Separation and culture method of adipose-derived mesenchymal stem cells

Abstract

The invention provides a method for separating and culturing adipose-derived mesenchymal stem cells. The method comprises the following steps: a, acquiring adipose; b, separating mesenchymal stem cells from the obtained fat; c, culturing the separated mesenchymal stem cells 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 fat 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 mesenchymal stem cells from adipose tissue. Background Art

[0002] Mesenchymal stem cells can come from different tissues, such as skin, internal organs (such as liver), placenta, adipose tissue, bone marrow, dental pulp, peripheral blood, umbilical cord blood and umbilical cord. However, the specific molecular phenotype, cell activity and differentiation potential of mesenchymal stem cells from different tissues are different. Adipose-derived mesenchymal stem cells are mostly positive for CD105, CD29, and CD90, while CD34 and CD45 are negative.

[0003] As one of the many sources of mesenchymal stem cells, adipose tissue has the characteristics of easy collection, low immunogenicity and multidirectional differentiation potential. At present, adipose-derived mesenchymal stem cells have been widely used in the treatment of many diseases, such as epithelial regeneration, and have achieved good results.

[0004] Adipose-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. The cell activity of adipose-derived stem cells has the advantages of high activity, strong division ability, and high safety.

[0005] Obesity is a common disease at present, and the incidence rate is increasing year by year both at home and abroad, and the number of liposuction surgeries in plastic surgery is also increasing year by year. Therefore, a large amount of fat tissue obtained is usually discarded, so it can be turned into treasure and used in clinical practice.

[0006] Obtaining mesenchymal stem cells from fat is a way of turning 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 adipose tissue are large in number, have strong proliferation ability, have a strong immune regulation effect, and secrete a high amount of active factors.

[0007] Adipose-derived mesenchymal stem cells can first be used for skin repair. Adipose-derived mesenchymal stem cells are derived from the mesoderm during embryonic development, and their differentiation into terminal cells requires cross-germ layer induction. Trottier et al. used adipose-derived mesenchymal stem cells to induce a complete three-layer skin substitute: epidermis, dermis, and subcutaneous fat, which can replace skin function.

[0008] In addition, adipose-derived mesenchymal stem cells can be used to treat diabetes, immune regulation, arthritis, retinal vascular repair, enteritis, solid tumors, etc.

[0009] How to prepare mesenchymal stem cells with high homogeneity and activity from adipose tissue is a key factor in the therapeutic effect of mesenchymal stem cells. In the prior art, the mesenchymal stem cells separated from adipose tissue are usually not homogeneous and their activity decreases with the increase of passage number. Summary of the invention

[0010] 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 adipose tissue, which has simple operation, high uniformity and strong activity.

[0011] Specifically, in a first aspect, the present invention provides a novel method for preparing mesenchymal stem cells from adipose tissue, wherein the mesenchymal stem cells separated from adipose are cultured and passaged in a culture medium supplemented with peptide 1 (KSKCVRDI SHYL) and / or peptide 2 (VAALEFVRDNNGL).

[0012] The novel method for preparing mesenchymal stem cells of the present invention further comprises any one, two, three, or four of the following steps, or any combination thereof:

[0013] a. Obtain adipose tissue;

[0014] b. isolating primary mesenchymal stem cells from the obtained adipose tissue;

[0015] c. culturing the separated mesenchymal stem cells in a complete medium supplemented with peptide 1 and peptide 2 to obtain mesenchymal stem cells;

[0016] d. Subculture.

[0017] 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.

[0018] 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.

[0019] Furthermore, the complete culture medium is α-MEM culture medium or DMEM culture medium.

[0020] In the complete medium, the inoculation density of subculture is 1000-5X10 5 ce ll / cm 2 , preferably 5000 cel l / cm 2 .

[0021] In step a, the adipose tissue is obtained from adipose tissue with consent from the liposuction patient and approval from the ethics committee of the hospital.

[0022] In step b, under sterile conditions, the obtained fat is repeatedly rinsed with a phosphate buffer containing double antibodies (penicillin and streptomycin) to remove blood vessels and connective tissue cells, and then digested with collagenase and centrifuged to obtain a cell precipitate, which is sieved to remove tissue that has not been enzymatically hydrolyzed to obtain fine fat granule cells. The fat granule cells are adherently cultured in a culture medium to obtain primary adipose-derived mesenchymal stem cells. Wherein, the culture medium in step b does not contain peptide 1 and / or peptide 2.

[0023] In step b, the digestion is performed by shaking with 0.2-0.5% collagenase at 37° C. on a constant temperature shaker for 1-2 hours.

[0024] In step b, the centrifugation refers to centrifugation at 600-1000 r / min for 8-20 min.

[0025] In step b, the cells are suspended in complete medium, inoculated into a culture flask, and cultured in a 37° C., 5% CO 2 incubator.

[0026] In step c, after adding peptide 1 and peptide 2 to the complete medium, peptide 1 and peptide 2 should be fully mixed with the complete medium. The primary mesenchymal stem cells obtained in step b are cultured in the complete medium supplemented with peptide 1 and peptide 2, and the medium is changed once every 2-3 days. When the cells are close to 60-90% confluence, they are passaged in time. If non-adherent cells are found, they need to be removed.

[0027] In step d, the subculture is carried out in a 37° C., 5% CO 2 incubator.

[0028] Optionally, single mesenchymal stem cells separated from adipose tissue are cultured adherently and isolated and purified when the confluence reaches 70-90%.

[0029] 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.

[0030] Current research has found that mesenchymal stem cells isolated from adipose tissue samples have better vitality. Moreover, if a lot of adipose tissue is not collected, it will be discarded. Therefore, isolating mesenchymal stem cells from adipose tissue is also a desirable way to turn waste into treasure.

[0031] 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.

[0032] 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.

[0033] 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 adipose tissue.

[0034] Illustratively, the present invention provides use of peptide 1 in preparing a kit for isolating mesenchymal stem cells from adipose tissue.

[0035] Furthermore, the present invention provides the use of peptide 2 in preparing a kit for isolating mesenchymal stem cells from adipose tissue.

[0036] 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 adipose tissue.

[0037] 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.

[0038] 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.

[0039] In a fourth aspect, the present invention provides a complete culture medium containing peptide 1 and peptide 2.

[0040] 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.

[0041] 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.

[0042] In a fifth aspect, the present invention also provides a composition comprising peptide 1 and peptide 2.

[0043] 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.

[0044] 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.

[0045] In a specific embodiment, the adipose-derived 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.

[0046] In addition, the adipose-derived stem cells of the present invention can treat autoimmune diseases, such as systemic lupus erythematosus, atopic dermatitis, etc. Adipose-derived stem cells can regulate immune function, prevent the release of inflammatory factors, and further regulate various immune cells and inhibit the overactivity of the immune system.

[0047] The adipose-derived stem cells of the present invention can also be used to treat diseases including diabetes, arteriosclerosis obliterans, thrombosis occlusive disease, etc. Adipose-derived 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.

[0048] Furthermore, the adipose-derived mesenchymal stem cells of the present invention can also be used for neuroprotection and promotion of neuroregeneration, 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.

[0049] 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.

[0050] 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.

[0051] 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 adipose mesenchymal stem cells. In addition, the isolated and cultured adipose mesenchymal stem cells can be identified by detecting cell surface markers by flow cytometry and detecting cell expression genes by PCR.

[0052] 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.

[0053] 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).

[0054] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the mesenchymal stem cells.

[0055] 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.

[0056] 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.

[0057] 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

[0058] / 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.

[0059] In certain embodiments, the subject for which the pharmaceutical composition is directed is a mammal, such as a human.

[0060] 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.

[0061] 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 3×104 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.

[0062] In certain embodiments, the subject is a mammal, such as a human.

[0063] 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.

[0064] Beneficial Effects

[0065] 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

[0066] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be described in further detail below.

[0067] Example 1

[0068] Adipose tissue was obtained from healthy patients who underwent liposuction surgery at the Ninth People's Hospital of Shanghai. The content of this study was approved by the ethics committee of the hospital and the informed consent of the fat donors. After the fat was collected, primary mesenchymal stem cells were isolated from the obtained adipose tissue. Specifically, under sterile conditions, the obtained fat was repeatedly rinsed with a phosphate buffer containing double antibodies (penicillin and streptomycin) to remove blood vessels and connective tissue cells, and then digested with collagenase and centrifuged to obtain a cell precipitate after continued rinsing. After sieving, the tissue that was not enzymatically hydrolyzed was removed to obtain fine fat granular cells. The fat granular cells were adherently cultured in a culture medium to obtain primary adipose-derived mesenchymal stem cells. In the process of obtaining primary mesenchymal stem cells, the culture medium did not contain peptide 1 and / or peptide 2; the digestion was performed by shaking and digesting with 0.5% collagenase at 37°C constant temperature shaker for 1 hour; the centrifugation was performed at 800r / min for 12min; the cells were suspended with α-MEM complete culture medium, inoculated in a culture bottle, and cultured in a 37°C, 5% CO2 incubator; and separation and purification were performed when the confluence reached 70-90%.

[0069] The isolated primary mesenchymal stem cells were subcultured in α-MEM complete medium supplemented with peptide 1 and peptide 2 for 10 generations.

[0070] In the secondary culture, the complete medium contained 5 μg / mL peptide 1 (KSKCVRDI SHYL) and 10 μg / mL peptide 2 (VAALEFVRDNNGL), but not in the primary culture. 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.

[0071] Table 1. Results of subculture cycle detection of adipose mesenchymal stem cells (mean ± SE, n = 3)

[0072]

[0073] Example 2

[0074] Adipose tissue was obtained from healthy patients who underwent liposuction surgery at the Ninth People's Hospital of Shanghai. The content of this study was approved by the ethics committee of the hospital and the informed consent of the fat donors. After the fat was collected, primary mesenchymal stem cells were isolated from the obtained adipose tissue. Specifically, under sterile conditions, the obtained fat was repeatedly rinsed with a phosphate buffer containing double antibodies (penicillin and streptomycin) to remove blood vessels and connective tissue cells, and then digested with collagenase and centrifuged to obtain a cell precipitate after continued rinsing. After sieving, the tissue that was not enzymatically hydrolyzed was removed to obtain fine fat granular cells. The fat granular cells were adherently cultured in a culture medium to obtain primary adipose-derived mesenchymal stem cells. In the process of obtaining primary mesenchymal stem cells, the culture medium did not contain peptide 1 and / or peptide 2; the digestion was performed by shaking and digesting with 0.5% collagenase at 37°C constant temperature shaker for 1 hour; the centrifugation was performed at 800r / min for 12min; the cells were suspended with α-MEM complete culture medium, inoculated in a culture bottle, and cultured in a 37°C, 5% CO2 incubator; and separation and purification were performed when the confluence reached 70-90%.

[0075] The isolated primary mesenchymal stem cells were subcultured in α-MEM complete medium supplemented with peptide 1 and peptide 2 for 10 generations.

[0076] In the secondary culture, the complete medium contains 10 μg / mL peptide 1 (KSKCVRDI SHYL) and 20 μg / mL peptide 2 (VAALEFVRDNNGL), but not in the primary culture. Flow cytometry was performed on the subcultured cells, and the results were similar to those in Example 1: it showed that most 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.

[0077] Example 3

[0078] Adipose tissue was obtained from healthy patients who underwent liposuction surgery at the Ninth People's Hospital of Shanghai. The content of this study was approved by the ethics committee of the hospital and the informed consent of the fat donors. After the fat was collected, primary mesenchymal stem cells were isolated from the obtained adipose tissue. Specifically, under sterile conditions, the obtained fat was repeatedly rinsed with a phosphate buffer containing double antibodies (penicillin and streptomycin) to remove blood vessels and connective tissue cells, and then digested with collagenase and centrifuged to obtain a cell precipitate after continued rinsing. After sieving, the tissue that was not enzymatically hydrolyzed was removed to obtain fine fat granular cells. The fat granular cells were adherently cultured in a culture medium to obtain primary adipose-derived mesenchymal stem cells. In the process of obtaining primary mesenchymal stem cells, the culture medium did not contain peptide 1 and / or peptide 2; the digestion was performed by shaking and digesting with 0.5% collagenase at 37°C constant temperature shaker for 1 hour; the centrifugation was performed at 800r / min for 12min; the cells were suspended with α-MEM complete culture medium, inoculated in a culture bottle, and cultured in a 37°C, 5% CO2 incubator; and separation and purification were performed when the confluence reached 70-90%.

[0079] The isolated primary mesenchymal stem cells were subcultured in α-MEM complete medium supplemented with peptide 1 and peptide 2 for 10 generations.

[0080] In the secondary culture, the complete medium contains 50 μg / mL peptide 1 (KSKCVRDI SHYL) and 100 μg / mL peptide 2 (VAALEFVRDNNGL), but not in the primary culture. Flow cytometry was performed on the subcultured cells, and the results were similar to those in Example 1: it showed that most 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 generations increased, there was no significant increase before the 6th generation, and then gradually increased.

[0081] Example 4

[0082] Adipose tissue was obtained from healthy patients who underwent liposuction surgery at the Ninth People's Hospital of Shanghai. The content of this study was approved by the ethics committee of the hospital and the informed consent of the fat donors. After the fat was collected, primary mesenchymal stem cells were isolated from the obtained adipose tissue. Specifically, under sterile conditions, the obtained fat was repeatedly rinsed with a phosphate buffer containing double antibodies (penicillin and streptomycin) to remove blood vessels and connective tissue cells, and then digested with collagenase and centrifuged to obtain a cell precipitate after continued rinsing. After sieving, the tissue that was not enzymatically hydrolyzed was removed to obtain fine fat granular cells. The fat granular cells were adherently cultured in a culture medium to obtain primary adipose-derived mesenchymal stem cells. In the process of obtaining primary mesenchymal stem cells, the culture medium did not contain peptide 1 and / or peptide 2; the digestion was performed by shaking and digesting with 0.5% collagenase at 37°C constant temperature shaker for 1 hour; the centrifugation was performed at 800r / min for 12min; the cells were suspended with α-MEM complete culture medium, inoculated in a culture bottle, and cultured in a 37°C, 5% CO2 incubator; and separation and purification were performed when the confluence reached 70-90%.

[0083] The isolated primary mesenchymal stem cells were subcultured in α-MEM complete medium supplemented with peptide 1 and peptide 2 for 10 generations.

[0084] In the secondary culture, the complete medium contains 30 μg / mL peptide 1 (KSKCVRDI SHYL) and 60 μg / mL peptide 2 (VAALEFVRDNNGL), but not in the primary culture. Flow cytometry was performed on the subcultured cells, and the results were similar to those in Example 1: it showed that most 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.

[0085] Comparative Example 1

[0086] Adipose tissue was obtained from healthy patients who underwent liposuction surgery at the Ninth People's Hospital of Shanghai. The content of this study was approved by the ethics committee of the hospital and the informed consent of the fat donors. After the fat was collected, primary mesenchymal stem cells were isolated from the obtained adipose tissue. Specifically, under sterile conditions, the obtained fat was repeatedly rinsed with a phosphate buffer containing double antibodies (penicillin and streptomycin) to remove blood vessels and connective tissue cells. After further rinsing, it was digested with collagenase and centrifuged to obtain a cell precipitate. After sieving, the tissue that was not enzymatically hydrolyzed was removed to obtain tiny fat granule cells. The fat granule cells were adherently cultured in a culture medium to obtain primary adipose-derived mesenchymal stem cells. In the process of obtaining primary mesenchymal stem cells, the culture medium does not contain peptide 1 and / or peptide 2; the digestion is performed by shaking with 0.5% collagenase at 37°C constant temperature shaker for 1 hour; the centrifugation is performed at 800r / min for 12min; the cells are suspended in α-MEM complete culture medium containing 50μg / mL peptide 1 (KSKCVRDI SHYL) and 100μg / mL peptide 2 (VAALEFVRDNNGL), inoculated in a culture bottle, and cultured in a 37°C, 5% CO2 incubator; and separation and purification are performed when the confluence reaches 70-90%.

[0087] The isolated primary mesenchymal stem cells were subcultured in α-MEM complete medium without peptide 1 and peptide 2 for 10 generations.

[0088] In the secondary culture, the complete medium does not contain peptide 1 and peptide 2, but contains them in the primary culture. The flow cytometry test of the subcultured cells showed significant differences from that of Example 1: only about 75% of the cells were in the G0 / G1 phase, the cell division activity was high, and the number of cells in the S phase and G2 / M phase increased significantly with the increase of the number of generations.

[0089] Comparative Example 2

[0090] Adipose tissue was obtained from healthy patients who underwent liposuction surgery at the Ninth People's Hospital of Shanghai. The content of this study was approved by the ethics committee of the hospital and the informed consent of the fat donors. After the fat was collected, primary mesenchymal stem cells were isolated from the obtained adipose tissue. Specifically, under sterile conditions, the obtained fat was repeatedly rinsed with a phosphate buffer containing double antibodies (penicillin and streptomycin) to remove blood vessels and connective tissue cells. After further rinsing, it was digested with collagenase and centrifuged to obtain a cell precipitate. After sieving, the tissue that was not enzymatically hydrolyzed was removed to obtain tiny fat granule cells. The fat granule cells were adherently cultured in a culture medium to obtain primary adipose-derived mesenchymal stem cells. In the process of obtaining primary mesenchymal stem cells, the culture medium does not contain peptide 1 and / or peptide 2; the digestion is performed by shaking with 0.5% collagenase at 37°C constant temperature shaker for 1 hour; the centrifugation is performed at 800r / min for 12 minutes; the cells are suspended with α-MEM complete culture medium without peptide 1 (KSKCVRDI SHYL) and peptide 2 (VAALEFVRDNNGL), inoculated in a culture bottle, and cultured in a 37°C, 5% CO2 incubator; and separation and purification are performed when the confluence reaches 70-90%.

[0091] The isolated primary mesenchymal stem cells were subcultured in α-MEM complete medium without peptide 1 and peptide 2 for 10 generations.

[0092] That is, in the primary and secondary cultures, the complete medium does not contain peptide 1 and peptide 2. The flow cytometry test of the subcultured cells showed no significant difference from that of Comparative Example 1: it showed that only about 70% or more of the cells were in the G0 / G1 phase, the cell division activity was high, and the number of cells in the S phase and G2 / M phase increased slowly with the increase of the generation number. The results are shown in Table 2.

[0093] Table 2. Results of subculture cycle detection of adipose mesenchymal stem cells (mean ± SE, n = 3)

[0094]

[0095] 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 fat, comprising the following steps: a. Obtain adipose tissue; b. isolating primary mesenchymal stem cells from the obtained adipose tissue; c. culturing the separated mesenchymal stem cells in a complete medium supplemented with peptide 1 and peptide 2 to obtain mesenchymal stem cells; d. Continue to subculture in complete medium containing peptide 1 and peptide 2. 2 . The method according to claim 1 , wherein peptide 1 and / or peptide 2 is not administered in isolating primary mesenchymal stem cells. The method according to claim 1 or 2, wherein the complete culture medium is α-MEM culture medium or DMEM 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 preparing a kit for isolating mesenchymal stem cells from adipose tissue.

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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