Use of adipose tissue-derived mesenchymal stem cells cultured in a hypoxic atmosphere for the treatment of premature ovarian failure

Abstract

The present invention relates to hypoxic atmosphere culture of adipose tissue-derived mesenchymal stem cells and their use for the treatment of premature ovarian failure. In particular, the invention relates to a method for the isolation and subculturing of adipose tissue-derived mesenchymal stem cells comprising (a) isolation and primary culture of adipose tissue-derived mesenchymal stem cells and (b) subculturing of adipose tissue-derived mesenchymal stem cells, wherein step (b) is the subculturing of adipose tissue-derived mesenchymal stem cells in a 5% oxygen, 5% CO2, 37°C three gas incubator. The invention further relates to a method for the treatment of premature ovarian failure using hypoxic atmosphere culture of adipose tissue-derived mesenchymal stem cells as described above and to the use of a cell preparation of hypoxic atmosphere cultured adipose tissue-derived mesenchymal stem cells as described above for the treatment of premature ovarian failure.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biotechnology, and relates to a method for culturing adipose-derived mesenchymal stem cells, particularly a method for improving the proliferation efficiency of adipose-derived mesenchymal stem cells, especially a method for culturing adipose-derived mesenchymal stem cells in a hypoxic atmosphere to improve their proliferation efficiency. In addition, this invention also relates to a method for treating premature ovarian failure by culturing adipose-derived mesenchymal stem cells in the aforementioned hypoxic atmosphere, and the use of cell preparations of adipose-derived mesenchymal stem cells cultured in the aforementioned hypoxic atmosphere for the treatment of premature ovarian failure. Background Technology

[0002] Premature ovarian failure (POF) refers to the condition where ovarian function fails, leading to amenorrhea and infertility in women before the age of 40. POF is characterized by amenorrhea, infertility, estrogen deficiency, decreased follicle count, and elevated gonadotropins, accompanied by a range of low-estrogen symptoms such as hot flashes, night sweats, facial flushing, and decreased libido, severely impacting women's physical and mental health. Furthermore, women with POF have an increased risk of osteoporosis and cardiovascular disease. POF is a significant cause of female infertility. The incidence of POF in women of reproductive age is approximately 1-3%, and it is showing an increasing trend and affecting younger women.

[0003] According to the guidelines of the European Society of Human Reproduction and Embryology (ESHRE), the diagnostic criteria for premature ovarian failure (POF) are: oligomenorrhea or amenorrhea for at least 4 months, and two FSH levels elevated >40 IU / L at an interval of more than 4 weeks. The cause of POF is unknown, but it may be related to genetic and autoimmune diseases, environmental factors, and iatrogenic and idiopathic conditions. There is currently no effective treatment. Hormone replacement therapy (HRT) is one of the most common treatments for POF, but its effectiveness is not ideal and it has been shown to increase the risk of venous thrombosis, breast cancer, and ovarian cancer. In addition to oligomenorrhea, amenorrhea, and infertility, POF can also cause menopausal symptoms such as hot flashes, night sweats, anxiety, depression, palpitations, and insomnia. It may accelerate female aging, leading to postmenopausal diseases such as osteoporosis, cardiovascular disease, and dementia, affecting women's quality of life and lifespan.

[0004] The etiology of POF is complex and not fully understood. It may be related to autoimmune responses, infections, genetic factors, the effects of treatments such as chemotherapy, radiotherapy, and surgery, as well as endocrine dysfunction. Currently, there is no effective treatment for POF. The most common treatment for POF is hormone replacement therapy (HRT). While this therapy can alleviate some of the clinical symptoms of POF, HRT cannot fundamentally repair damaged ovaries or restore ovarian function. Furthermore, studies have shown that long-term HRT treatment increases the risk of heart disease and stroke, and may increase the risk of breast and ovarian cancer. Therefore, new treatment strategies are needed to restore ovarian function in patients with POF.

[0005] Mesenchymal stem cells (MSCs), such as human MSCs, were first isolated from bone marrow. They are a type of tissue stem cell derived from the mesoderm, possessing multi-lineage differentiation potential and self-renewal capacity. Under specific in vivo and in vitro conditions, they can differentiate into various adult cell types, including osteoblasts, chondrocytes, adipocytes, endothelial cells, nerve cells, muscle cells, and hepatocytes (Caplan AI. Mesenchymal stem cells. JOrthop Res. 1991, 9: 641-650. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284: 143-147). Recent studies have shown that mesenchymal stem cells have immunomodulatory and hematopoietic support functions and are easily introduced and expressed with exogenous genes. Therefore, mesenchymal stem cells (MSCs) are not only seed cells in tissue-engineered bone, cartilage, and myocardium construction, and important carrier cells in gene therapy, but also have broad application prospects in hematopoietic stem cell transplantation and organ transplantation due to their functions in promoting hematopoietic reconstitution and inhibiting graft-versus-host disease. MSCs possess the characteristic of adherent growth in vitro, and utilizing this characteristic, MSCs have been successfully isolated and cultured from various tissues such as liver, kidney, pancreas, muscle, cartilage, skin, and peripheral blood.

[0006] A type of stem cell with multipotent differentiation potential has been isolated from adipose tissue and successfully differentiated into adipocytes, osteoblasts, chondrocytes, and myocytes in a specialized in vitro culture system. These differentiateable stem cells are recognized as adipose-derived mesenchymal cells, or adipose-derived stem cells (ADSCs). ADSCs are a type of stem cell with multipotent differentiation potential isolated from adipose tissue in recent years. They possess the characteristic of differentiating into multiple germ layers, including the ability to differentiate into mesoderm, endoderm, and ectoderm cells, and can secrete many potentially beneficial growth factors and cytokines. Cell-based therapies to promote wound healing are rapidly developing. Adipose-derived stem cells are a group of pluripotent mesenchymal stem cells that can differentiate into other cell lines. A large number of adipose-derived cells exist in the non-adipose (mesenchymal) fragments of adipose tissue and are easily isolated and obtained.

[0007] Adipose-derived stem cells (ADSCs) are recommended as an adjunctive treatment for various tissue defects. Angiogenesis is a crucial step in wound healing; the formation of new blood vessels is essential for supporting granulation tissue formation and the survival and expansion of keratinocytes to heal wounds. Therefore, it is believed that ADSCs promote wound healing by rapidly inducing angiogenesis. Furthermore, ADSCs have also shown to promote wound healing through cell differentiation and the secretion of long-term factors that promote collagen synthesis and dermal fibroblast migration.

[0008] Adipose-derived stem cells (ADSCs) have been combined with extracellular matrix scaffold components for use in animal skin and soft tissue wounds, revealing that ADSCs promote scaffold vascularization. Studies have shown that the wound-healing-promoting effect of ADSCs is related to their ability to promote epithelialization and granulation tissue formation, and ADSCs also possess potential epidermal cell differentiation capabilities. Further research suggests that the distant differentiation capacity of ADSCs is a response of pluripotent ADSCs to the wound microenvironment. ADSCs contribute to local vascular remodeling through direct differentiation into vascular endothelial cells and the secretion of angiogenesis factors, both of which benefit vascular regeneration. In other words, ADSCs promote wound healing through cell differentiation and angiogenesis. Furthermore, ADSCs have been combined with platelet-derived growth factor (PDGF), a well-known factor involved in normal wound healing, for wound treatment. Research indicates a synergistic effect, potentially promoting wound healing by increasing PGF levels.

[0009] In theory, cell therapy holds great promise because pluripotent stem cells or progenitor cells promote wound healing by regenerating missing tissue and continuously providing numerous beneficial factors. Nevertheless, the mechanisms by which adipose-derived stem cells contribute to wound healing still require further investigation. The remodeling of local blood vessels by adipose-derived stem cells includes direct differentiation into vascular endothelial cells and the secretion of angiogenesis factors, both of which are beneficial for vascular regeneration.

[0010] The application prospects of adipose-derived mesenchymal stem cells (ADSCs) have been validated through their use in various animal models of diseases. They have been used to intervene in the treatment of diabetes, liver damage repair, bladder reconstruction, type 2 diabetic eczema, myocardial infarction, cerebral infarction, cognitive impairment, stroke, intervertebral disc repair, glioblastoma treatment, bone defects, post-traumatic angiogenesis, rheumatoid arthritis, cleft lip and palate, heart failure, colitis, and urinary incontinence. Some researchers have transformed ADSCs into cardiomyocytes, reprogramming mature stem cells and improving the treatment of heart disease. Others have injected ADSCs, combined with fibrin glue, into the left ventricular wall of rats with myocardial infarction; ADSCs show great promise for clinical tissue engineering treatment of myocardial infarction. Furthermore, ADSCs, used as seed cells in a mesh scaffold, successfully repaired skull injuries in dogs, providing a potential clinical treatment for skull injuries. Studies have shown that injecting adipose-derived mesenchymal stem cells (ADMSCs) from the abdominal fat of heart patients into their hearts reduced cardiac damage and increased blood flow. Six months after the injection, the patients' hearts showed improved oxygenation, and the left ventricle pumped 3.5 times more blood. Spelt imaging demonstrated a 5.7% increase in cardiac pumping capacity in patients receiving ADMSCs, and MRI scans showed a decrease in the average myocardial scar area from 31.6% to 15.4%. These findings were presented at the 2010 American Heart Association Scientific Sessions. During tissue repair, ADMSCs can regulate growth hormones and cytokines in surrounding tissues, preventing apoptosis. Experiments have shown that ADMSCs can secrete various skin growth factors, such as basic fibroblast growth factor (bFGF), to promote fibroblast proliferation and possess anti-photoaging, antioxidant, anti-wrinkle, and anti-ultraviolet radiation effects. Research using ADMSCs to treat complex anal ulcers has entered Phase II clinical trials. Furthermore, human adipose-derived mesenchymal stem cells have been transplanted into the breasts of patients with breast atrophy, uneven size, or those requiring breast augmentation. The resulting breasts are symmetrical and balanced in size. Mammograms show that the repaired breasts are naturally soft, completely free of calcification, cysts, and obvious injection scars. Recent clinical studies have shown that intramuscular injection of adipose-derived mesenchymal stem cells also has some therapeutic effect on diabetic foot and arteriosclerosis obliterans. Six months after injection, patients experienced relief of resting pain, a significant increase in painless walking distance, and no complications were observed. Trials have shown that adipose-derived mesenchymal stem cells induced into islet-like cells and reinfused into diabetic patients, with a 23-month follow-up, resulted in a decrease in exogenous insulin usage, an increase in average weight, and no discomfort or side effects.

[0011] The strong and rapid adhesion of adipose-derived mesenchymal stem cells to the extracellular matrix has enabled the development of specific methods for screening and culturing human adipose-derived mesenchymal stem cells both domestically and internationally. Furthermore, the biological properties of stem cells allow for enhanced function, avoidance of potentially harmful genes, and control over differentiation time and different differentiation directions. These advantages provide irreplaceable guidance in basic research, clinical treatment, stem cell therapy research, skin regeneration and repair, and the construction of highly tissue-engineered skin.

[0012] Adipose-derived mesenchymal stem cells (ADSCs), as mesenchymal stem cells derived from fat, are easy to obtain, with an extraction efficiency 40 times higher than that of bone marrow mesenchymal stem cells, and they proliferate faster under in vitro culture conditions. They possess multi-lineage differentiation capabilities, capable of differentiating into adipocytes, osteoblasts, chondrocytes, cardiomyocytes, and even nerve cells. Current Phase I and Phase II clinical trials have demonstrated that ADSCs are safe and effective in repairing injuries to various organs, including the heart, rectum, and breast. Furthermore, studies have confirmed that ADSCs promote skin regeneration and repair.

[0013] ADSCs exhibit inherent advantages in several aspects. Belonging to the monocyte family, ADSCs are a type of adult stem cell. Derived from processed adipose tissue, they grow in cell culture dishes using adherent and adhesive methods and possess diverse differentiation capabilities. They can differentiate not only into adipocytes but also into myocytes, chondrocytes, nerve cells, vascular endothelial cells, and osteoblasts, thus finding widespread application in tissue engineering. For example, ADSCs can self-replicate and differentiate into adipocytes, playing a role in tissue regeneration; ADSCs can differentiate into vascular endothelial cells or peripheral cells; ADSCs can secrete angiogenesis factors under hypoxic or other conditions; when ADSCs are transplanted together with adipocytes, they can differentiate into endothelial cells, preventing fibrosis and fat necrosis, and improving fat survival rate; recent studies have shown that ADSCs can express various cytokines such as VEGF, IGF, TGF-β1, bFGF, and EGF, and also prevent apoptosis and maintain adipocyte survival rate by expressing angiogenesis factors such as VEGF and IGF-1.

[0014] Numerous methods for culturing adipose-derived mesenchymal stem cells (ADMSCs) have been disclosed in the prior art. For example, CN106479970A (201611050980.1) discloses a method for large-scale culture of human ADMSCs. The method for large-scale culture of human ADMSCs described in this invention involves adding ADMSC culture medium to a glass culture flask and incubating at 35°C for 30 minutes in an incubator with 95% humidity and 5% CO2. The temperature of the ADMSC culture medium is then adjusted to 35°C and pH 7.05, and pretreated microcarriers and ADMSCs are added. The mixture is stirred at 5-20 RPM for 2 hours. The temperature is then raised to 37°C and cultured at 15-40 RPM and 5% CO2 for 96 hours, with supplemental ADMSC culture medium added during this period. Compared with existing methods, this invention is believed to yield more cells after large-scale culture of human ADMSCs, with stronger cell viability and proliferative capacity, and it effectively maintains the morphology and good stem cell characteristics of ADMSCs.

[0015] CN104762260A (201510197879.8) relates to a method for preparing adipose-derived mesenchymal stem cells, characterized by the following steps: Step 1: Obtaining fat, pretreatment, enzymatic hydrolysis, centrifugation, collecting the precipitate, and obtaining adipose-derived mesenchymal stem cells; Step 2: Culturing the adipose-derived mesenchymal stem cells; when the adipose-derived mesenchymal stem cells grow to 80% confluence, discarding the culture medium, washing with PBS, then digesting with EDTA-trypsin solution, terminating digestion, centrifuging, and passage culture. This invention is believed to have the following advantages: 1. High purity of the cultured stem cells; 2. Recovery of various factors secreted during the culture of adipose-derived mesenchymal stem cells, which can significantly promote the proliferation of epidermal cells, indicating that it can accelerate the regeneration and turnover of epidermal cells, achieving a significant anti-aging effect.

[0016] CN104974984A (application number 201510173453.9) discloses a method for expanding and culturing mesenchymal stem cells derived from adipose tissue, comprising the following steps: resuspending isolated mesenchymal stem cells derived from adipose tissue in adipose stem cell culture medium to obtain a resuspension containing mesenchymal stem cells; seeding the resuspension into a stem cell culture device for expansion culture; replacing the adipose stem cell culture medium after 24 hours of expansion culture, and then replacing the adipose stem cell culture medium every two days thereafter, until the growth and confluence reach 80-90%, digesting with trypsin and then passage culture; wherein, the adipose stem cell culture medium contains 0.1-10 v / v% fetal bovine serum, 1-10 v / v% gentamicin and 80-98.9 v / v% high glucose DMEM medium; the stem cell culture device is a stem cell culture device coated with fibronectin. The invention is believed to be a method for expanding and culturing adipose-derived mesenchymal stem cells by coating a solid support on which the adipose-derived mesenchymal stem cells depend for growth with fibronectin. It utilizes the synergistic effect of low-concentration fetal bovine serum and high-concentration gentamicin to screen and culture the cells, enabling adipose-derived mesenchymal stem cells to expand 400-500 times within two weeks with high purity and the ability to be directed to differentiate into adipose tissue.

[0017] CN106520686A (application number 201610887111.8) discloses a method for culturing adipose-derived mesenchymal stem cells. The method comprises the following steps: primary adipose-derived mesenchymal stem cells are seeded into a stem cell culture medium for culturing, wherein the culture medium contains stem cell factors and interleukin-3, and the ratio of stem cell factors to interleukin-3 is (5-20 μmol / L):(5-20 μg / ml). This method enhances the proliferation capacity of adipose-derived mesenchymal stem cells and achieves higher migration ability.

[0018] CN101984049A (application number 201010580537.1) discloses a method for isolating mesenchymal stem cells from adipose tissue, characterized by the following steps: (1) Obtaining adipose tissue: the adipose tissue is waste material obtained by liposuction using the tumescent method in a professional hospital or beauty salon, and is stored at 4-20℃ for less than 48 hours under sterile conditions; (2) Preliminary removal of red blood cells: after the fat extract is left to stand and separate into layers, the lower layer of liquid is carefully removed, and the extract is washed multiple times with D-Hanks solution until the wash liquid is clear; (3) Digesting the fat extract: type I collagenase prepared with D-Hanks solution is added to 0.01-2g / 100ml. l, digest at 37℃ with shaking at 20-400 rpm for 15-120 minutes; (4) Obtaining adipose stem cells: centrifuge the digested product at 4℃ and 450g for 5 minutes, resuspend the adipose stem cell culture medium, filter with a 100μm pore size filter membrane to remove impurities and finally obtain adipose stem cells; (5) Remove red blood cells again by repeated washing and red blood cell lysis; (6) Count, detect activity and culture of adipose stem cells: take 100μl of isolated cells and count the cells with a cell counting plate; at the same time, take 100μl of cells and count the viable cells by trypan blue staining. According to the results of the two counts, follow the 3*10 4 / cm 2 The cells were seeded at a density in T-75 culture flasks and cultured at 37°C, 5% CO2, and 100% humidity. This method is believed to be able to obtain large quantities of well-formed, multi-directionally differentiated adipose-derived stem cells from a small amount of adipose tissue, and is simple, easy to perform, and highly reproducible. However, the method in this literature targets mesenchymal stem cells simultaneously during cell lysis, which is detrimental to stem cell survival.

[0019] In the culture method for efficiently obtaining adipose-derived mesenchymal stem cells disclosed in CN106222134A (application number 201610605996.8), only the fat particles in the middle layer after centrifugation are extracted during fat separation, and the cell precipitate at the bottom of the centrifuge is not extracted.

[0020] CN102329783A (application number 201110310461.5) discloses a method for separating adipose-derived mesenchymal stem cells from the tumescent fluid during liposuction, comprising the following steps: (1) Collection of tumescent fluid: Before liposuction, an excess of hypotonic tumescent fluid is injected into the area to be extracted. Then, the fat and tumescent fluid obtained during the liposuction process are placed at room temperature for 10 minutes. After the fat and tumescent fluid are completely separated, the red liquid under the yellow fat is aspirated with a pipette, which is the tumescent fluid containing adipose-derived stem cells; (2) Processing of tumescent fluid: The collected tumescent fluid is dispensed into 50ml centrifuge tubes and centrifuged at 1500rpm for 5 minutes. Centrifuge and discard the liquid at the top of the centrifuge tube, collect the precipitate, suspend each tube in PBS, add red blood cell lysis buffer and let stand at room temperature for 5 minutes, centrifuge at 1200 rpm for 3 minutes, and discard the supernatant; (3) Washing the cells: Add 5 ml of DMEM (without serum) to disperse the precipitate, filter with a cell filter, centrifuge the filtered liquid at 1200 rpm for 3 minutes, and the precipitate is the adipose mesenchymal stem cells to be obtained. Add DMEM (without serum) and repeat the above operation 3 times; (4) Cell culture: Seed the obtained cells into a 75 cm2 cell culture flask, add mesenchymal stem cell culture medium, and culture at 37°C with 5% CO2. However, CN102329783A only targets the separation of stem cells in the swelling solution, not the separation of stem cells in the fat solution mixed with the swelling solution.

[0021] As described in CN112522193B (Chinese Patent Application No. CN2020115689421), the general method for obtaining human adipose-derived mesenchymal stem cells includes two stages: (a) isolating and culturing primary adipose-derived mesenchymal stem cells and (b) passage culturing adipose-derived mesenchymal stem cells. The passage culturing stage (b) is usually carried out in an incubator atmosphere with a normal oxygen concentration of 5% carbon dioxide and the remainder being air (approximately 21% oxygen and approximately 78% nitrogen).

[0022] MSCs for clinical research and application require in vitro culture and expansion. Different culture environments can affect the characteristics of MSCs, such as immunomodulatory function, secretion levels of active factors, and cell differentiation capacity. How to select a suitable culture environment for the application of MSCs to produce cells that are more beneficial to clinical use is an urgent problem to be solved. Currently, MSCs are usually cultured in vitro in a normoxic environment (20% oxygen), but in reality, the O2 concentration in the physiological microenvironment of MSCs is much lower [Nekanti U, et al. Increased proliferation and analysis of differential gene expression in human Wharton's jelly-derived mesenchymal stromal cells underhypoxia. Int J Biol Sci, 2010, 6(5): 499]. In vivo, the O2 level of tissues is maintained within a small range called "normooth oxygen," usually 2% to 9%.

[0023] Currently, numerous studies have selected a 5% oxygen environment (i.e., an atmosphere of 5% carbon dioxide, 5% oxygen, and a balance of nitrogen) as the intermediate value of "physiological normoxic" for hypoxic culture, achieving some promising results, particularly in significantly shortening cell doubling time (also known as population doubling time, PDT, measured in hours) by reducing oxygen concentration. However, it is well known that a shortened cell doubling time implies accelerated cell senescence, which is undesirable in certain situations for clinical research and applications of mesenchymal stem cells. If a simple method could be used to adjust culture conditions and thus regulate the cell doubling time of MSCs, thereby improving the proliferation efficiency of adipose-derived mesenchymal stem cells, it would have immense clinical application value and is something that those skilled in the art eagerly anticipate. Summary of the Invention

[0024] One object of the present invention is to provide a method for isolating and culturing adipose-derived mesenchymal stem cells (ADMSCs), or to provide a method for improving the proliferation efficiency of ADMSCs, or to provide a method for culturing ADMSCs in a hypoxic environment to improve their proliferation efficiency; or, one object of the present invention is to use ADMSCs obtained by the method of the present invention for the treatment of premature ovarian failure. It has been unexpectedly found that the stem cell preparations of the present invention exhibit satisfactory results in the treatment of premature ovarian failure. The present invention is based on these findings.

[0025] Therefore, the first aspect of the present invention provides a method for isolating and passage-culturing adipose-derived mesenchymal stem cells, comprising two stages: (a) isolating and culturing primary adipose-derived mesenchymal stem cells and (b) passage-culturing adipose-derived mesenchymal stem cells, wherein: (a) The stage of isolating and culturing primary adipose-derived mesenchymal stem cells includes the following steps: (a1) Process fat samples transported to the laboratory via a cold chain at 2-8°C in a biosafety cabinet; (a2) Centrifuge the fat sample, remove the upper adipose tissue, wash it with D-Hanks solution, centrifuge it again, remove the adipose tissue, add 1% type II collagenase and shake to digest. (a3) After digestion, dilute with an equal volume of D-hanks solution, centrifuge, and collect the cell pellet at the bottom for the next step; (a4) Take the cell pellet obtained in step (3), add primary supplemental culture medium to resuspend it, take a sample and count it, and inoculate it into a culture flask according to the specified cell amount; place it in a CO2 incubator for culture; (a5) After culturing for 3 days, the culture medium was completely replaced once. When the cell confluence reached more than 80%, the old culture medium was removed, the cells were washed with D-Hanks solution, and recombinant trypsin solution was added to digest the cells to cause them to detach. The cells were diluted with D-Hanks solution, centrifuged, and the cell pellet was resuspended in primary supplemented culture medium to obtain primary adipose mesenchymal stem cells, i.e., P0 generation. (b) The passage culture of adipose-derived mesenchymal stem cells includes the following steps: P0 generation cells are washed with PBS, digested with recombinant trypsin solution until most of the cells detach, digestion is stopped by adding complete culture medium, the cells are transferred to centrifuge tubes, centrifuged, the supernatant is discarded, the cells are resuspended in passage culture medium, counted and seeded into culture flasks, and passaged in a tri-gas incubator of 5% oxygen, 5% CO2, and 37°C. When the cell confluence reaches more than 80%, digestion is performed with digestive enzymes to obtain P1 generation cells; then the same procedure is followed to passage the cells for each generation to obtain cell suspensions of adipose-derived mesenchymal stem cells.

[0026] According to the method of the first aspect of the present invention, the passage culture medium comprises: 12% fetal bovine serum, 1% L-glutamine, and 12 μg / mL epidermal growth factor (EGF). Zinc monomethionine 15~120μg / mL, for example, 15~100μg / mL, for example, 15~75μg / mL, for example, 50μg / mL, DMEM-F12 added to 100%.

[0027] According to the method of the first aspect of the present invention, the PBS is a disodium hydrogen phosphate / sodium dihydrogen phosphate buffer with a phosphate ion concentration of 0.025M and a pH of 6.8.

[0028] According to the method of the first aspect of the invention, the complete culture medium is DMEM-F12 medium containing 12% fetal bovine serum.

[0029] According to the method of the first aspect of the present invention, in step (b), a cell suspension of adipose-derived mesenchymal stem cells of passages P1-P15, such as P1-P9, is obtained by passage.

[0030] According to the method of the first aspect of the present invention, the D-Hanks solution is formulated as follows: 8.0g of NaCl, 0.4g of KCl, 0.06g of KH2PO4, 0.08g of Na2HPO4·12H2O, 0.35g of NaHCO3, and water to 1000ml.

[0031] According to the method of the first aspect of the present invention, the primary supplemental culture medium is prepared with DMEM-F12 medium as the matrix and supplemented with: 1% platelet lysate, 1% human serum albumin, 2 μg / ml recombinant insulin, 15 ng / ml EGF, 25 ng / ml bFGF, 0.12% thioglycerol, and 1% fructose.

[0032] According to the method of the first aspect of the present invention, in step (a2), both centrifugations are performed at 100 x g for 5 min.

[0033] According to the method of the first aspect of the present invention, in step (a2), 1% type II collagenase is added at twice the volume and shaken for 30 min for digestion.

[0034] According to the method of the first aspect of the present invention, in step (a3), the centrifugation is performed at a speed of 100 x g for 5 min.

[0035] According to the method of the first aspect of the present invention, in step (a4), seeding cells into the culture flask according to the specified cell amount means seeding cells at a amount of 1~5×10⁶ cells / year. 4 / cm 2 Inoculate into T75 vials.

[0036] According to the method of the first aspect of the present invention, in step (a4), seeding the culture flask with the prescribed cell amount means seeding the culture flask with a cell amount of 2 × 10⁻⁶ cells / year. 4 / cm 2 Inoculate into T75 vials.

[0037] According to the method of the first aspect of the present invention, in step (a4), the conditions for culturing in the CO2 incubator are: 5% CO2, 37°C, and saturated humidity.

[0038] According to the method of the first aspect of the present invention, in step (a5), 2 ml of recombinant trypsin solution is added to each bottle to digest the cells for 2 min.

[0039] According to the method of the first aspect of the present invention, in step (a5), 10 ml of D-hanks solution is added to each bottle for dilution.

[0040] According to the method of the first aspect of the present invention, in step (a5), centrifugation is performed at 100 x g for 10 min.

[0041] According to the method of the first aspect of the present invention, the DMEM-F12 culture medium is formulated as follows: anhydrous calcium chloride 116.6 mg, L-leucine 59.05 mg, linoleic acid 0.042 mg, copper sulfate pentahydrate 0.0013 mg, L-lysine hydrochloride 91.25 mg, lipoic acid 0.105 mg, ferric nitrate nonahydrate 0.05 mg, L-methionine 17.24 mg, phenol red 8.1 mg, ferrous sulfate heptahydrate 0.417 mg, L-phenylalanine 35.48 mg, 1,4-butanediol... Diamine dihydrochloride 0.081 mg, potassium chloride 311.8 mg, L-serine 26.25 mg, sodium pyruvate 55 mg, magnesium chloride 28.64 mg, L-threonine 53.45 mg, vitamin H 0.0035 mg, anhydrous magnesium sulfate 48.84 mg, L-alanine 4.45 mg, D-calcium pantothenate 2.24 mg, sodium chloride 7000 mg, L-asparagine 7.5 mg, choline chloride 8.98 mg, anhydrous sodium dihydrogen phosphate 54.35 mg, L-aspartic acid 6.6 mg. 5mg, folic acid 2.65mg, disodium hydrogen phosphate 71.02mg, L-cysteine ​​hydrochloride 17.56mg, i-inositol 12.6mg, zinc sulfate heptahydrate 0.432mg, L-glutamate 7.35mg, nicotinamide 2.02mg, L-arginine hydrochloride 147.5mg, L-proline 17.25mg, pyridoxal hydrochloride 2mg, L-cysteine ​​hydrochloride 31.29mg, L-tryptophan 9.02mg, pyridoxine hydrochloride 0.031mg, L-glutamine 365mg Ingredients: L-tyrosine 38.4 mg, riboflavin 0.219 mg, glycine 18.75 mg, L-valine 52.85 mg, thiamine hydrochloride 2.17 mg, L-histidine hydrochloride 31.48 mg, D-glucose 3151 mg, thymidine 0.365 mg, L-isoleucine 54.47 mg, hypoxanthine 2 mg, vitamin B12 0.68 mg, and water to a final volume of 1000 mL. Preparation: Dissolve all ingredients in 1000 mL of water and filter through a 0.22 µm microporous membrane for sterilization.

[0042] According to the method of the first aspect of the present invention, it further includes testing the primary adipose-derived mesenchymal stem cells obtained by isolation and culture, and identifying cell morphology and / or immunophenotype.

[0043] According to the method of the first aspect of the present invention, the immunophenotypic identification refers to the detection of CD73, CD90, CD105, CD19, CD11b, CD31, CD45, HLADR, and CD34.

[0044] According to the method of the first aspect of the present invention, in step (b), the cells are passaged in a three-gas incubator containing 5% oxygen, 5% CO2, and 37°C; as is well known, this three-gas incubator is a saturated humidity condition. Furthermore, the gas composition in the aforementioned three-gas incubator essentially means 5% oxygen, 5% carbon dioxide, and the remainder nitrogen. Additionally, as shown in step (a), the gas environment for culturing cells in a CO2 incubator at 37°C, 5% CO2, and saturated humidity is 5% carbon dioxide and the remainder air (approximately 21% oxygen and approximately 78% nitrogen). When only the carbon dioxide concentration is specified in the incubator without specifying the oxygen concentration, it refers to an environment of 5% carbon dioxide and the remainder air, to distinguish it from the 5% oxygen and 5% CO2 three-gas culture environment of step (5). In short, a three-gas incubator refers to a total of 100% nitrogen, oxygen, and carbon dioxide.

[0045] According to the method of the first aspect of the present invention, in step (b), the cells are passaged in an incubator until the cell fusion rate reaches 80% to 90%.

[0046] According to the method of the first aspect of the present invention, in step (b), the digestive enzyme is TrypLE Express digestive enzyme.

[0047] According to the method of the first aspect of the present invention, in step (b), the seeding density of cells in the culture flask is 6000-10000 cells / cm². 2 For example, 8000 cells / cm³ 2 .

[0048] According to the method of the first aspect of the present invention, step (b) is performed as follows: P0 generation cells are washed with PBS, digested with 2 ml of recombinant trypsin solution for 2-5 min until most cells detach, digestion is terminated with 5 ml of complete culture medium, cells are transferred to centrifuge tubes, centrifuged at 1400 rpm for 5 min, the supernatant is discarded, the cells are resuspended in 5 ml of passage culture medium, and then counted and seeded into culture flasks at a cell density of 8000 cells / cm³. 2 The cells were passaged in a tri-gas incubator containing 5% oxygen, 5% CO2, and 37°C. When the cell fusion rate reached 80%~90%, the cells were digested with TrypLE Express for 3 minutes to obtain P1 generation cells. Then, the same procedure was followed to pass the cells to the next generation, and cell suspensions of each generation of adipose-derived mesenchymal stem cells were obtained.

[0049] According to the method of the first aspect of the present invention, the cell purity of each generation of placental mesenchymal stem cells obtained in step (b) is greater than 90%.

[0050] Furthermore, a second aspect of the present invention provides adipose-derived mesenchymal stem cells, which are obtained by isolation and passage culture using the method described in any embodiment of the first aspect of the present invention.

[0051] Furthermore, a third aspect of the present invention provides a cell preparation for injection, the composition of which is: 2 × 10-1 adipose-derived mesenchymal stem cells. 6 The ingredients are: 1, 9.0 mg sodium chloride, magnesium citrate (to achieve a magnesium ion concentration of 5 mmol / L), 0.2 mg soybean lecithin for injection, and water to a final volume of 1 ml; the adipose-derived mesenchymal stem cells are P1 to P9 generation cells, and are obtained by isolation and passage culture using the method described in any embodiment of the first aspect of the present invention.

[0052] According to a third aspect of the present invention, the cell preparation is prepared by a method comprising the following steps: dissolving sodium chloride, magnesium citrate and phospholipid in an appropriate amount of water to obtain a saline solution; transferring mesenchymal stem cells obtained from passage culture into a centrifuge tube and centrifuging (e.g., centrifuging at 2000 rpm for 5 min), discarding the supernatant, adding saline solution to resuspend the cells, thereby obtaining the cell preparation.

[0053] Furthermore, the fourth aspect of the present invention provides the use of adipose-derived mesenchymal stem cells described in any aspect of the present invention, or adipose-derived mesenchymal stem cells isolated and passaged by the method described in any aspect of the present invention, or cell preparations described in any aspect of the present invention in the preparation of a medicament for treating premature ovarian failure.

[0054] Although the specific steps described in the methods and steps described in various aspects of this invention may differ in some details or language from the steps described in the examples in the detailed embodiments section below, those skilled in the art can fully grasp the above-described methods and steps based on the detailed disclosure of the entire invention.

[0055] Any embodiment of any aspect of the present invention can be combined with other embodiments, as long as they do not contradict each other. Furthermore, any technical feature in any embodiment of any aspect of the present invention can be applied to the same technical feature in other embodiments, as long as they do not contradict each other. The present invention will now be further described.

[0056] All references cited in this invention are incorporated herein by reference in their entirety, and in the event of any inconsistency between the meanings expressed in these references and those expressed herein, the meanings expressed herein shall prevail. Furthermore, the various terms and phrases used in this invention have their general meanings known to those skilled in the art; however, this invention still seeks to provide a more detailed explanation and interpretation of these terms and phrases, and in the event of any inconsistency between the mentioned terms and their known meanings and those expressed herein, the meanings expressed herein shall prevail.

[0057] In this invention, the term "adipose-derived mesenchymal stem cells" refers to mesenchymal stem cells derived from adipose tissue. Therefore, in this invention, and particularly in the context of this invention, the term "adipose-derived mesenchymal stem cells" may be used interchangeably with "adipose-derived stem cells," "stem cells," and "mesenchymal stem cells," unless otherwise expressly specified.

[0058] Mesenchymal stem cells have regenerative potential and immunomodulatory capabilities. They have been found in bone marrow, adipose tissue, umbilical cord, umbilical cord blood, amniotic fluid, placenta, dental pulp, tendons, synovium, and skeletal muscle [Widowati W, et al.]. Hypoxia in mesenchymal stem cell[M / OL]. Licensee InTech, 2017. Doi:10.5772 / 62960;Yin JQ, et al. Manufacturing of primed mesenchymal stromalcells for therapy [J]. Nat Biomed Eng, 2019, 3(2): 90-104]. Umbilical cords, once considered waste, are favored by researchers and users due to their wide availability, lack of ethical controversy, ease of separation, and low immunogenicity. Numerous researchers are currently evaluating the effectiveness and safety of their application in clinical treatment. In 2006, the International Society for Cellular Therapy updated the minimum criteria for mesenchymal stem cells: (1) adherent growth in a standard culture environment; (2) positive expression of CD73, CD90, and CD105 with an expression rate ≥95%, and negative expression of CD34, CD45, CD14 or CD11b, CD79α or CD19, and HLA-DR with an expression rate ≤2%; (3) able to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro [Dominici M, et al. Minimalcriteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement [J]. Cytotherapy, 2006, 8(4): 315-317]. Nevertheless, mesenchymal stem cells prepared by researchers from different laboratories still show significant differences in in vitro proliferation capacity, immunomodulatory capacity, cytokine and growth factor expression levels and secretion capacity, and further exploration is needed to find more suitable in vitro culture protocols for mesenchymal stem cells [Yin JQ, et al.Manufacturing of primed mesenchymal stromal cells for therapy [J]. Nat BiomedEng, 2019, 3(2): 90-104].

[0059] The method for isolating and passage-culturing adipose-derived mesenchymal stem cells of the present invention exhibits one or more superior effects as described in the context herein. For example, the method of the present invention uses 5% oxygen for the isolation and passage-culturing of adipose-derived mesenchymal stem cells, which can enhance the proliferative capacity of adipose-derived mesenchymal stem cells while maintaining the original phenotype, differentiation capacity, and lymphocyte proliferation inhibition capacity of adipose-derived mesenchymal stem cells. Attached Figure Description

[0060] Figure 1 This is a typical microscopic image of adipose-derived mesenchymal stem cells.

[0061] Figure 2 These are typical staining patterns of osteogenic, adipogenic, and chondrogenic differentiation of adipose-derived mesenchymal stem cells, respectively. Figure 2 A, Figure 2 B Figure 2 C. Detailed Implementation

[0062] The invention will be further described through the following embodiments; however, the scope of the invention is not limited to the embodiments described below. Those skilled in the art will understand that various changes and modifications can be made to the invention without departing from its spirit and scope. The invention provides a general and / or specific description of the materials and methods used in the experiments. Although many materials and methods of operation used to achieve the objectives of the invention are well known in the art, the invention is still described in as much detail as possible herein. The following embodiments further illustrate the invention, but are not intended to limit it.

[0063] Unless otherwise stated, DMEM / F12 was used as the basal culture medium in this invention. Epidermal growth factor, fetal bovine serum and DMEM / F12 were purchased from GIBCO brand of Thermo Fisher Scientific. EGF was purchased from PeproTech. L-glutamine, gentamicin, zinc monomethionine and digestive enzyme TrypLE Express were all purchased from Sigma-Aldrich.

[0064] Unless otherwise stated, the complete culture medium used in this invention is DMEM-F12 medium containing 12% fetal bovine serum.

[0065] Unless otherwise specified, the Hank's equilibrium salt solution used in this invention consists of: 8.0 g / L NaCl, 0.4 g / L KCl, 0.1 g / L MgSO4·7H2O, 0.1 g / L MgCl2·6H2O, 0.06 g / L Na2HPO4·2H2O, 0.06 g / L KH2PO4, 1.0 g / L glucose, 0.14 g / L CaCl2, 0.35 g / L NaHCO3, 0.2 g / L phenol red, and hydrochloric acid or sodium hydroxide adjusted to pH 7.4.

[0066] Some steps of the method used in the embodiment of the isolation and passage of adipose mesenchymal stem cells of the present invention are performed with reference to the relevant operations described in the Chinese patent application CN112522193A (CN2020115689421) of the applicant team.

[0067] Unless otherwise specified, the DMEM-F12 culture medium used in this invention comprises the following components: anhydrous calcium chloride 116.6 mg, L-leucine 59.05 mg, linoleic acid 0.042 mg, copper sulfate pentahydrate 0.0013 mg, L-lysine hydrochloride 91.25 mg, lipoic acid 0.105 mg, ferric nitrate nonahydrate 0.05 mg, L-methionine 17.24 mg, phenol red 8.1 mg, ferrous sulfate heptahydrate 0.417 mg, L-phenylalanine 35.48 mg, 1,4- Butylene diamine dihydrochloride 0.081 mg, potassium chloride 311.8 mg, L-serine 26.25 mg, sodium pyruvate 55 mg, magnesium chloride 28.64 mg, L-threonine 53.45 mg, vitamin H 0.0035 mg, anhydrous magnesium sulfate 48.84 mg, L-alanine 4.45 mg, D-calcium pantothenate 2.24 mg, sodium chloride 7000 mg, L-asparagine 7.5 mg, choline chloride 8.98 mg, anhydrous sodium dihydrogen phosphate 54.35 mg, L-aspartic acid 6 mg. 65mg, folic acid 2.65mg, disodium hydrogen phosphate 71.02mg, L-cysteine ​​hydrochloride 17.56mg, i-inositol 12.6mg, zinc sulfate heptahydrate 0.432mg, L-glutamate 7.35mg, nicotinamide 2.02mg, L-arginine hydrochloride 147.5mg, L-proline 17.25mg, pyridoxal hydrochloride 2mg, L-cysteine ​​hydrochloride 31.29mg, L-tryptophan 9.02mg, pyridoxine hydrochloride 0.031mg, L-glutamine 365mg Ingredients: L-tyrosine 38.4 mg, riboflavin 0.219 mg, glycine 18.75 mg, L-valine 52.85 mg, thiamine hydrochloride 2.17 mg, L-histidine hydrochloride 31.48 mg, D-glucose 3151 mg, thymidine 0.365 mg, L-isoleucine 54.47 mg, hypoxanthine 2 mg, vitamin B12 0.68 mg, and water to a final volume of 1000 mL. Preparation: Dissolve all ingredients in 1000 mL of water and filter through a 0.22 µm microporous membrane for sterilization.

[0068] In this invention, the platelet lysate used in the experiment can be readily purchased from the market. Unless otherwise specified, the platelet lysate used in this experiment is PLTGold Human Platelet Lysate purchased from Sigma-Aldrich, catalog number SCM151.

[0069] In this invention, the type II collagenase used in the experiment can be readily purchased from the market. Unless otherwise specified, the collagenase used in this experiment was purchased from Gibco.

[0070] In this invention, the bFGF (basic fibroblast growth factor) used in the experiment can be readily purchased from the market. Unless otherwise specified, the bFGF used in this experiment was purchased from Sigma-Aldrich, with catalog number GF003.

[0071] In this invention, the EGF (epidermal growth factor) used in the experiment can be readily purchased from the market. Unless otherwise specified, the EGF used in this experiment was purchased from Gibco, with catalog number PHG0311L.

[0072] In this invention, the recombinant insulin used in the experiment is readily available from the market. Unless otherwise specified, the insulin used in this experiment was purchased from Solarbio, catalog number I8830.

[0073] In this invention, the recombinant trypsin solution used in the experiment can be readily purchased from the market. Unless otherwise specified, the recombinant trypsin solution with a concentration of 2000u / ml purchased from Lamborghini, catalog number RT2S01, was used in the experiment.

[0074] In this invention, unless otherwise specified, the D-Hanks solution used in the experiment has the following formula and preparation method: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4·12H2O, 0.35g NaHCO3, and water to 1000ml; Preparation: Dissolve each material in 1000ml, filter and sterilize through a 0.22µm microporous membrane to obtain the solution.

[0075] In this invention, unless otherwise specified, the primary supplemental culture medium used in the experiment is prepared with DMEM-F12 medium as the matrix and contains: 1% platelet lysate, 1% human serum albumin, 2 μg / ml recombinant insulin, 15 ng / ml EGF, 25 ng / ml bFGF, 0.12% thioglycerol, and 1% fructose.

[0076] In this invention, unless otherwise specified, PBS is a sodium hydrogen phosphate / sodium dihydrogen phosphate buffer with a phosphate ion concentration of 0.025M and a pH of 6.8.

[0077] In the specific experiments of this invention, a sample of a certain generation of stem cells was taken, and nucleated cells, namely MSC cells, were counted using a Sysmex hematology analyzer. Cell viability was detected by trypan blue staining, and samples were taken for microbial detection.

[0078] Example 1: Isolation and culture of primary adipose-derived mesenchymal stem cells (1) Processing fat donated by volunteers that has been transported to the laboratory via a cold chain at 2-8°C in a biosafety cabinet; (2) Centrifuge at 100xg for 5 min, transfer the upper adipose tissue, wash it once with D-Hanks solution, centrifuge at 100xg for 5 min, transfer the adipose tissue, add 2 times the volume of 1% type II collagenase and shake to digest for 30 min. (3) After digestion, dilute with an equal volume of D-hanks solution, centrifuge at 100xg for 5 minutes, and collect the cell pellet at the bottom for the next operation; (4) Take the cell pellet obtained in step (3), resuspend it in primary culture medium, take samples and count them, according to 2×10 4 / cm 2 Inoculate into T75 flasks; incubate in a CO2 incubator (5% CO2, 37℃, saturated humidity); (5) After culturing for 3 days, the culture medium is completely replaced once. When the cell confluence reaches more than 80% (around 5 days), the old culture medium is removed, and the cells are washed with D-Hanks solution. 2 ml of recombinant trypsin solution is added to each bottle to digest the cells for 2 min to detach the cells. 10 ml of D-Hanks solution is added to each bottle for dilution, and the cells are centrifuged at 100 x g for 10 min. The cell pellet is resuspended in primary supplemented culture medium to obtain primary adipose mesenchymal stem cells (i.e., P0 generation), which can be used for subsequent passage. [In this example 1, the above steps (1) to (5) are performed with 2 × 10⁻⁶ cells per 100 ml culture medium in step (4).] 4 / cm 2 After inoculation into T75 flasks, the number of nucleated cells obtained per flask (average of 5 replicates) was 5.72 × 10^6 (n=5) (in this paper, this data can be referred to as cell harvest), and the cell viability was 92.2% (n=5)]. (6) Cell passage: Wash P0 generation cells with PBS, add 2 ml of recombinant trypsin solution and digest for 2-5 min until most cells detach. Add 5 ml of complete culture medium to stop digestion, transfer cells to centrifuge tubes, centrifuge at 1400 rpm for 5 min, discard the supernatant, resuspend in 5 ml of passage medium, count and seed into culture flasks, with a cell density of 8000 cells / cm³. 2 The cells were passaged in a tri-gas incubator at 37°C with 5% oxygen and 5% CO2. When the cell confluence reached 80%–90%, the cells were digested with TrypLE Express for 3 minutes to obtain P1 generation cells. This process was then repeated for subsequent passages to obtain P2–P9 generation adipose-derived mesenchymal stem cell suspensions. The passage medium used in this example contained: 12% fetal bovine serum, 1% L-glutamine, and 12 μg / mL epidermal growth factor (EGF). Zinc monomethionine Add 50 μg / mL of DMEM-F12 to 100%. Zinc monomethionine CAS number 56329-42-1, purchased from Sigma-Aldri ch.

[0079] Experimental Example 1: Identification of Biological Characteristics of Adipose-Derived Mesenchymal Stem Cells The biological characteristics of the P1-P9 passage adipose-derived mesenchymal stem cells obtained by the method in Example 1 were identified, and the results are as follows: Each generation of cells exhibited typical stem cell morphology: spindle-shaped, plump, and adherent. The typical microscopic morphology of P3 generation mesenchymal stem cells is as follows: Figure 1 As shown.

[0080] Flow cytometry phenotyping of P1-P9 passages showed that the positive expression of CD73, CD90, and CD105 was >98%, for example, the positive expression of CD73, CD90, and CD105 in P6 passage cells was 99.72%, 99.33%, and 99.58%, respectively. Flow cytometry phenotyping results also showed that the levels of CD19, CD11b, CD31, CD45, HLADR, and CD34 in all passages of adipose-derived mesenchymal stem cells were less than 2% (e.g., in example P5). The adipose-derived mesenchymal stem cells (ADMSCs) had CD19=0.33%, CD11b=0.18%, CD31=0.12%, CD45=0.43%, HLADR=0.36%, and CD34=0.24% respectively. Directed differentiation potential assays showed that all generations of ADMSCs possessed osteogenic, chondrogenic, and adipogenic differentiation potentials. For example, the microscopic results of the osteogenic, chondrogenic, and adipogenic differentiation potentials of a batch of P6 generation ADMSCs obtained in Example 1 are shown below. Figure 2 As shown, these results indicate that the cells isolated and cultured from fat using the method of this embodiment are mesenchymal stem cells with high purity.

[0081] In addition, the growth cycle of cells from generation P1 to P9 was measured. The results showed that the number of cells in the G2 phase was <1% and the number of cells in the S phase was >10% in all generations. For example, the number of cells in the G2 phase of generation P5 was 0.63% and the number of cells in the S phase was 12.48%. These results indicate that the cells harvested in Example 1 have strong proliferative capacity and have not entered the division phase.

[0082] In other embodiments of the present invention, the P1 to P9 generation cells obtained also basically exhibited the above-mentioned cell characteristics.

[0083] Experiment Example 2: Cell Proliferation Experiment This experiment used population doubling time (PDT) as a parameter to evaluate the proliferation of adipose-derived mesenchymal stem cells. Specifically, the P1 generation or other generation cell suspensions obtained in Example 1 were respectively divided into 5x10... 4Cells were seeded at a density of 1 cell / ml into 30 culture dishes with a diameter of 35 mm, and randomly divided into 10 groups of 3 dishes each. The culture medium described in Example 1 was used, and the dishes were cultured in a tri-gas incubator with 5% oxygen, 5% CO2, and 37°C. Each day, at the same time, one group (3 dishes) was digested with TrypLE Express enzyme for 3 minutes to obtain a cell suspension. Cells were counted (using a cell counting chamber), and the average of the three dishes was taken as the total cell count for that day. This process continued until the 10th group (10 days). A cell growth curve was plotted with cell count as the ordinate and culture time (h) as the abscissa. The cell population doubling time (PDT, h) was calculated using the following formula: PDT = Δt × Lg2 / (LgNt - LgN0) In the formula, Δt is the culture time (in hours) during which cell proliferation reaches the plateau phase. N0 represents the initial number of cells seeded during culture. Nt represents the number of cells that have reached the plateau phase of cell proliferation.

[0084] Using the above method, the population doubling time (PDT) of adipose-derived mesenchymal stem cells (ADSs) from passages P1, P3, P5, P7, and P9 obtained in Example 1 was measured. The results were 27.86 h, 28.16 h, 28.82 h, 27.23 h, and 28.35 h, respectively, with a mean ± SD of 28.08 ± 0.53. This indicates that the population doubling time of cells from different passages is basically the same.

[0085] The adipose-derived mesenchymal stem cells obtained using the method in Example 1 were subjected to cell proliferation experiments using the method in Experiment 2. The inventors unexpectedly discovered that the cell population doubling time showed a certain trend when the amount of zinc monomethionine added to the passage medium was different. The specific experiments and results are as follows.

[0086] Example 2: Isolation and passage of adipose-derived mesenchymal stem cells This embodiment refers to Embodiment 1 for related operations.

[0087] (1) Processing fat donated by volunteers that has been transported to the laboratory via a cold chain at 2-8°C in a biosafety cabinet; (2) Centrifuge at 100xg for 5 min, transfer the upper adipose tissue, wash it once with D-Hanks solution, centrifuge at 100xg for 5 min, transfer the adipose tissue, add 2 times the volume of 1% type II collagenase and shake to digest for 30 min. (3) After digestion, dilute with an equal volume of D-hanks solution, centrifuge at 100xg for 5 minutes, and collect the cell pellet at the bottom for the next operation; (4) Take the cell pellet obtained in step (3), resuspend it in primary culture medium, take samples and count them, according to 2×10 4 / cm 2 Inoculate into T75 flasks; incubate in a CO2 incubator (5% CO2, 37℃, saturated humidity); (5) After culturing for 3 days, the culture medium is completely replaced once. When the cell confluence reaches more than 80% (around 5 days), the old culture medium is removed, and the cells are washed with D-Hanks solution. 2 ml of recombinant trypsin solution is added to each bottle to digest the cells for 2 min to detach the cells. 10 ml of D-Hanks solution is added to each bottle to dilute the cells. The cells are centrifuged at 100 x g for 10 min. The cell pellet is resuspended in primary supplemented culture medium to obtain primary adipose mesenchymal stem cells (i.e., P0 generation), which can be used for subsequent passage. (6) Cell passage: Wash P0 generation cells with PBS, add 2 ml of recombinant trypsin solution and digest for 2-5 min until most cells detach. Add 5 ml of complete culture medium to stop digestion, transfer cells to centrifuge tubes, centrifuge at 1400 rpm for 5 min, discard the supernatant, resuspend in 5 ml of passage medium, count and seed into culture flasks, with a cell density of 8000 cells / cm³. 2 The cells were passaged in a tri-gas incubator at 37°C with 5% oxygen and 5% CO2. When the cell confluence reached 80%–90%, the cells were digested with TrypLE Express for 3 minutes to obtain P1 generation cells. This process was then repeated for subsequent passages to obtain P2–P9 generation adipose-derived mesenchymal stem cell suspensions. The passage medium used in this example contained: 12% fetal bovine serum, 1% L-glutamine, 12 μg / mL epidermal growth factor (EGF), 80 μg / mL zinc monomethionine, and DMEM-F12 to 100%.

[0088] Example 3: Isolation and passage of adipose-derived mesenchymal stem cells This embodiment refers to Embodiment 1 for related operations.

[0089] (1) Processing fat donated by volunteers that has been transported to the laboratory via a cold chain at 2-8°C in a biosafety cabinet; (2) Centrifuge at 100xg for 5 min, transfer the upper adipose tissue, wash it once with D-Hanks solution, centrifuge at 100xg for 5 min, transfer the adipose tissue, add 2 times the volume of 1% type II collagenase and shake to digest for 30 min. (3) After digestion, dilute with an equal volume of D-hanks solution, centrifuge at 100xg for 5 minutes, and collect the cell pellet at the bottom for the next operation; (4) Take the cell pellet obtained in step (3), resuspend it in primary culture medium, take samples and count them, according to 2×10 4 / cm 2 Inoculate into T75 flasks; incubate in a CO2 incubator (5% CO2, 37℃, saturated humidity); (5) After culturing for 3 days, the culture medium is completely replaced once. When the cell confluence reaches more than 80% (around 5 days), the old culture medium is removed, and the cells are washed with D-Hanks solution. 2 ml of recombinant trypsin solution is added to each bottle to digest the cells for 2 min to detach the cells. 10 ml of D-Hanks solution is added to each bottle to dilute the cells. The cells are centrifuged at 100 x g for 10 min. The cell pellet is resuspended in primary supplemented culture medium to obtain primary adipose mesenchymal stem cells (i.e., P0 generation), which can be used for subsequent passage. (6) Cell passage: Wash P0 generation cells with PBS, add 2 ml of recombinant trypsin solution and digest for 2-5 min until most cells detach. Add 5 ml of complete culture medium to stop digestion, transfer cells to centrifuge tubes, centrifuge at 1400 rpm for 5 min, discard the supernatant, resuspend in 5 ml of passage medium, count and seed into culture flasks, with a cell density of 8000 cells / cm³. 2 The cells were passaged in a tri-gas incubator at 37°C with 5% oxygen and 5% CO2. When the cell confluence reached 80%–90%, the cells were digested with TrypLE Express for 3 minutes to obtain P1 generation cells. This process was then repeated for subsequent passages to obtain P2–P9 generation adipose-derived mesenchymal stem cell suspensions. The passage medium used in this example contained: 12% fetal bovine serum, 1% L-glutamine, 12 μg / mL epidermal growth factor (EGF), 120 μg / mL zinc monomethionine, and DMEM-F12 to 100%.

[0090] Example 4: Isolation and passage of adipose-derived mesenchymal stem cells This embodiment refers to Embodiment 1 for related operations.

[0091] (1) Processing fat donated by volunteers that has been transported to the laboratory via a cold chain at 2-8°C in a biosafety cabinet; (2) Centrifuge at 100xg for 5 min, transfer the upper adipose tissue, wash it once with D-Hanks solution, centrifuge at 100xg for 5 min, transfer the adipose tissue, add 2 times the volume of 1% type II collagenase and shake to digest for 30 min. (3) After digestion, dilute with an equal volume of D-hanks solution, centrifuge at 100xg for 5 minutes, and collect the cell pellet at the bottom for the next operation; (4) Take the cell pellet obtained in step (3), resuspend it in primary culture medium, take samples and count them, according to 2×10 4 / cm 2 Inoculate into T75 flasks; incubate in a CO2 incubator (5% CO2, 37℃, saturated humidity); (5) After culturing for 3 days, the culture medium is completely replaced once. When the cell confluence reaches more than 80% (around 5 days), the old culture medium is removed, and the cells are washed with D-Hanks solution. 2 ml of recombinant trypsin solution is added to each bottle to digest the cells for 2 min to detach the cells. 10 ml of D-Hanks solution is added to each bottle to dilute the cells. The cells are centrifuged at 100 x g for 10 min. The cell pellet is resuspended in primary supplemented culture medium to obtain primary adipose mesenchymal stem cells (i.e., P0 generation), which can be used for subsequent passage. (6) Cell passage: Wash P0 generation cells with PBS, add 2 ml of recombinant trypsin solution and digest for 2-5 min until most cells detach. Add 5 ml of complete culture medium to stop digestion, transfer cells to centrifuge tubes, centrifuge at 1400 rpm for 5 min, discard the supernatant, resuspend in 5 ml of passage medium, count and seed into culture flasks, with a cell density of 8000 cells / cm³. 2 The cells were passaged in a tri-gas incubator at 37°C with 5% oxygen and 5% CO2. When the cell confluence reached 80%–90%, the cells were digested with TrypLE Express for 3 minutes to obtain P1 generation cells. This process was then repeated for subsequent passages to obtain P2–P9 generation adipose-derived mesenchymal stem cell suspensions. The passage medium used in this example contained: 12% fetal bovine serum, 1% L-glutamine, 12 μg / mL epidermal growth factor (EGF), 30 μg / mL zinc monomethionine, and DMEM-F12 to 100%.

[0092] Example 5: Isolation and passage of adipose-derived mesenchymal stem cells This embodiment refers to Embodiment 1 for related operations.

[0093] (1) Processing fat donated by volunteers that has been transported to the laboratory via a cold chain at 2-8°C in a biosafety cabinet; (2) Centrifuge at 100xg for 5 min, transfer the upper adipose tissue, wash it once with D-Hanks solution, centrifuge at 100xg for 5 min, transfer the adipose tissue, add 2 times the volume of 1% type II collagenase and shake to digest for 30 min. (3) After digestion, dilute with an equal volume of D-hanks solution, centrifuge at 100xg for 5 minutes, and collect the cell pellet at the bottom for the next operation; (4) Take the cell pellet obtained in step (3), resuspend it in primary culture medium, take samples and count them, according to 2×10 4 / cm 2 Inoculate into T75 flasks; incubate in a CO2 incubator (5% CO2, 37℃, saturated humidity); (5) After culturing for 3 days, the culture medium is completely replaced once. When the cell confluence reaches more than 80% (around 5 days), the old culture medium is removed, and the cells are washed with D-Hanks solution. 2 ml of recombinant trypsin solution is added to each bottle to digest the cells for 2 min to detach the cells. 10 ml of D-Hanks solution is added to each bottle to dilute the cells. The cells are centrifuged at 100 x g for 10 min. The cell pellet is resuspended in primary supplemented culture medium to obtain primary adipose mesenchymal stem cells (i.e., P0 generation), which can be used for subsequent passage. (6) Cell passage: Wash P0 generation cells with PBS, add 2 ml of recombinant trypsin solution and digest for 2-5 min until most cells detach. Add 5 ml of complete culture medium to stop digestion, transfer cells to centrifuge tubes, centrifuge at 1400 rpm for 5 min, discard the supernatant, resuspend in 5 ml of passage medium, count and seed into culture flasks, with a cell density of 8000 cells / cm³. 2 The cells were passaged in a tri-gas incubator at 37°C with 5% oxygen and 5% CO2. When the cell confluence reached 80%–90%, the cells were digested with TrypLE Express for 3 minutes to obtain P1 generation cells. This process was then repeated for subsequent passages to obtain P2–P9 generation adipose-derived mesenchymal stem cell suspensions. The passage medium used in this example contained: 12% fetal bovine serum, 1% L-glutamine, 12 μg / mL epidermal growth factor (EGF), 15 μg / mL zinc monomethionine, and DMEM-F12 to 100%.

[0094] Example 6: Isolation and passage of adipose-derived mesenchymal stem cells This embodiment refers to Embodiment 1 for related operations.

[0095] (1) Processing fat donated by volunteers that has been transported to the laboratory via a cold chain at 2-8°C in a biosafety cabinet; (2) Centrifuge at 100xg for 5 min, transfer the upper adipose tissue, wash it once with D-Hanks solution, centrifuge at 100xg for 5 min, transfer the adipose tissue, add 2 times the volume of 1% type II collagenase and shake to digest for 30 min. (3) After digestion, dilute with an equal volume of D-hanks solution, centrifuge at 100xg for 5 minutes, and collect the cell pellet at the bottom for the next operation; (4) Take the cell pellet obtained in step (3), resuspend it in primary culture medium, take samples and count them, according to 2×10 4 / cm 2 Inoculate into T75 flasks; incubate in a CO2 incubator (5% CO2, 37℃, saturated humidity); (5) After culturing for 3 days, the culture medium is completely replaced once. When the cell confluence reaches more than 80% (around 5 days), the old culture medium is removed, and the cells are washed with D-Hanks solution. 2 ml of recombinant trypsin solution is added to each bottle to digest the cells for 2 min to detach the cells. 10 ml of D-Hanks solution is added to each bottle to dilute the cells. The cells are centrifuged at 100 x g for 10 min. The cell pellet is resuspended in primary supplemented culture medium to obtain primary adipose mesenchymal stem cells (i.e., P0 generation), which can be used for subsequent passage. (6) Cell passage: Wash P0 generation cells with PBS, add 2 ml of recombinant trypsin solution and digest for 2-5 min until most cells detach. Add 5 ml of complete culture medium to stop digestion, transfer cells to centrifuge tubes, centrifuge at 1400 rpm for 5 min, discard the supernatant, resuspend in 5 ml of passage medium, count and seed into culture flasks, with a cell density of 8000 cells / cm³. 2 The cells were passaged in a tri-gas incubator at 37°C with 5% oxygen and 5% CO2. When the cell confluence reached 80%–90%, the cells were digested with TrypLE Express for 3 minutes to obtain P1 generation cells. The same procedure was then followed to passage subsequent generations of adipose-derived mesenchymal stem cells (P2–P9). The passage medium used in this example contained: 12% fetal bovine serum, 1% L-glutamine, 12 μg / mL epidermal growth factor (EGF), 0 μg / mL zinc monomethionine, and DMEM-F12 to 100%.

[0096] Example 7: Isolation and passage of adipose-derived mesenchymal stem cells This embodiment refers to Embodiment 1 for related operations.

[0097] (1) Processing fat donated by volunteers that has been transported to the laboratory via a cold chain at 2-8°C in a biosafety cabinet; (2) Centrifuge at 100xg for 5 min, transfer the upper adipose tissue, wash it once with D-Hanks solution, centrifuge at 100xg for 5 min, transfer the adipose tissue, add 2 times the volume of 1% type II collagenase and shake to digest for 30 min. (3) After digestion, dilute with an equal volume of D-hanks solution, centrifuge at 100xg for 5 minutes, and collect the cell pellet at the bottom for the next operation; (4) Take the cell pellet obtained in step (3), resuspend it in primary culture medium, take samples and count them, according to 2×10 4 / cm 2 Inoculate into T75 flasks; incubate in a CO2 incubator (5% CO2, 37℃, saturated humidity); (5) After culturing for 3 days, the culture medium is completely replaced once. When the cell confluence reaches more than 80% (around 5 days), the old culture medium is removed, and the cells are washed with D-Hanks solution. 2 ml of recombinant trypsin solution is added to each bottle to digest the cells for 2 min to detach the cells. 10 ml of D-Hanks solution is added to each bottle to dilute the cells. The cells are centrifuged at 100 x g for 10 min. The cell pellet is resuspended in primary supplemented culture medium to obtain primary adipose mesenchymal stem cells (i.e., P0 generation), which can be used for subsequent passage. (6) Cell passage: Wash P0 generation cells with PBS, add 2 ml of recombinant trypsin solution and digest for 2-5 min until most cells detach. Add 5 ml of complete culture medium to stop digestion, transfer cells to centrifuge tubes, centrifuge at 1400 rpm for 5 min, discard the supernatant, resuspend in 5 ml of passage medium, count and seed into culture flasks, with a cell density of 8000 cells / cm³. 2 The cells were passaged in a tri-gas incubator at 37°C with 20% oxygen and 5% CO2. When the cell confluence reached 80%–90%, the cells were digested with TrypLE Express for 3 minutes to obtain P1 generation cells. This process was then repeated for subsequent passages to obtain P2–P9 generation adipose-derived mesenchymal stem cell suspensions. The passage medium used in this example contained: 12% fetal bovine serum, 1% L-glutamine, 12 μg / mL epidermal growth factor (EGF), 120 μg / mL, 80 μg / mL, 50 μg / mL, 15 μg / mL, or 0 μg / mL zinc monomethionine, and DMEM-F12 to 100%.

[0098] Experiment Example 3: Cell Proliferation Experiment Cell proliferation experiments were conducted according to the method in Example 2. Specifically, the cell population doubling time (PDT) of P1, P3, P5, P7, and P9 generations obtained in Examples 2-6 was tested and calculated. The cell population doubling time (PDT value, hours, mean ± sd, n=5, a smaller sd value indicates very small difference between the five generations, the same below) of the five generations of adipose-derived mesenchymal stem cells obtained in each example were as follows: (PDT value of Example 1 = 28.08 ± 0.53), PDT value of Example 2 = 25.27 ± 0.84, PDT value of Example 3 = 21.48 ± 1.32, PDT value of Example 4 = 33.17 ± 1.06, PDT value of Example 5 = 38.32 ± 0.95, PDT value of Example 6 = 44. The results indicate that, with a concentration of 0.58 ± 1.16, the population doubling time (PDT) of adipose-derived mesenchymal stem cells passaged and cultured in a medium containing zinc monomethionine showed a typical trend relationship with the zinc monomethionine concentration. Within the range of 0–80 μg / mL, the cell population doubling time decreased with increasing zinc monomethionine concentration, but remained essentially unchanged after the concentration increased to 120 μg / mL (in other experiments, the mean PDT values ​​obtained using concentrations of 100 μg / mL, 150 μg / mL, and 200 μg / mL were 23.18 h, 21.75 h, and 20.84 h, respectively, all within the range of 20–23 h, showing essentially no change). This discovery has considerable practical value. For example, the concentration of zinc monomethionine can be adjusted appropriately according to specific actual needs, especially the different requirements of cell growth rate. Preferably, the concentration of zinc monomethionine is in the range of 15~100μg / mL, and more preferably, the concentration of zinc monomethionine is in the range of 15~80μg / mL.

[0099] In another experiment, the method of Example 3 of this experiment was used to determine, Example 7, using normoxic subculture, showed that the population doubling time (PDT) values ​​of P1, P3, P5, P7, and P9 cells obtained from subculture medium containing 120 μg / mL zinc monomethionine were 43.41 ± 1.58. Example 7, using normoxic subculture, showed that the population doubling time (PDT) values ​​of P1, P3, P5, P7, and P9 cells obtained from subculture medium containing 80 μg / mL zinc monomethionine were 42.17 ± 1.01. Example 7, using normoxic subculture, showed that the population doubling time (PDT) values ​​of P1, P3, P5, P7, and P9 cells obtained from subculture medium containing 50 μg / mL zinc monomethionine were 43.82 ± 1.73. Example 7, using normoxic subculture, showed that the population doubling time (PDT) values ​​of P1, P3, P5, and P9 cells obtained from subculture medium containing 15 μg / mL zinc monomethionine were 41.57 ± 1.45. Example 7, using normoxic subculture medium, showed that the population doubling time (PDT) values ​​of P1, P3, P5, and P9 cells obtained from P1, P3, P5, and P9 generations were 42.82 ± 1.26. This indicates that the cell population doubling time of cells obtained by passage under normoxic conditions is long, and the concentration of zinc monomethionine has virtually no effect on the cell population doubling time.

[0100] Furthermore, it should be noted that although the research team of the inventors found that zinc glycine also exhibited the above-mentioned similar pattern when conducting similar experiments using umbilical cord mesenchymal stem cells and placental mesenchymal stem cells, when zinc glycine was used in the adipose mesenchymal stem cells of the present invention, the relationship between cell population doubling time and zinc monomethionine concentration mentioned above could not be obtained. For example, when zinc monomethionine was replaced with an equal amount of zinc glycine in Examples 1 to 6 of the present invention, the cell population doubling time (PDT) values ​​of P1, P3, P5, and P9 cells obtained from the passage culture medium were all within a small range of 27 to 33 hours.

[0101] Experimental Example 4: Detection of Surface Markers The P2, P5, and P8 generation adipose-derived mesenchymal stem cells obtained in Examples 1-6 were subjected to a 500× [method / treatment]. g Centrifuge for 5 min, resuspend in 2 mL of 1×PBS, and label with mouse anti-human CD73-PE, CD90-PE, CD105-PE (isotype control: mouse IgG1 antibody) and mouse anti-human CD34-FITC antibody, CD45-FITC antibody, HLA-DR-FITC antibody (isotype control: mouse IgG2a antibody), respectively, and detect by flow cytometry. All antibodies used in the experiment were purchased from BioVision, USA. The results showed that the expression of CD73, CD90, and CD105 in the three passages of adipose-derived mesenchymal stem cells obtained in the six examples was positive (>96%), while the expression of CD34, CD45, and HLA-DR in the three passages of adipose-derived mesenchymal stem cells obtained in the six examples was negative (<2%). For example, the expression levels of the surface markers CD73, CD90, and CD105 of the P5 generation adipose-derived mesenchymal stem cells obtained in Example 4 were 99.62%, 99.37%, and 99.58%, respectively, while the expression levels of the surface markers CD34, CD45, and HLA-DR of the P5 generation adipose-derived mesenchymal stem cells obtained in Example 4 were 0.226%, 0.173%, and 0.292%, respectively.

[0102] Experimental Example 5: Induction of Differentiation and Staining The P4 generation adipose-derived mesenchymal stem cells obtained in Examples 1-6 were seeded into 24-well plates at a specific cell density, with 3 wells for each cell type. Osteogenic / adipogenic differentiation was induced at a density of 3 × 10⁻⁶ cells / well. 4 Cells were seeded at a density of cells / cm² in 24-well plates, with the density halved for chondrogenic differentiation. After 24 hours of culture in DMEM / F12 containing 10% FBS, the medium was replaced with osteogenic, adipogenic, and chondrogenic differentiation media (all purchased from Biological Industriers). The medium was changed every 3 days. After 21 days of induction culture, Alizarin Red-S staining of calcified osteoblasts was used to detect osteogenic differentiation, Oil Red O staining of lipid droplets was used to detect adipogenic differentiation, and Alcian Blue 8GX staining of proteoglycans was used to detect chondrogenic differentiation (all reagents purchased from Sigma-Aldrich). Results showed that the P4 generation adipose-derived mesenchymal stem cells obtained in all six examples possessed excellent osteogenic, adipogenic, and chondrogenic differentiation capabilities. Typically, the staining images of osteogenic, adipogenic, and chondrogenic differentiation of P6 generation adipose-derived mesenchymal stem cells from Example 1 are shown below. Figure 2 A, Figure 2 B. Figure 2 C.

[0103] Experimental Example 6: Adipose-derived mesenchymal stem cells inhibit lymphocyte proliferation The P5 generation adipose-derived mesenchymal stem cells prepared in Examples 1-6 were used in 24-well plates at a density of 1 × 10⁶ cells per well. 5 Cells were seeded and cultured in a three-gas incubator with 5% oxygen, 5% CO2, and 37°C for 2 hours. Then, they were treated with 10 μg / mL mitomycin C (purchased from Sigma-Aldrich) for 3 hours to promote proliferation.

[0104] Freshly isolated peripheral blood mononuclear cells (PBMCs, prepared according to the method described in CN110551686A) were stained with 2.0 µmol / L carboxyfluorescein diacetate succinimide (CFSE, purchased from Beijing Zhijie Fangyuan Co., Ltd.) at 37°C for 10 min, followed by washing away excess CFSE. CFSE-stained PBMCs were co-cultured with mitomycin C-treated adipose-derived mesenchymal stem cells at a ratio of 10:1 for 5 days, and the cells were analyzed by flow cytometry.

[0105] The experiment was divided into three groups: a negative control group (PBMC group), a positive stimulation group (PBMC+PHA-P group), and an experimental group (adipose-derived mesenchymal stem cell inhibition group [PBMC+PHA-P+MSCs group]). Except for the PBMC group, all other groups were stimulated with 10 μg / mL phytohemagglutinin P (PHA-P, purchased from InvivoGen). The proportion of progeny cells was analyzed, and the inhibition rate was calculated using the following formula:

[0106] The proportion of progeny cells in the negative control group (PBMC group) was 2.27%, and the proportion in the positive stimulation group (PBMC+PHA-P group) was 85.47%. Furthermore, the inhibition rates of the P5 generation adipose-derived mesenchymal stem cells prepared in Examples 1-6 were calculated to be 53.25%, 57.41%, 60.36%, 48.47%, 43.62%, and 38.64%, respectively. Additionally, the inhibition rates of the P8 generation adipose-derived mesenchymal stem cells prepared in Examples 1-6 were measured using the same method, with results of 52.27%, 56.74%, 61.52%, 47.36%, 42.84%, and 39.76%, respectively. These results indicate that the higher the concentration of zinc monomethionine in the passage medium, the higher the inhibition rate of the adipose-derived mesenchymal stem cells, a finding that was entirely unexpected. In addition, following the method tested in Example 6 of this experiment, the inhibition rates of P5 and P8 generation adipose-derived mesenchymal stem cells obtained in Example 7 with passage medium containing 50 μg / mL zinc monomethionine were 40.35% and 41.18%, respectively; the inhibition rates of P4 and P7 generation adipose-derived mesenchymal stem cells obtained in Example 7 with passage medium containing 15 μg / mL zinc monomethionine were 39.53% and 38.84%, respectively; and the inhibition rates of P3 and P6 generation adipose-derived mesenchymal stem cells obtained in Example 7 with passage medium containing 0 μg / mL zinc monomethionine were 39.23% and 40.52%, respectively. These results indicate that compared to the passage conditions using 5% oxygen in Example 1, the inhibition rate of mesenchymal stem cells obtained under normoxic conditions, with or without zinc monomethionine, was significantly weaker, and the concentration of zinc monomethionine was independent of the inhibition rate under normoxic conditions.

[0107] Formulation Example 1: Formulation for preparing mesenchymal stem cells Prescription: 2 × 10T mesenchymal stem cells (P5 generation, obtained from Example 1) 6 One, 9.0 mg sodium chloride, magnesium citrate (to achieve a magnesium ion concentration of 5 mmol / L), 0.2 mg soybean lecithin for injection, and water to a final volume of 1 ml.

[0108] Preparation: Dissolve sodium chloride, magnesium citrate and phospholipids in an appropriate amount of water to obtain a saline solution; transfer the P5 generation mesenchymal stem cells obtained in Example 1 into a centrifuge tube, centrifuge at 2000 rpm for 5 min, discard the supernatant, add saline solution to resuspend the cells, and obtain the cell preparation.

[0109] Case Study 7: Efficacy of Mesenchymal Stem Cell Therapy for Premature Ovarian Failure (POF) This experimental case studies investigated the effectiveness of mesenchymal stem cell therapy for premature ovarian failure (POF) using the method described in CN109652366A (Chinese Patent Application No. 2018115685527) by the inventors' team.

[0110] (1) Six-week-old female C57BL / 6 mice were intraperitoneally injected with 50 mg / kg / day of cyclophosphamide (CTX) for 15 consecutive days at the same time each day to establish a POF mouse model. Ovarian reserve function was assessed by indicators such as follicle number, hormone levels, estrous cycle and fertility test.

[0111] (2) Six-week-old female C57BL / 6 mice were randomly divided into three groups: a control group (n=20), a POF model group (n=20), and an adipose-derived mesenchymal stem cell (PD-MSC) treatment group (n=20). Mice in the treatment group were injected twice with adipose-derived mesenchymal stem cell preparation on day 1 and day 14 after POF modeling, at a dose of 2 × 10⁻⁶ per mouse. 5 Adipose-derived mesenchymal stem cells were injected via the tail vein. The POF model group was injected with an equal volume of physiological saline.

[0112] In this experiment, the adipose-derived mesenchymal stem cell injection formulation used was the cell formulation obtained from Formulation Example 1.

[0113] (3) Hormone levels Fourteen and twenty-eight days after the second adipose-derived mesenchymal stem cell transplantation, ten mice from each group were harvested from their orbital sinuses, and serum was separated and stored at -20°C. Enzyme-linked immunosorbent assay (ELISA) was used to analyze the levels of estradiol (E2), follicle-stimulating hormone (FSH), and anti-Müllerian hormone (AMH) (specific methods were performed according to Xiang Li's paper). Results showed that compared with the POF model group, the serum E2 level in the adipose-derived mesenchymal stem cell group was significantly increased and the FSH level was significantly decreased at all time points (p<0.05). Specific results are shown in Table 1 below.

[0114] Table 1:

[0115] Compared with the POF model group on the same detection day, *p<0.05, **p<0.01.

[0116] (4) Follicle count in mouse ovarian tissue Twenty-eight days after the second transplantation of adipose-derived mesenchymal stem cells, ten mice from each group were sacrificed, and the left ovarian tissue of the mice was fixed in 4% paraformaldehyde. The fixed tissue was then dehydrated with alcohol, cleared with xylene, embedded in paraffin, serially sectioned, and sectioned to a thickness of 5 μm. The sections were stained with hematoxylin and eosin (HE) and observed under a microscope.

[0117] The results showed that compared with the control group, the number of primary, secondary, and mature follicles in the POF model group was significantly reduced, while the number of atretic follicles was significantly increased. Twenty-eight days after adipose-derived mesenchymal stem cell (ADMSC) treatment, the number of follicles at all levels recovered to varying degrees, granulosa cell growth increased, apoptosis decreased, ovarian epithelial cell morphology stabilized, and the number of primary, secondary, and mature follicles significantly increased, while the number of atretic follicles significantly decreased. The follicle counts at all levels 28 days after ADMSC transplantation were significantly different from those in the POF group (P<0.01). Specific results are shown in Table 2 below.

[0118] Table 2:

[0119] Compared with the POF model group, *p<0.05, **p<0.01.

[0120] (5) Observation of mouse fertility: On day 28 after adipose-derived mesenchymal stem cell transplantation, male and female mice were housed together at a ratio of 2:1. The fertility rate of the mice was recorded, and the number of offspring was compared to observe the restorative effect of adipose-derived mesenchymal stem cell transplantation on ovarian function in mice. There was a significant difference between the adipose-derived mesenchymal stem cell group and the POF group (P<0.01). The specific results are shown in Table 3 below.

[0121] Table 3:

[0122] Compared with the POF model group, *p<0.05, **p<0.01.

[0123] The results show that adipose-derived mesenchymal stem cell transplantation can significantly improve the ovarian reserve function of POF mice, increase the number of follicles, raise estrogen and progesterone levels, and restore fertility in some mice, indicating that adipose-derived mesenchymal stem cells can be applied to the clinical treatment of POF.

[0124] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A method for isolating and passage-culturing adipose-derived mesenchymal stem cells, comprising two stages: (a) isolating and culturing primary adipose-derived mesenchymal stem cells and (b) passage-culturing adipose-derived mesenchymal stem cells, wherein: (a) The stage of isolating and culturing primary adipose-derived mesenchymal stem cells includes the following steps: (a1) Process fat samples transported to the laboratory via a cold chain at 2-8°C in a biosafety cabinet; (a2) Centrifuge the fat sample, remove the upper adipose tissue, wash it with D-Hanks solution, centrifuge it again, remove the adipose tissue, add 1% type II collagenase and shake to digest. (a3) After digestion, dilute with an equal volume of D-hanks solution, centrifuge, and collect the cell pellet at the bottom for the next step; (a4) Take the cell pellet obtained in step (a3), add primary supplemented culture medium to resuspend it, take samples to count, and seed it into culture flasks according to the specified cell amount; place it in a CO2 incubator for culture; (a5) After culturing for 3 days, the culture medium was completely replaced once. When the cell confluence reached more than 80%, the old culture medium was removed, the cells were washed with D-Hanks solution, recombinant trypsin solution was added to digest the cells to cause them to detach, D-Hanks solution was added to dilute, centrifuged, and the cell pellet was resuspended in primary supplemented culture medium to obtain primary adipose mesenchymal stem cells, i.e., P0 generation. (b) The passage culture of adipose-derived mesenchymal stem cells includes the following steps: P0 generation cells are washed with PBS, digested with recombinant trypsin solution until most cells detach, digestion is stopped by adding complete culture medium, the cells are transferred to centrifuge tubes, centrifuged, the supernatant is discarded, the cells are resuspended in passage culture medium, counted and seeded into culture flasks, and passaged in a tri-gas incubator of 5% oxygen, 5% CO2, and 37°C. When the cell confluence reaches more than 80%, digestion is performed with digestive enzymes to obtain P1 generation cells; then the same procedure is followed for subsequent passages to obtain cell suspensions of adipose-derived mesenchymal stem cells of each generation. The passage culture medium contained: 12% fetal bovine serum, 1% L-glutamine, and 12 μg / mL epidermal growth factor. single egg Zinc amino acids 15~100μg / mL, DMEM-F12 added to 100%.

2. The method according to claim 1, wherein the subculture medium contains... Zinc monomethionine The concentration is 15~75μg / mL.

3. The method according to claim 1, wherein the subculture medium contains... Zinc monomethionine The concentration is 50 μg / mL.

4. The method according to claim 1, wherein the PBS is a disodium hydrogen phosphate / sodium dihydrogen phosphate buffer with a phosphate ion concentration of 0.025M and a pH of 6.

8.

5. The method according to claim 1, wherein the complete culture medium is DMEM-F12 medium containing 12% fetal bovine serum.

6. The method according to claim 1, wherein in step (b) the cell suspensions of adipose-derived mesenchymal stem cells of passages P1 to P9 are obtained through passage.

7. The method according to claim 1, wherein the D-Hanks solution is formulated as follows: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4·12H2O, 0.35g NaHCO3, and water to 1000ml.

8. The method according to claim 1, wherein the primary supplemental culture medium is prepared using DMEM-F12 medium as the substrate and supplemented with: 1% platelet lysate, 1% human serum albumin, 2 μg / ml recombinant insulin, 15 ng / ml EGF, 25 ng / ml bFGF, 0.12% thioglycerol, and 1% fructose.

9. According to the method of claim 1, in step (a2), both centrifugations are performed at 100 x g for 5 min.

10. According to the method of claim 1, in step (a2), add 2 times the volume of 1% type II collagenase and shake to digest for 30 min.

11. In the method of claim 1, in step (a3), the centrifugation is performed at 100 x g for 5 min.

12. According to the method of claim 1, in step (a4), seeding cells into the culture flask according to the specified cell amount means seeding cells at a amount of 1~5×10⁻⁶. 4 / cm 2 Inoculate into T75 vials.

13. According to the method of claim 1, in step (a4), seeding cells into the culture flask according to the specified cell amount means seeding cells at a amount of 2 × 10⁻⁶ cells / flask. 4 / cm 2 Inoculate into T75 vials.

14. According to the method of claim 1, in step (a4), the conditions for cultivation in the CO2 incubator are: 5% CO2, 37°C, and saturated humidity.

15. According to the method of claim 1, in step (a5), 2 ml of recombinant trypsin solution is added to each bottle to digest the cells for 2 min.

16. According to the method of claim 1, in step (a5), 10 ml of D-hanks solution is added to each bottle for dilution.

17. In the method of claim 1, in step (a5), centrifugation is performed at 100 x g for 10 min.

18. The method according to claim 1, wherein the DMEM-F12 culture medium is formulated as follows: anhydrous calcium chloride 116.6 mg, L-leucine 59.05 mg, linoleic acid 0.042 mg, copper sulfate pentahydrate 0.0013 mg, L-lysine hydrochloride 91.25 mg, lipoic acid 0.105 mg, ferric nitrate nonahydrate 0.05 mg, L-methionine 17.24 mg, phenol red 8.1 mg, ferrous sulfate heptahydrate 0.417 mg, L-phenylalanine 35.48 mg, 1,4-butanediamine dihydrochloride 0.081 mg, potassium chloride 311.8 mg, L-serine 26.25 mg, sodium pyruvate 55 mg, magnesium chloride 28.64 mg, L-threonine 53.45 mg, and vitamin H. 0.0035 mg, anhydrous magnesium sulfate 48.84 mg, L-alanine 4.45 mg, D-calcium pantothenate 2.24 mg, sodium chloride 7000 mg, L-asparagine 7.5 mg, choline chloride 8.98 mg, anhydrous sodium dihydrogen phosphate 54.35 mg, L-aspartic acid 6.65 mg, folic acid 2.65 mg, disodium hydrogen phosphate 71.02 mg, L-cysteine ​​hydrochloride 17.56 mg, i-inositol 12.6 mg, zinc sulfate heptahydrate 0.432 mg, L-glutamate 7.35 mg, nicotinamide 2.02 mg, L-arginine hydrochloride 147.5 mg, L-proline 17.25 mg, pyridoxal hydrochloride 2 mg. L-Cystine hydrochloride 31.29 mg, L-Tryptophan 9.02 mg, Pyridoxine hydrochloride 0.031 mg, L-Glutamine 365 mg, L-Tyrosine 38.4 mg, Riboflavin 0.219 mg, Glycine 18.75 mg, L-Valine 52.85 mg, Thiamine hydrochloride 2.17 mg, L-Histidine hydrochloride 31.48 mg, D-Glucose 3151 mg, Thymidine 0.365 mg, L-Isoleucine 54.47 mg, Hypoxanthine 2 mg, Vitamin B12 0.68 mg, and water added to 1000 mL; Preparation: Dissolve all materials in 1000 mL of water, filter through a 0.22 µm microporous membrane for sterilization, and the product is ready.

19. The method of claim 1, wherein: It also includes testing the primary adipose-derived mesenchymal stem cells obtained from isolation and culture, and identifying cell morphology and / or immunophenotype. The immunophenotypic identification refers to the detection of CD73, CD90, CD105, CD19, CD11b, CD31, CD45, HLADR, and CD34.

20. According to the method of claim 1, in step (b), the cells are passaged in an incubator until the cell fusion rate reaches 80% to 90%.

21. The method according to claim 1, wherein in step (b), the digestive enzyme is TrypLE Express digestive enzyme.

22. According to the method of claim 1, in step (b), the cell seeding density in the culture flask is 6000-10000 cells / cm². 2 .

23. According to claim 1, step (b) is performed as follows: P0 generation cells are washed with PBS, digested with 2 ml of recombinant trypsin solution for 2-5 min until most cells detach, digestion is terminated with 5 ml of complete culture medium, cells are transferred to centrifuge tubes, centrifuged at 1400 rpm for 5 min, the supernatant is discarded, the cells are resuspended in 5 ml of passage culture medium, and then counted and seeded into culture flasks at a cell density of 8000 cells / cm³. 2 The cells were passaged in a tri-gas incubator containing 5% oxygen, 5% CO2, and 37°C. When the cell fusion rate reached 80%~90%, the cells were digested with TrypLE Express for 3 minutes to obtain P1 generation cells. Then, the same procedure was followed to pass the cells to the next generation, and cell suspensions of each generation of adipose-derived mesenchymal stem cells were obtained.

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