Method for separating and culturing umbilical cord-derived mesenchymal stem cells

Abstract

The invention provides a method for separating and culturing umbilical cord-derived mesenchymal stem cells. The method comprises the following steps: a, obtaining an umbilical cord; b, separating Wharton's jelly from the obtained umbilical cord; c, culturing the separated Wharton's jelly in a complete culture medium added with the peptide 1 and the peptide 2; d, digesting the primarily cultured mesenchymal stem cells by using an enzyme; and e, carrying out subculture on the enzyme digested mesenchymal stem cells. The mesenchymal stem cells separated from the umbilical cord are high in uniformity and high in activity.

Description

Technical Field

[0001] The present invention relates to the field of mesenchymal stem cell preparation, and in particular to a method for separating and culturing umbilical cord stem cells from umbilical cords. Background Art

[0002] Mesenchymal stem cells can come from different tissues, such as skin, internal organs (such as liver), placenta, fat, bone marrow, dental pulp, peripheral blood, umbilical cord blood, and umbilical cord. Red blood cells, white blood cells, and platelets are continuously added to the blood. However, the specific molecular phenotypes, cell activity, and differentiation potential of mesenchymal stem cells from different tissue sources are different. Umbilical cord-derived mesenchymal stem cells highly express CD44 and CD105, but lowly express CD34 and CD45.

[0003] Umbilical cord is one of the many sources of mesenchymal stem cells, which has the characteristics of easy collection, low immunogenicity, strong growth capacity and multidirectional differentiation. At present, mesenchymal stem cells derived from umbilical cord have been widely used in the treatment of many diseases and have achieved good results.

[0004] Umbilical cord-derived mesenchymal stem cells can migrate to damaged areas in the body and specifically differentiate into tissue cells in that area, while releasing a large number of active factors or exosomes, thereby repairing damaged areas and regulating immune capacity. Compared with skin or fat-derived stem cells, umbilical cord-derived stem cells have the advantages of high activity, strong division ability, and high safety.

[0005] The umbilical cord is shaped like a rope, with a smooth and transparent surface. It contains connective tissue, an umbilical vein, and a pair of umbilical arteries. It is the connecting structure between the fetus and the placenta. Umbilical cord mesenchymal stem cells (hUC-MSCs) are a type of mesenchymal stem cells that exist in the Wharton's jelly and perivascular tissues of the umbilical cord.

[0006] Obtaining mesenchymal stem cells from the umbilical cord is a way to turn waste into treasure because it is easy to obtain, and there is no moral or ethical controversy. The mesenchymal cells that can be obtained from the umbilical cord are large in number, have strong proliferation ability, have a strong immune regulation effect, and secrete a high amount of active factors.

[0007] Umbilical cord mesenchymal stem cells have a good therapeutic effect on advanced liver diseases. Umbilical cord mesenchymal stem cells can differentiate into hepatocyte-like cells in damaged liver tissue, and can also produce active factors or exosomes in damaged liver tissue. These active factors or exosomes can promote endogenous hepatocyte proliferation and angiogenesis by regulating the body's immune response, resisting hepatocyte apoptosis, and resisting liver fibrosis.

[0008] In addition, umbilical cord mesenchymal stem cells can treat autoimmune diseases such as systemic lupus erythematosus, atopic dermatitis, etc. Umbilical cord mesenchymal stem cells can regulate immune function, prevent the release of inflammatory factors, and then regulate various immune cells and inhibit the excessive activity of the immune system.

[0009] Umbilical cord mesenchymal stem cells can also be used to treat diseases including diabetes, arteriosclerosis obliterans, thrombosis occlusive disease, etc. Umbilical cord mesenchymal stem cells can differentiate into endothelial cells and smooth muscle cells, and home to damaged blood vessels, where they directly differentiate into new blood vessels.

[0010] Furthermore, replacing mesenchymal stem cells has the effect of neuroprotection and promoting neural regeneration, and is particularly important in the treatment of diseases such as neurodegeneration and Alzheimer's disease, so as to reduce disease activity and lower the levels of indicators such as proteinuria, blood creatinine, and urea nitrogen.

[0011] How to prepare mesenchymal stem cells with high homogeneity and activity from umbilical cord is a key factor in the therapeutic effect of mesenchymal stem cells. So far, many countries and regions have built umbilical cord mesenchymal cell banks that meet the standards. In the prior art, mesenchymal stem cells isolated from umbilical cord are usually not homogeneous and their activity decreases with the increase of passage number. Summary of the invention

[0012] The present inventors have overcome the above-mentioned shortcomings through in-depth research and provided a method for isolating and culturing mesenchymal stem cells from umbilical cords with simple operation, high uniformity and strong activity.

[0013] Specifically, in a first aspect, the present invention provides a novel method for preparing mesenchymal stem cells from umbilical cord, wherein the separated Wharton's jelly is cultured in a culture medium supplemented with peptide 1 (KSKCVRDI SHYL) and / or peptide 2 (VAALEFVRDNNGL), and the mesenchymal stem cells are isolated.

[0014] The novel method for preparing mesenchymal stem cells of the present invention further comprises any one, two, three, four or five of the following steps, or any combination thereof:

[0015] a. Obtain the umbilical cord;

[0016] b. separating Wharton's jelly from the harvested umbilical cord;

[0017] c. culturing the isolated Wharton's jelly in complete medium supplemented with peptide 1 and peptide 2;

[0018] d. digesting primary cultured mesenchymal stem cells with enzymes;

[0019] e. Subculturing the enzyme-digested mesenchymal stem cells.

[0020] In an embodiment of the present invention, the concentration of peptide 1 is 1-100 μg / mL: the concentration of peptide 2 is 1-200 μg / mL. Exemplarily, the concentration of peptide 1 is 5-50 μg / mL, for example, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 μg / mL. L; the concentration of peptide 2 is 10-200 μg / mL, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 μg / mL.

[0021] Further preferably, the concentration of peptide 1 is 5 to 30 μg / mL, and the concentration of peptide 2 is 20 to 60 μg / mL. Exemplarily, when the concentration of peptide 1 is 5 to 30 μg / mL, the concentration of peptide 2 is 10, 20, 30, 40, 50, 60 μg / mL. Alternatively, when the concentration of peptide 2 is 10 to 60 μg / mL, the concentration of peptide 1 is 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30 μg / mL. The concentrations of peptide 1 and peptide 2 can be arbitrarily combined, for example, the concentration of peptide 1 is 10 μg / mL, and the concentration of peptide 2 is 20 μg / mL; or, the concentration of peptide 1 is 20 μg / mL, and the concentration of peptide 2 is 40 μg / mL.

[0022] Furthermore, the complete culture medium is DMEM / F-12 culture medium.

[0023] In the complete medium, the inoculation density of subculture is 1000-5X10 5 ce ll / cm 2 , preferably 5000 cel l / cm 2 .

[0024] In step a, the umbilical cord is obtained from the mother with consent and approved by the ethics committee of the hospital.

[0025] In step b, the umbilical cord is first washed with PBS or saline, then disinfected with 75% alcohol, and then washed with PBS or saline to remove residual alcohol. The umbilical cord is cut into small pieces and washed with PBS or saline until there is no blood stain. The umbilical vein and two umbilical arteries are then removed to separate the Wharton's jelly.

[0026] In step c, after adding peptide 1 and peptide 2 to the complete medium, peptide 1 and peptide 2 should be thoroughly mixed with the complete medium.

[0027] In step d, the enzyme digestion is trypsin digestion, so that the cells are separated into single cells.

[0028] In step e, the subculture is carried out at 37°C and 5% CO 2 Culture in an incubator.

[0029] Optionally, single mesenchymal stem cells isolated from the umbilical cord are cultured adherently and isolated and purified when the confluence reaches 70-90%.

[0030] The method for preparing mesenchymal stem cells adopted in the present invention will not cause any interference to the subject, and thus has good compliance for the subject, because, compared with other approaches, the method of the present invention will not cause any damage to the body of the subject.

[0031] Current research has found that mesenchymal stem cells isolated from umbilical cord samples have better vitality. Moreover, many umbilical cords will be discarded if they are not collected. Therefore, isolating mesenchymal stem cells from umbilical cords is also a desirable way to turn waste into treasure.

[0032] In a second aspect, the present invention further provides a mesenchymal stem cell, which is prepared by the culture and separation method of the present invention.

[0033] Optionally, the stem cells are mesenchymal stem cells that are further passaged and purified from mesenchymal stem cells prepared by primary culture and separation. Preferably, the passage number is 6-10 generations, more preferably 6 generations.

[0034] In a third aspect, the present invention provides use of peptide 1 and / or peptide 2 in preparing a kit for isolating mesenchymal stem cells from umbilical cord.

[0035] Illustratively, the present invention provides use of peptide 1 in preparing a kit for isolating mesenchymal stem cells from umbilical cord.

[0036] Furthermore, the present invention provides the use of peptide 2 in preparing a kit for isolating mesenchymal stem cells from umbilical cord.

[0037] Alternatively, the present invention provides a use of a combination of Peptide 1 and Peptide 2 in preparing a kit for isolating mesenchymal stem cells from umbilical cord.

[0038] Illustratively, the present invention further provides a kit or culture medium comprising peptide 1 and peptide 2 provided in combination, wherein the ratio (mass) of peptide 1 to peptide 2 can be: about 1:0.01 to about 1:99, preferably about 1:1, about 1:1.5, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90 and the range of ratios between the ratios; or about 0.1:1 to about 100:1, for example, about 0.1:1, about 0.2:1 , about 0.3:1, about 0.4:1, about 0.45:1, about 0.5:1, about 0.55:1, about 0.6:1, about 0.65:1, about 0.7:1, about 0.75:1, about 0.8:1, about 0.85:1, about 0.9:1, about 0.95:1, about 1:1, about 1:1.1, about 1.2:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, and ranges of ratios between the above ratios. Preferably, the mass ratio of peptide 1 to peptide 2 is 1:1-1:3, more preferably 1:2.

[0039] Further, the present invention also provides a kit or culture medium, which comprises peptide 1 and peptide 2 provided separately, wherein the ratio (mass) of peptide 1 to peptide 2 can be: about 1:0.01 to about 1:99, preferably about 1:1, about 1:1.5, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90 and the ratio range between the said ratios; or about 0.1:1 to about 100:1, for example, about 0.1:1, about 0.2:1 , about 0.3:1, about 0.4:1, about 0.45:1, about 0.5:1, about 0.55:1, about 0.6:1, about 0.65:1, about 0.7:1, about 0.75:1, about 0.8:1, about 0.85:1, about 0.9:1, about 0.95:1, about 1:1, about 1:1.1, about 1.2:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, and ranges of ratios between the above ratios. Preferably, the mass ratio of peptide 1 to peptide 2 is 1:1-1:3, more preferably 1:2.

[0040] In a fourth aspect, the present invention provides a complete culture medium containing peptide 1 and peptide 2.

[0041] In one embodiment, the concentration of peptide 1 is 1-100 μg / mL: the concentration of peptide 2 is 1-200 μg / mL. Exemplarily, the concentration of peptide 1 is 5-50 μg / mL, for example 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 μg / mL L; the concentration of peptide 2 is 10-200 μg / mL, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 μg / mL.

[0042] Further preferably, the concentration of peptide 1 is 5 to 30 μg / mL, and the concentration of peptide 2 is 20 to 60 μg / mL. Exemplarily, when the concentration of peptide 1 is 5 to 30 μg / mL, the concentration of peptide 2 is 10, 20, 30, 40, 50, 60 μg / mL. Alternatively, when the concentration of peptide 2 is 10 to 60 μg / mL, the concentration of peptide 1 is 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30 μg / mL. The concentrations of peptide 1 and peptide 2 can be arbitrarily combined, for example, the concentration of peptide 1 is 10 μg / mL, and the concentration of peptide 2 is 20 μg / mL; or, the concentration of peptide 1 is 20 μg / mL, and the concentration of peptide 2 is 40 μg / mL.

[0043] In a fifth aspect, the present invention also provides a composition comprising peptide 1 and peptide 2.

[0044] Exemplarily, the ratio (mass) of peptide 1 to peptide 2 can be: about 1:0.01 to about 1:99, preferably about 1:1, about 1:1.5, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90 and the range of ratios therebetween; or about 0.1:1 to about 100:1, for example, about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1, about 0.7:1, about 0.8:1, about 0.9:1, about 10:10 1, about 0.45:1, about 0.5:1, about 0.55:1, about 0.6:1, about 0.65:1, about 0.7:1, about 0.75:1, about 0.8:1, about 0.85:1, about 0.9:1, about 0.95:1, about 1:1, about 1:1.1, about 1.2:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, and the range of ratios between the above ratios. Preferably, the mass ratio of peptide 1 to peptide 2 is 1:1-1:3, more preferably 1:2.

[0045] In a sixth aspect, the present invention provides use of mesenchymal stem cells separated or cultured by the method of the present invention in diseases.

[0046] In a specific embodiment, the umbilical cord mesenchymal stem cells of the present invention can differentiate into hepatocyte-like cells in damaged liver tissue, and can also produce active factors or exosomes in damaged liver tissue. These active factors or exosomes can promote endogenous hepatocyte proliferation and angiogenesis by regulating the body's immune response, resisting hepatocyte apoptosis, and resisting liver fibrosis.

[0047] In addition, the umbilical cord mesenchymal stem cells of the present invention can treat autoimmune diseases, such as systemic lupus erythematosus, atopic dermatitis, etc. Umbilical cord mesenchymal stem cells can regulate immune function, prevent the release of inflammatory factors, and then regulate various immune cells and inhibit the excessive activity of the immune system.

[0048] The umbilical cord mesenchymal stem cells of the present invention can also be used to treat diseases including diabetes, arteriosclerosis obliterans, thrombosis occlusive disease, etc. Umbilical cord mesenchymal stem cells can differentiate into endothelial cells and smooth muscle cells, and home to damaged blood vessels, and directly differentiate into new blood vessels therein.

[0049] Furthermore, the umbilical cord mesenchymal stem cells of the present invention can also be used for neuroprotection and promotion of neural regeneration, especially for the treatment of diseases such as neurodegeneration and Alzheimer's disease, so as to reduce disease activity and lower the levels of indicators such as proteinuria, blood creatinine, and urea nitrogen.

[0050] The mesenchymal stem cell population of the present invention can be formulated and administered as a pharmaceutical composition. Exemplarily, the pharmaceutical composition is any form known in the medical field, such as an injection (e.g., an injection, a lyophilized powder). In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution (e.g., an isotonic saline solution or a physiological saline solution), a dispersion, a suspension, or an emulsion.

[0051] In a seventh aspect, the present invention provides a drug for treating leukemia or immune diseases, comprising mesenchymal stem cells separated or cultured by the method of the present invention.

[0052] Exemplarily, the growth curve of the isolated mesenchymal stem cells is measured by WST method, MTT method, ATP detection method, DNA content detection method, etc. to evaluate the growth activity of umbilical cord mesenchymal stem cells. In addition, the isolated and cultured umbilical cord mesenchymal stem cells can be identified by detecting cell surface markers by flow cytometry and detecting cell expression genes by PCR.

[0053] In an eighth aspect, the present invention provides a pharmaceutical composition comprising the mesenchymal stem cells separated or cultured according to the present invention. In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient, or other auxiliary materials that may be required.

[0054] In this article, the term "excipient" refers to a substance other than the active ingredient (mesenchymal stem cells) that is necessary for the preparation or formulation of a pharmaceutical preparation. Generally speaking, these substances have no physiological activity and do not affect the efficacy and content determination of the drug in the pharmaceutical preparation, nor the stability of the pharmaceutical composition. The main purpose of adding an excipient is to facilitate the preparation of the pharmaceutical composition and facilitate clinical application. Preferably, the excipient used in the pharmaceutical composition of the present invention is medicinal and does not conflict with the active ingredient (mesenchymal stem cells).

[0055] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the mesenchymal stem cells.

[0056] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable biomaterial, including but not limited to collagen, gelatin, amino-modified gelatin, chitosan, cellulose, fibrin, polylactic acid, cellulose polylactic acid, polyurethane, tropoelastin, hyaluronic acid, sodium alginate, polyethylene glycol, polylactic acid glycolic acid, polycaprolactone, silicate, silicone rubber, extracellular matrix or any combination thereof, etc. In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution (e.g., isotonic saline solution or physiological saline), dispersion, emulsion or suspension.

[0057] In certain embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (e.g., injections, lyophilized powders), etc. In certain preferred embodiments, the pharmaceutical composition is an injection (including injections, lyophilized powders) or an aerosol.

[0058] Further illustratively, in the case where the pharmaceutical composition is an injectable preparation, in certain embodiments, the dosage of the mesenchymal stem cells is at least 10 5 / mL (e.g. at least 1×10 5 / ml, at least 3×10 5 / ml, at least 5×10 5 / ml, at least 7×10 5 / ml, at least 1×106 5 indivual

[0059] / ml, at least 3×10 6 / ml, at least 5×10 6 / ml, at least 7×10 6 / ml, at least 1×10 7 / ml, at least 3×10 7 / ml, at least 5×10 7 / ml, at least 7×10 7 / ml, at least 1×10 8 / ml, at least 3×10 8 / ml, at least 5×10 8 / ml, at least 7×10 8 / ml, at least 1×10 9 / ml, at least 3×10 9 / ml, at least 5×10 9 / ml, at least 7×10 9 Pieces / ml.

[0060] In certain embodiments, the subject for which the pharmaceutical composition is directed is a mammal, such as a human.

[0061] In a ninth aspect, the present invention provides a method for treating a disease, comprising administering a pharmaceutical composition comprising the mesenchymal stem cells of the present invention to a subject.

[0062] In some embodiments, the drug is in a unit dosage form, and the unit dosage (e.g., per milliliter) of the drug contains at least 1×10 4 (e.g. at least 1×10 4 At least 3×10 4 At least 5×10 4 At least 7×10 4 At least 1×10 5 At least 3×10 5 At least 5×10 5 At least 7×10 5 At least 1×10 6 At least 3×10 6 At least 5×10 6 At least 7×10 6 At least 1×10 7 At least 3×10 7 At least 5×10 7 At least 7×10 7 At least 1×10 8 At least 3×10 8 At least 5×10 8 At least 7×10 8 At least 1×10 9 At least 3×10 9 At least 5×10 9 At least 7×10 9 At least 1×10 10 At least 3×10 10 At least 5×10 10 or at least 7×10 10 , preferably 3 to 6 × 10 6 ) of the mesenchymal stem cells.

[0063] In certain embodiments, the subject is a mammal, such as a human.

[0064] In certain embodiments, a preventively and / or therapeutically effective amount of mesenchymal stem cells is administered to a subject by the following routes: injection, mucosal administration, cavity administration, oral administration, respiratory tract administration, or skin administration.

[0065] Beneficial Effects

[0066] Unexpectedly, the addition of peptide 1 and peptide 2 increased the activity of the mesenchymal stem cells obtained by the method of the present invention. DETAILED DESCRIPTION

[0067] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention are described in further detail below.

[0068] Example 1

[0069] Umbilical cord tissue was obtained from the fetuses of healthy pregnant women in the Department of Obstetrics and Gynecology of Renji Hospital. This study was approved by the Ethics Committee of the hospital and the informed consent of the umbilical cord donors. After the umbilical cord was collected, it was carefully placed in an umbilical cord preservation bottle (containing umbilical cord preservation solution) and stored at low temperature (4°C).

[0070] First, wash the umbilical cord three times with PBS, then disinfect it with 75% alcohol for 60 seconds, and then wash it three times with PBS to remove the residual alcohol. Cut the umbilical cord into small pieces of about 1-2 cm and wash it with PBS until there is no blood stain. Then remove the umbilical vein and two umbilical arteries to separate Wharton's jelly.

[0071] The separated Wharton's gel was cut into pieces and placed in a culture bottle with DMEM / F12 medium (containing 10% fetal bovine serum and 1% penicillin + 1% streptomycin) and incubated at 37°C and 5% CO. 2 The cells were cultured and subcultured in an incubator, and when the cells were full or 80% full, they were digested into single cells with trypsin and continued to be subcultured for 10 generations. In the primary culture, the complete medium contained 5 μg / mL peptide 1 (KSKCVRD I SHYL) and 10 μg / mL peptide 2 (VAALEFVRDNNGL), but not in the subculture. Flow cytometry was performed on the subcultured cells, and the results showed that most of the cells were in the G0 / G1 phase, with high cell division activity, and fewer cells in the S phase and G2 / M phase. As the number of generations increased, there was no significant increase before the 6th generation, and then gradually increased. The results are shown in Table 1.

[0072] Table 1. Results of subculture cycle detection of umbilical cord mesenchymal stem cells (mean ± SE, n = 3)

[0073]

[0074]

[0075] Example 2

[0076] Umbilical cord tissue was obtained from the fetuses of healthy pregnant women in the Department of Obstetrics and Gynecology of Renji Hospital. This study was approved by the Ethics Committee of the hospital and the informed consent of the umbilical cord donors. After the umbilical cord was collected, it was carefully placed in an umbilical cord preservation bottle (containing umbilical cord preservation solution) and stored at low temperature (4°C).

[0077] First, wash the umbilical cord three times with PBS, then disinfect it with 75% alcohol for 60 seconds, and then wash it three times with PBS to remove the residual alcohol. Cut the umbilical cord into small pieces of about 1-2 cm and wash it with PBS until there is no blood stain. Then remove the umbilical vein and two umbilical arteries to separate Wharton's jelly.

[0078] The separated Wharton's gel was cut into pieces and placed in a culture bottle with DMEM / F12 medium (containing 10% fetal bovine serum and 1% penicillin + 1% streptomycin) and incubated at 37°C and 5% CO. 2 The cells were cultured and passaged in an incubator, and when the cells were full or 80% full, they were digested with trypsin into single cells and continued to be passaged for 10 generations. In the primary culture, the complete medium contained 10 μg / mL peptide 1 (KSKCVRDI SHYL) and 20 μg / mL peptide 2 (VAALEFVRDNNGL), but not in the secondary culture. Flow cytometry was performed on the passaged cells, and the results showed that similar to Example 1, most of the cells were in the G0 / G1 phase (more than 95%) before the 6th generation, and the cell division activity was high. There were fewer cells in the S phase and G2 / M phase, which increased with the number of generations.

[0079] Example 3

[0080] Umbilical cord tissue was obtained from the fetuses of healthy pregnant women in the Department of Obstetrics and Gynecology of Renji Hospital. This study was approved by the Ethics Committee of the hospital and the informed consent of the umbilical cord donors. After the umbilical cord was collected, it was carefully placed in an umbilical cord preservation bottle (containing umbilical cord preservation solution) and stored at low temperature (4°C).

[0081] First, wash the umbilical cord three times with PBS, then disinfect it with 75% alcohol for 60 seconds, and then wash it three times with PBS to remove the residual alcohol. Cut the umbilical cord into small pieces of about 1-2 cm and wash it with PBS until there is no blood stain. Then remove the umbilical vein and two umbilical arteries to separate Wharton's jelly.

[0082] The separated Wharton's gel was cut into pieces and placed in a culture bottle with DMEM / F12 medium (containing 10% fetal bovine serum and 1% penicillin + 1% streptomycin) and incubated at 37°C and 5% CO. 2The cells were cultured and passaged in an incubator, and when the cells were full or 80% full, they were digested into single cells with trypsin and continued to be passaged for 10 generations. In the primary culture, the complete medium contained 50 μg / mL peptide 1 (KSKCVRDI SHYL) and 100 μg / mL peptide 2 (VAALEFVRDNNGL), but not in the secondary culture. Flow cytometry was performed on the passaged cells, and the results showed that only about 75% of the cells were in the G0 / G1 phase in the first 6 generations. The number of cells in the S phase and G2 / M phase increased significantly compared to low concentrations of polypeptides.

[0083] Example 4

[0084] Umbilical cord tissue was obtained from the fetuses of healthy pregnant women in the Department of Obstetrics and Gynecology of Renji Hospital. This study was approved by the Ethics Committee of the hospital and the informed consent of the umbilical cord donors. After the umbilical cord was collected, it was carefully placed in an umbilical cord preservation bottle (containing umbilical cord preservation solution) and stored at low temperature (4°C).

[0085] First, wash the umbilical cord three times with PBS, then disinfect it with 75% alcohol for 60 seconds, and then wash it three times with PBS to remove the residual alcohol. Cut the umbilical cord into small pieces of about 1-2 cm and wash it with PBS until there is no blood stain. Then remove the umbilical vein and two umbilical arteries to separate Wharton's jelly.

[0086] The separated Wharton's gel was cut into pieces and placed in a culture bottle with DMEM / F12 medium (containing 10% fetal bovine serum and 1% penicillin + 1% streptomycin) and incubated at 37°C and 5% CO. 2 The cells were cultured and passaged in an incubator, and when the cells were full or 80% grown, they were digested with trypsin into single cells and continued to be passaged for 10 generations. In the primary culture, the complete medium contained 30 μg / mL peptide 1 (KSKCVRDI SHYL) and 60 μg / mL peptide 2 (VAALEFVRDNNGL), but not in the secondary culture. Flow cytometry was performed on the passaged cells, and the results were similar to those of Examples 1 and 2. The results showed that only about 93% of the cells were in the G0 / G1 phase in the first 6 generations. The number of cells in the S phase and G2 / M phase gradually increased with the increase in the number of generations. It can be seen that when peptide 1 does not exceed 30 μg / mL and peptide 2 does not exceed 60 μg / mL, basically satisfactory results can be achieved.

[0087] Comparative Example 1

[0088] Umbilical cord tissue was obtained from the fetuses of healthy pregnant women in the Department of Obstetrics and Gynecology of Renji Hospital. This study was approved by the Ethics Committee of the hospital and the informed consent of the umbilical cord donors. After the umbilical cord was collected, it was carefully placed in an umbilical cord preservation bottle (containing umbilical cord preservation solution) and stored at low temperature (4°C).

[0089] First, wash the umbilical cord three times with PBS, then disinfect it with 75% alcohol for 60 seconds, and then wash it three times with PBS to remove the residual alcohol. Cut the umbilical cord into small pieces of about 1-2 cm and wash it with PBS until there is no blood stain. Then remove the umbilical vein and two umbilical arteries to separate Wharton's jelly.

[0090] The separated Wharton's gel was cut into pieces and placed in a culture bottle with DMEM / F12 medium (containing 10% fetal bovine serum and 1% penicillin + 1% streptomycin) and incubated at 37°C and 5% CO. 2 The cells were cultured and subcultured in an incubator, wherein when the cells were full or 80% grown, they were digested with trypsin into single cells and continued to be subcultured for 10 generations. In the primary culture, the complete medium contained 5 μg / mL peptide 1 (KSKCVRD I SHYL) and 10 μg / mL peptide 2 (VAALEFVRDNNGL). In the subculture, the culture medium was the same as the primary culture, i.e., it contained the same peptide combination as the primary culture. Flow cytometry was performed on the subcultured cells, and the results showed that the cells in the G0 / G1 phase decreased by 15% relative to Example 1, and decreased rapidly after 6 generations of subculture.

[0091] Comparative Example 2

[0092] Umbilical cord tissue was obtained from the fetuses of healthy pregnant women in the Department of Obstetrics and Gynecology of Renji Hospital. This study was approved by the Ethics Committee of the hospital and the informed consent of the umbilical cord donors. After the umbilical cord was collected, it was carefully placed in an umbilical cord preservation bottle (containing umbilical cord preservation solution) and stored at low temperature (4°C).

[0093] First, wash the umbilical cord three times with PBS, then disinfect it with 75% alcohol for 60 seconds, and then wash it three times with PBS to remove the residual alcohol. Cut the umbilical cord into small pieces of about 1-2 cm and wash it with PBS until there is no blood stain. Then remove the umbilical vein and two umbilical arteries to separate Wharton's jelly.

[0094] The separated Wharton's gel was cut into pieces and placed in a culture bottle with DMEM / F12 medium (containing 10% fetal bovine serum and 1% penicillin + 1% streptomycin) and incubated at 37°C and 5% CO. 2 The cells were cultured and subcultured in an incubator, and when the cells were full or 80% grown, they were digested with trypsin into single cells and subcultured for 10 generations. That is, in the primary and subcultures, the complete medium did not contain peptide 1 and peptide 2. The flow cytometry test was performed on the subcultured cells, and the results showed that the cells in the G0 / G1 phase decreased relative to Example 1, at a level of more than 83%, and decreased rapidly after 6 generations of subculture. The results are shown in Table 2

[0095] Table 2. Results of subculture cycle detection of umbilical cord mesenchymal stem cells (mean ± SE, n = 3)

[0096]

[0097] It can be seen that the culture medium added with the peptide combination can improve the activity of mesenchymal stem cells, but the concentration is preferably not more than 60 μg / mL. As the concentration increases, the effect becomes worse.

[0098] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for isolating mesenchymal stem cells from umbilical cord, comprising the following steps: a. Obtain the umbilical cord; b. separating Wharton's jelly from the harvested umbilical cord; c. The isolated Wharton's jelly was cultured in complete medium supplemented with peptide 1 and peptide 2.

2. The method according to claim 1, further comprising the steps of: d. digesting primary cultured mesenchymal stem cells with enzymes; e. Subculturing the enzyme-digested mesenchymal stem cells.

3. The method according to claim 1 or 2, wherein the complete culture medium is DMEM / F-12 culture medium.

4. The method according to claim 1 or 2, wherein the inoculation density of the complete medium for subculture is 1000 to 5X10 5 cell / cm 2 , preferably 5000 cel l / cm 2 .

5. The method according to claim 1 or 2, comprising further subculturing the separated mesenchymal stem cells under the following conditions: culturing in an incubator at 37°C and 5% CO2.

6. Use of peptide 1 and / or peptide 2 in the preparation of a kit for isolating mesenchymal stem cells from umbilical cord.

7. Complete culture medium containing peptide 1 and peptide 2. 8 . The culture medium according to claim 7 , wherein the concentration of peptide 1 is 1 to 100 μg / mL, and the concentration of peptide 2 is 1 to 200 μg / mL.

9. Use of mesenchymal stem cells separated or cultured by the method according to any one of claims 1 to 5 in treating diseases.

10. A pharmaceutical composition comprising mesenchymal stem cells separated or cultured by the method according to any one of claims 1 to 5, optionally further comprising a pharmaceutically acceptable carrier or excipient, or other auxiliary materials.

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