece of umbilical cord tissue was then directly inoculated into a T75 culture bottle and placed in a 5% CO2 incubator at a temperature of 38°C for 24 hours. No cell culture medium was added during this process. After the incubation, the culture bottle containing the umbilical cord tissue was placed in a 5% CO2, 2% O2 incubator at 37°C, 5 mL of complete culture medium 1 was added to the culture bottle, and 10 mL of complete culture medium 1 was added after overnight culture. Thereafter, the culture medium was changed every 3-5 days. After the cell fusion degree reached 80%, the cells were digested with pancreatic enzyme digestion solution to obtain P0 generation human umbilical cord mesenchymal stem cells.

[0097] (2) The P0 generation human umbilical cord mesenchymal stem cells obtained in step (1) were centrifuged at 300-400 g x 5 min, the supernatant was discarded, and complete culture medium 2 with a H2 concentration of 1.5 mg / L was added, and the cells were subcultured in an incubator at 37° C. in a 5% CO2 + 10% O2 environment for 72 hours, which was recorded as P1 generation. The preparation method of complete culture medium 2 with a H2 concentration of 1.5 mg / L was the same as in Example 1.

[0098] (3) The P1 human umbilical cord mesenchymal stem cells obtained in step (2) were centrifuged at 400 g for 5 min, the supernatant was discarded, complete culture medium 3 was added, and the cells were placed in an incubator at 37° C. with a 5% CO 2 environment and 18% O 2 for subculture for 72 hours, which was recorded as the P2 generation.

[0099] (4) The P2 human umbilical cord mesenchymal stem cells obtained in step (3) were centrifuged at 400 g for 5 min, the supernatant was discarded, a portion was tested, and the other portion was frozen, and the cells that passed the test were used as a seed bank.

[0100] (5) Take the seed bank cells described in step (4), continue to culture to P5 generation and freeze, the culture system and environment are: complete culture medium 3, 37°C, 5% CO2.

[0101] (6) The P5 mesenchymal stem cells frozen in step (5) are tested as a reference seed bank, and cells that pass the test are kept as a working bank, denoted as W2, and unqualified cells are discarded.

[0102] The complete culture medium 1 is: 10% v / v FBS, 10 μg / L bFGF, 5 μg / L EGF, 200 μg / L SDF-1α and DMEM / F12 (DF12) culture medium;

[0103] The complete culture medium 2 is: 10% v / v FBS, 10 μg / L bFGF, 5 μg / L EGF, 200 μg / L SDF-1α, 25 μg / L glutathione, 1.5 mg / L hydrogen and DMEM / F12 (DF12) culture medium;

[0104] The complete culture medium 3 comprises: 10% v / v FBS, 10 μg / L bFGF, 5 μg / L EGF, 200 μg / L SDF-1α, 5 mg / L vitamin C, 25 μg / L glutathione and DMEM / F12 (DF12) culture medium.

[0105] Comparative Example 1

[0106] The difference from Example 1 is that bFGF is not added to the complete culture medium 1-3, and the working bank cells are recorded as W3.

[0107] Comparative Example 2

[0108] The difference from Example 1 is that EGF is not added to the complete culture medium 1-3, and the working bank cells are recorded as W4.

[0109] Comparative Example 3

[0110] The difference from Example 1 is that EGF and bFGF are not added to the complete culture medium 1-3, and the working bank cells are recorded as W5.

[0111] Comparative Example 4

[0112] The difference from Example 1 is that glutathione is not added to the complete culture medium 3, and the working bank cells are recorded as W6.

[0113] Comparative Example 5

[0114] The difference from Example 1 is that the complete culture medium-2 does not contain H2, and the working bank cells are recorded as W7.

[0115] Comparative Example 6

[0116] The difference from Example 1 is that vitamin C is not added to the complete culture medium 3, and the working bank cells are recorded as W8.

[0117] Comparative Example 7

[0118] The difference from Example 1 is that vitamin C and glutathione are not added to the complete culture medium 3, and the working bank cells are recorded as W9.

[0119] Experimental Example 1 Detection of Mesenchymal Stem Cell Working Bank Cells

[0120] The mesenchymal stem cell working banks W1-W9 prepared above were taken for testing of cell morphology, cell phenotype, cell proliferation, osteogenic and adipogenic differentiation ability, biological efficacy (HGF, KGF, IGF-1); and bacterial, mycoplasma, and viral safety.

[0121] 1.1 Cell morphology detection

[0122] Take the working bank cells W1-W9 and add 1×10 4 / cm2 were inoculated into T75 culture flasks and cultured at 37°C and 5% CO2. The cell status was observed using an inverted microscope. The measurement results were as follows: Figure 1 It showed that W1-W9 cells were in good condition and the cells were spindle-shaped.

[0123] 1.2 Cell flow cytometry phenotyping

[0124] The working bank cells W1-W9 obtained in Examples 1-2 and Comparative Examples 1-7 were subjected to the following operations: 5×10 6 Umbilical cord mesenchymal stem cells were evenly divided into 10 centrifuge tubes and centrifuged at 300 g for 5 minutes, then the supernatant was discarded. The following antibodies were added respectively: PE-IgG1, PE-CD73, PE-CD90, PE-CD105, PE-CD45, PE-HLA-DR, PE-CD11b, FITC-IgG1, FITC-CD34, and FITC-CD19. Cell surface antigens were detected using a BD Calibur flow cytometer.

[0125] The results are shown in Table 2: Mesenchymal stem cell working bank cells W1-W10 all highly expressed CD73, CD90, and CD105, but did not express CD19, CD34, CD45, CD11b, and HLA-DR.

[0126] Table 2 Cell flow cytometry phenotyping results

[0127]

[0128] 1.3 Cell proliferation assay

[0129] Take W1-W9 working bank cells and inoculate 5000 cells per well into a 96-well plate. Use CCK-8 method to detect cell proliferation at 0h, 24h, 48h, 72h, and 96h after inoculation. The results are as follows: Figure 2 The results showed that W1 and W2 had the strongest cell proliferation ability, among which W1 had the strongest proliferation ability, followed by W6, W4 and W8, while W3 and W7 had poorer cell proliferation ability. Among the remaining W5 and W9 groups, W9 had the poorest cell proliferation ability.

[0130] 1.4 Osteogenic and adipogenic differentiation ability detection

[0131] The working bank cells W1-W9 obtained in Examples 1-2 and Comparative Examples 1-7 were used for the following operations: 2×10 4 / well inoculated in a 24-well plate, and when the cell confluence reaches about 80%, the adipogenic and osteogenic induction medium is replaced every 2-3 days. On the 21st day of induction culture, the cells are fixed with 4% paraformaldehyde solution at room temperature for 30 minutes, and then stained with 10% Oil Red-O to observe the adipogenic differentiation under a microscope, and the osteogenic differentiation is observed under a microscope after staining with Alizarin Red solution. The measurement results are shown in Figure 3 As shown, each group of cells has the ability to differentiate into osteoblasts and adipocytes. Among them, the mesenchymal stem cells of W1 and W2 have the strongest ability to differentiate into osteoblasts and adipocytes, followed by W4, W6 and W8. The differentiation ability of cells in W3 and W7 groups is weaker, and the differentiation ability of mesenchymal stem cells in W5 and W9 groups is the weakest.

[0132] 1.5 Biological efficacy test

[0133] The working bank cells W1-W9 obtained in Examples 1-2 and Comparative Examples 1-7 were taken at 1×10 4 / cm 2 Inoculate 6-well plates, harvest the supernatant after 72 hours of culture, and digest the cells and count them. The ELISA method was used to detect the content of HGF, KGF, and IGF-1 in the supernatant according to the instructions. The results are shown in Table 3. The mesenchymal stem cells in the working bank W1-W9 can secrete HGF, KGF, and IGF-1. Among them, W1 and W2 secrete the highest HGF, KGF, and IGF-1, followed by W4, W6, and W8, and W3 and W7 are relatively low. W5 and W9 secrete the lowest HGF, KGF, and IGF-1. Compared with W5, W9 has the lowest factor secretion.

[0134] Table 3 Results of HGF, KGF, and IGF-1 in the mesenchymal stem cell working bank

[0135]

[0136]

[0137] 1.6 Sterility, mycoplasma and virus safety testing

[0138] The working bank cells W1-W10 obtained in Examples 1-2 and Comparative Examples 1-5 were subjected to safety tests for bacteria, mycoplasma, viruses, etc. The specific detection methods are as follows:

[0139] Sterility testing was performed according to the sterility test method in General Rules 1101 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0140] Mycoplasma testing was performed according to the first method in General Rules 3301 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0141] According to the kit instructions, PCR method was used to complete the detection of EBV, HCMV, HIV and other viruses in the working library cells.

[0142] The test results showed that bacteria, mycoplasma, virus and other tests were all negative.

[0143] Experimental Example 2 Treatment of TNBS-induced IBD model with umbilical cord mesenchymal stem cell injection

[0144] 2.1 Preparation of Mesenchymal Stem Cell Injection 1, 2 (P1, P2):

[0145] The mesenchymal stem cell working bank cells W1 and W2 obtained in Example 1 and Example 2 were resuscitated and cultured to 80% cell confluence, then digested with trypsin, centrifuged, and washed with a compound electrolyte solution three times. The cells were resuspended in a compound electrolyte solution containing 10 mg / mL human albumin and 50 IU / mL heparin to prepare mesenchymal stem cell injection 1 (P1) and mesenchymal stem cell injection 2 (P2), respectively.

[0146] 2.2 Construction of TNBS-induced IBD model

[0147] 60 8-week-old male SD rats were fasted for 24 hours. After anesthesia, a 2mm diameter rubber hose was inserted into the colon 8cm from the anal verge, and 0.5mL of TNBS ethanol solution was slowly pushed in. The TNBS dose was 30mg / mL of 40% ethanol solution, which was given once a week for 7 consecutive weeks. A satellite group (3 rats) was set up. At the 5th week of modeling, the colon was dissected for gross observation to determine whether the modeling was successful.

[0148] 2.3 Animal grouping and drug administration

[0149] After the model was established, the rats in the model group were randomly divided into 4 groups according to the DAI score: model group (G3), mesenchymal stem cell injection treatment group (G4: P1 group, G5: P2 group) and positive drug group (G6: mesalazine sustained-release granules gavage), with 12 rats in each group. A sham operation group (saline solution enema, G1, N = 10) and an ethanol control group (ethanol solution enema, G2, N = 12) were also set up. The hUC-MSC injection treatment group was intravenously injected with 1×10 7 / kg dose), the sham operation group, ethanol control group and model group were intravenously injected with equal volume of normal saline at the same time, once a week, for a total of 2 times; the positive drug group was given mesalazine sustained-release granules (0.155g / kg dose) by gavage every day from the 6th week for two consecutive weeks; samples were collected at the 8th week.

[0150] Table 4 Animal grouping and dosing regimen

[0151]

[0152] 2.4 Test indicators and results

[0153] 2.4.1 General observations and mortality statistics

[0154] The rats in the sham operation group had shiny fur, good mental state, normal water and food intake, and no death was observed. The rats in the model group had diarrhea, bloody stools, semi-loose stools, redness and swelling around the anus, and the cage litter was obviously damp, with semi-loose stools, laziness, dull fur, less food and water intake, and weight loss the next day after TNBS modeling.

[0155] After the last modeling, 3 rats died in the model group, 1 rat died in each of the hUC-MSC (P1, P2) groups, and 3 rats died in the positive drug mesalazine group. The dead rats showed obvious blood in the stool and obvious inflammatory changes in the colon. The cause of death was considered to be severe intestinal inflammation. At the end of the experiment, the survival rates of the sham operation group, ethanol control group, model group, hUC-MSCP1, P2 group, and mesalazine group were 100%, 100%, 75%, 91.67%, 91.67%, and 75%, respectively; the survival rate of animals in the hUC-MSC (P1, P2) group was higher than that in the model group and the positive drug group. See Table 5 for details. Figure 4 .

[0156] Table 5 Effect on the survival rate of rats (Mean±SD, n=9~12)

[0157] Group Survival number / total number Survival rate% Sham operation group 10 / 10 100.00 Ethanol control group 12 / 12 100.00 Model Group 9 / 12 75 hUC-MSC P1 group 11 / 12 91.67 hUC-MSC P2 group 11 / 12 91.67 Mesalazine group 9 / 12 75

[0158] 2.4.2 Weight and DAI score

[0159] (1) Body weight: Weigh once before and after administration.

[0160] (2) DAI score: Before and after drug administration, the evaluation was conducted once a week. The scoring method is shown in Table 6:

[0161] Table 6 DAI scoring method

[0162] Scoring Weight loss% Stool consistency score Blood in stool score 0 0 normal Negative 1 1-5 Soft stools occult blood 2 5-10 Heavy, semi-loose stools Occult blood 3 10-15 Heavy, mucous stools Obvious occult blood 4 ＞15 Thin liquid stool Naked bloody stool

[0163] Note: If there is mucus or feces in the anal fur area, it is considered as diarrhea. If there is no obvious sign of diarrhea, it is scored as normal (0 point). If there is diarrhea, it is scored as semi-loose stool (2 points). If the diarrhea covers the skin from the tail to the abdomen, it is considered as massive diarrhea and scored as loose stool (4 points).

[0164] The results are as follows Figure 5 As shown, at 1 week of administration, the body weight of the rats in the model group was significantly reduced compared with that of the rats in the ethanol control group one week after modeling (P<0.05); at 2 weeks of administration, the body weight of the rats in each treatment group increased, but no significant difference was observed, among which the body weight of the rats in the hUC-MSC P1 group increased by 5.8% to 7.0%.

[0165] like Figure 6 As shown, compared with the sham operation group, the DAI score of rats in the ethanol control group at 0w of administration was significantly increased; compared with the sham operation group and the ethanol control group, the DAI score of rats in the model group was significantly increased at all observation time points (P<0.05 or P<0.001); compared with the model group, the DAI score of rats in the hUC-MSC P1 group was significantly decreased at 1 and 2w of administration (P<0.05 or P<0.01).

[0166] 2.4.3 Colon macroscopic scoring

[0167] Colon macroscopic scoring: including rectum and cecum. At the end of the test, the colon of the rat was dissected, 10 cm of the intestine was cut from the anus upward, cleaned, scored, and photographed. The scoring method is shown in Table 7:

[0168] Table 7 Colonic mucosal macroscopic scores

[0169] Scoring Scoring Criteria 0 healthy 1 Local congestion, slight thickening, no ulcer 2 Congestion, marked thickening, no ulceration 3 Ulcers and inflammation are only seen in one area 4 Ulcers and inflammation in two or more areas 5 Ulcers and inflammation in two or more areas or ulcer lesions > 1 cm in length 6-10 If the length of the ulcer lesion is > 2 cm, one point will be added for every additional cm.

[0170] The results are as follows Figure 7 As shown in the figure, compared with the model group, the colon macroscopic scores of rats in the hUC-MSC P1, P2 and mesalazine groups were significantly reduced (P<0.001). Among them, the hUC-MSC P1 group had the best effect in reducing the colon macroscopic scores of rats (P<0.01).

[0171] 2.4.4 Colon weight and intestinal coefficient testing

[0172] After the test period, the colon weight was measured.

[0173] After washing, the mice were weighed and the intestinal coefficient was calculated: intestinal coefficient = colon weight / body weight × 100%.

[0174] The results are as follows Figure 8 , 9 As shown in the figure, compared with the sham operation group, the colon weight and intestinal coefficient of rats in the ethanol control group, model group, hUC-MSC P1, P2 group, and mesalazine group were significantly increased (P<0.01 or P<0.001). Compared with the model group, the colon weight and intestinal coefficient of rats in the hUC-MSCP1, P2 group, and mesalazine group were significantly decreased (P<0.01 or P<0.001). Among them, the hUC-MSCP1 group had the best therapeutic effect on the colon weight and intestinal coefficient of rats.

[0175] Experimental Example 3 Treatment of CCl4-induced chronic liver injury model with umbilical cord mesenchymal stem cell injection

[0176] 3.1 Preparation of injection solution:

[0177] Prepare the injection solution according to 2.1.

[0178] 3.2 Construction of CCl4-induced chronic liver injury model

[0179] Sixty 8-week-old male SD rats were selected. In addition to 10 normal control group animals given the solvent (corn oil), 50 model induction group animals were given 20% CCl4 / corn oil solution by intraperitoneal injection, the injection volume was 5mL / kg, twice a week for 6 consecutive weeks, and the first model induction day was D1. During the model induction period, all experimental animals were routinely fed with normal diet and drinking water.

[0180] 3.3 Treatment groups:

[0181] After two weeks of CCl4 induction, the model-induced animals were randomly divided into three groups according to body weight parameters, namely, model control group, hUC-MSCs injection treatment group (P1, P2 group); hUC-MSC treatment was started from the third week, the treatment frequency was once every two weeks, a total of 3 times, and the treatment dose was 1×10 7 cells / kg, administered by tail vein injection, the administration volume was 10mL / kg; the normal control group and the model control group were given the same volume of normal saline at the same time; the 8th week of the experiment was the end point of the experiment.

[0182] 3.4 Animal weight

[0183] The animals were weighed twice a week for 6 consecutive weeks during the test period to evaluate the disease progression and therapeutic effects of the test article. Fig.10The results showed that the weight of animals in the normal control group showed normal and stable growth, while the weight growth of animals in the model control group was significantly inhibited after continuous CCl4 induction, which was statistically different from the normal control group during the same period (P<0.0001). The weight growth inhibition began to appear in the first week of CCl4 induction and continued until the end of the experiment. The hUC-MSCs injection treatment group (P1 and P2 groups) had no significant effect on the CCl4-induced weight growth inhibition under the set treatment plan. Compared with the animals in the model control group during the same period, the weight growth rate of animals in the hUC-MSC P1 and P2 treatment groups did not show significant improvement.

[0184] 3.5 Blood biochemical test

[0185] The experimental animals were anesthetized with sodium pentobarbital and blood samples were collected through the jugular vein or abdominal aorta. After the serum was separated, the liver function ALT, AST, ALB, ALP and other tests were performed using a blood biochemical analyzer. The blood biochemical test results before treatment were as follows: Fig.11 The results showed that the animals in the model control group showed obvious liver damage after continuous CCl4 induction for 2 weeks. The serum ALT, AST and T-BIL levels were significantly higher than those in the normal control group during the same period, and the differences were statistically significant (P<0.0001). At the same time, the serum ALP and ALB levels also increased to a certain extent, but the difference was not statistically significant (P>0.05). Compared with the normal control group during the same period, the animals in the hUC-MSCs injection treatment group (P1, P2) showed obvious liver damage before treatment, and the damage manifestations and degree were consistent with those of the model control group during the same period, showing a significant increase in serum ALT, AST and T-BIL levels, and a slight increase in ALP and ALB, the degree of increase was consistent with that of the model control group, indicating that the model animals had already shown progressive liver disease before group treatment, and the degree of liver lesions in each experimental animal was basically the same.

[0186] The blood biochemical results of the mid-term hUC-MSCs treatment showed that the liver damage of the animals in the model control group continued to progress, and their serum ALT, AST, ALP and T-BIL levels increased significantly, while the serum ALB level decreased significantly. The degree of change was statistically significant compared with the animals in the normal control group during the same period, indicating that the animals in the model control group had progressive liver damage and jaundice at this stage. The hUC-MSCs injection treatment group (P1, P2) had a significant improvement on the liver damage process of the model animals under the set treatment plan. Compared with the model control group during the same period, the serum ALT, AST, ALP and T-BIL of the animals in the hUC-MSCs injection treatment group (P1, P2) decreased to a certain extent, while the serum ALB increased. The treatment degree of the hUC-MSCs injection P1 treatment group was the most obvious.

[0187] The results of blood biochemical tests at the end of hUC-MSCs treatment showed that at the end of the experiment, the liver damage and jaundice symptoms of the animals in the model control group continued to progress, and their serum ALT, AST, ALP and T-BIL levels continued to increase, while the serum ALB level decreased significantly, and the degree of change was statistically significant compared with the normal control group animals in the same period. The hUC-MSCs injection treatment group (P1, P2) had a significant therapeutic effect on the liver damage process of the model animals under the set treatment plan for 4 weeks of continuous intervention. Compared with the model control group in the same period, the serum ALT, AST, ALP and T-BIL of the animals in the hUC-MSCs injection treatment group (P1, P2) decreased significantly, and the serum ALB showed a significant increase. The degree of improvement was statistically different, and the therapeutic effect of the hUC-MSCs injection P1 treatment group was more obvious.

[0188] 3.6 Liver histopathology

[0189] Tissue sampling: liver tissues of all experimental animals planned for measurement;

[0190] Tissue processing: 10% formaldehyde fixation, paraffin embedding, cutting into 3μm thick sections, then dewaxing in xylene, hydrating with gradient ethanol, H&E staining, dehydration, transparency, sealing, observation and photography of liver tissue pathological changes under a microscope; Pathological diagnosis and analysis: Based on the results of liver H&E staining, liver cell necrosis, inflammation, fibrosis and related lesions of animals in each test group were analyzed. Fig.12 The results showed that the animals in the model control group developed obvious histological features of cirrhosis (pseudolobular structure and extensive fibrous bridging) after 6 weeks of continuous CCl4 induction. Their liver necrosis, inflammatory infiltration, fibrosis score, liver collagen volume fraction and liver HYP content were significantly increased compared with those of the animals in the normal control group during the same period, and the differences were statistically significant.

[0191] Compared with the degree of liver histopathological lesions in the model control group of the same period, the degree of liver necrosis, inflammatory infiltration and fibrosis in the hUC-MSCs injection treatment group (P1, P2) showed significant relief. Under the same diagnostic analysis conditions, the diagnostic scores of various histological lesions and the degree of fibrosis in the liver of the hUC-MSCs injection treatment group (P1, P2) showed a significant decrease. The improvement effects on liver confluent necrosis, inflammatory infiltration, liver fibrosis score and hydroxyproline level were statistically significant. The improvement of liver collagen volume fraction was only statistically significant under the treatment conditions of hUC-MSCs injection treatment group P1. Among them, the efficacy of hUC-MSCs injection treatment group P1 was the most obvious.

Claims

1. A method for preparing mesenchymal stem cells, characterized in that: The steps of the preparation method are as follows: (1) incubating the treated tissue and adding complete culture medium 1 for culturing; culturing until the cell confluence reaches 70-90%, digesting with digestion solution to obtain P0 generation mesenchymal stem cells; (2) centrifuging the P0 generation mesenchymal stem cells, discarding the supernatant, and adding complete culture medium 2 for the first subculture to obtain P1 generation mesenchymal stem cells; (3) centrifuging the P1 generation mesenchymal stem cells, discarding the supernatant, and adding complete culture medium 3 for a second subculture to obtain P2 generation mesenchymal stem cells; (4) centrifuging the P2 mesenchymal stem cells, discarding the supernatant, harvesting the mesenchymal stem cells, and obtaining seed bank cells; (5) adding the seed bank cells to complete culture medium 3 and culturing to P5 to obtain mesenchymal stem cells; The complete culture solution 1 is composed of FBS, bFGF, EGF, SDF-1α and basal culture medium; The complete culture medium 2 consists of FBS, bFGF, EGF, SDF-1α, glutathione, hydrogen and basal culture medium; The complete culture medium 3 consists of FBS, bFGF, EGF, SDF-1α, vitamin C, glutathione and basal culture medium.

2. The preparation method according to claim 1, characterized in that: The culture conditions in step (1) include 5% CO2, 1%-2% O2; The conditions of the first subculture in step (2) include 5% CO2, 5%-10% O2; The conditions of the second subculture in step (3) include 5% CO2 and 18%-20% O2; The culture conditions in step (5) include 5% CO2.

3. The preparation method according to claim 1, characterized in that: The basic culture medium includes one or more of MesenCult-XFMedium, Essential 8 Medium, StemPro hESC SFM, CTS Essential 6 Medium, DMEM / F12 or DMEM.

4. The preparation method according to claim 3, characterized in that: The basic culture medium is DMEM / F12.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The complete culture solution 1 consists of 10-15% v / v FBS, 5-10 μg / L bFGF, 5-10 μg / L EGF, 100-200 μg / L SDF-1α and DMEM / F12 culture medium; The complete culture solution 2 consists of 10-15% v / v FBS, 5-10 μg / L bFGF, 5-10 μg / L EGF, 100-200 μg / L SDF-1α, 1-25 μg / L glutathione, 0.5-1.5 mg / L hydrogen and DMEM / F12 culture medium; The complete culture medium 3 consists of 10-15% v / v FBS, 5-10 μg / L bFGF, 5-10 μg / L EGF, 100-200 μg / L SDF-1α, 5-10 mg / L vitamin C, 1-25 μg / L glutathione and DMEM / F12 culture medium.

6. The preparation method according to claim 5, characterized in that: The mesenchymal stem cells are derived from bone marrow, adipose tissue, umbilical cord, umbilical cord blood or placental tissue.

7. The preparation method according to claim 6, characterized in that: The mesenchymal stem cells are derived from the umbilical cord.

8. Mesenchymal stem cells obtained by the preparation method according to any one of claims 1 to 6.

9. The mesenchymal stem cell according to claim 8, characterized in that The concentration of the mesenchymal stem cells is 1×10 6 -1×10 8 cells / mL.

10. A culture, characterized in that The culture comprises the mesenchymal stem cells according to claim 8 or 9, and the biological efficacy detection indexes thereof comprise the levels of HGF, IGF-1 and KGF secreted by the mesenchymal stem cells.

11. Use of the mesenchymal stem cells according to claim 8 or 9 or the culture according to claim 10 in the preparation of a medicament for treating a digestive system disease.

12. The use according to claim 11, characterized in that: The digestive system diseases include inflammatory bowel disease, chronic liver fibrosis and acute liver failure.

13. The use according to claim 11, characterized in that: The dosage form of the drug is injection, gel, cell membrane, patch, microsphere, microparticle or composite material.

14. The method for quality control of mesenchymal stem cells according to claim 8 or 9, characterized in that: The method comprises performing one or more of the following tests on the cell morphology, cell flow cytometry phenotype, cell proliferation, osteogenic and adipogenic differentiation ability or biological efficacy of mesenchymal stem cells.

15. The quality control method according to claim 14, characterized in that: The cell flow cytometry phenotype includes one or more of CD73, CD90, CD105, CD19, CD34, CD45, CD11b or HLA-DR.

16. The quality control method according to claim 14, characterized in that: The biological efficacy assay includes one or more of HGF, KGF or IGF-1.

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