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CN117305236A - Adipose-derived mesenchymal stem cell low-oxygen atmosphere culture and application thereof in treating premature ovarian failure - Google Patents

Adipose-derived mesenchymal stem cell low-oxygen atmosphere culture and application thereof in treating premature ovarian failure Download PDF

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CN117305236A
CN117305236A CN202311275612.7A CN202311275612A CN117305236A CN 117305236 A CN117305236 A CN 117305236A CN 202311275612 A CN202311275612 A CN 202311275612A CN 117305236 A CN117305236 A CN 117305236A
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cells
adipose
mesenchymal stem
stem cells
cell
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王正
肖海蓉
刘冰
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BOYALIFE Inc
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BOYALIFE Inc
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Abstract

The invention relates to a low-oxygen atmosphere culture of adipose-derived mesenchymal stem cells and application thereof in treating premature ovarian failure. In particular, the method for separating and subculturing the adipose-derived mesenchymal stem cells comprises two stages of (a) separating and culturing primary adipose-derived mesenchymal stem cells and (b) subculturing adipose-derived mesenchymal stem cells, wherein in the step (b), the adipose-derived mesenchymal stem cells are subjected to 5% oxygen and 5% CO 2 Subculturing in a three-gas incubator at 37 ℃. The invention also relates to a method for treating premature ovarian failure by using the low-oxygen environment-cultured adipose-derived mesenchymal stem cells and application of the cell preparation of the low-oxygen environment-cultured adipose-derived mesenchymal stem cells in treating premature ovarian failure.

Description

Adipose-derived mesenchymal stem cell low-oxygen atmosphere culture and application thereof in treating premature ovarian failure
Technical Field
The invention belongs to the technical field of medical biology, relates to a method for culturing adipose-derived mesenchymal stem cells, in particular to a method for improving proliferation efficiency of adipose-derived mesenchymal stem cells, and particularly relates to a method for culturing adipose-derived mesenchymal stem cells by using a low-oxygen atmosphere environment so as to improve proliferation efficiency of adipose-derived mesenchymal stem cells.
Background
Premature ovarian failure (Premature ovarian failure, POF) refers to the phenomenon of amenorrhea and infertility in women before age 40 due to ovarian failure. POF is a disease characterized by amenorrhea, infertility, estrogen deficiency, follicular reduction, and gonadotrophin elevation, accompanied by a range of low estrogen symptoms such as: hot flashes, excessive sweating, flushing of the face, hyposexuality, etc., seriously affect the physical and mental health of women. Furthermore, women with POF have an increased risk of developing osteoporosis and cardiovascular disease. POF is one of the important causes of female infertility. The incidence of POF in women of childbearing age is about 1-3%, and there is a trend toward rising and younger.
Diagnostic criteria for POF according to the guidelines of the European Society for Human Reproduction and Embryology (ESHRE): at least 4 months of menorrhagia or amenorrhea, two FSH levels, more than 4 weeks apart, are elevated by >40IU/L. Premature ovarian failure is of unknown etiology, and may be associated with genetic and autoimmune diseases, environmental factors, and iatrogenic and idiopathic conditions, there is no effective treatment. Hormone Replacement Therapy (HRT) is one of the most common treatments for POF, but is not ideal and has been shown to increase the risk of venous thrombosis, breast and ovarian cancer. POF can also show symptoms of hectic fever, hyperhidrosis, anxiety, depression, palpitation, insomnia and other climacteric symptoms besides the symptoms of amenorrhea, infertility and the like, and can accelerate female aging, so that postmenopausal diseases such as osteoporosis, cardiovascular diseases, dementia and the like can be caused, and the quality of life and the service life of females can be influenced.
The etiology of POF is complex and not yet fully elucidated, and may be related to autoimmune response, infection, genetic factors, therapeutic effects of chemotherapy, radiotherapy, surgery, etc., and endocrine dysfunctions, and there is no effective treatment. Currently, the most common treatment for POF is hormone replacement therapy (hormone replacement therapy, HRT). Although the therapy has a certain relieving effect on the clinical symptoms of POF, HRT can not fundamentally repair damaged ovaries and restore the ovarian function. Furthermore, studies have shown that long-term HRT treatment increases the risk of heart disease and stroke, possibly increasing the risk of breast and ovarian cancer. Thus, new therapeutic strategies are needed to restore ovarian function in POF patients.
Mesenchymal stem cells (Mesenchymal stem cell, MSC), such as human Mesenchymal stem cells, were originally isolated from bone marrow, were derived from a class of tissue stem cells of mesoderm having multipotent differentiation potential and self-renewal ability, and were capable of differentiating into various adult cells such as osteoblasts, chondrocytes, adipocytes, endothelial cells, neural cells, myocytes, hepatocytes, etc., under specific conditions in vivo and in vitro (Caplan AI. Mesenchyal stem cells J ortho 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 immunoregulatory and hematopoietic support effects and are easy for exogenous gene expression. Therefore, the mesenchymal stem cells not only tissue engineer seed cells in bone, cartilage and myocardial construction and carrier cells important in gene therapy, but also have wide application prospects in hematopoietic stem cell transplantation and organ transplantation because the mesenchymal stem cells promote hematopoietic reconstruction and inhibit graft versus host reaction functions. Mesenchymal stem cells have the characteristic of in vitro adherent growth, and by utilizing the characteristic, people have successfully isolated and cultured the mesenchymal stem cells from various tissues such as liver, kidney, pancreas, muscle, cartilage, skin, peripheral blood and the like.
A class of stem cells having multipotent differentiation potential has been isolated from adipose tissue and successfully differentiated into adipocytes, osteoblasts, chondrocytes, myocytes, etc. in an in vitro culture special system, and this differentiable stem cell is recognized as adipose-derived mesenchymal cells, i.e., adipose stem cells or adipose mesenchymal stem cells (adipose-derived stem cells, ADSCs), which have been isolated from adipose tissue in recent years, have multipotent characteristics of differentiated polyblast, including the ability of mesoderm, endoderm and ectoderm differentiated cells, capable of secreting many potentially advantageous growth factors and cytokines, and a cell-based therapeutic approach to promote wound healing has been rapidly developed. Adipose-derived stem cells are a group of multifunctional mesenchymal stem cells that can differentiate into other cell lines, which have a large number of adipose-derived cells in non-adipose (mesenchymal) fragments of adipose tissue, and are easy to isolate and obtain.
Adipose-derived stem cells are recommended as an adjuvant treatment method for various tissue defects. In the wound healing process, revascularization is an important step in the formation of new blood vessels, a necessary condition to support granulation tissue formation and keratinocyte survival expansion healing wounds. It is therefore believed that adipose-derived stem cells promote wound healing by rapidly promoting revascularization. In addition, adipose-derived stem cells have been shown to promote wound healing by cell differentiation and secretion of long factors that promote collagen synthesis and dermal fibroblast migration.
Adipose-derived stem cells have been applied to the skin soft tissue wound surface of animals in combination with extracellular matrix scaffold components to find adipose-derived stem cells to promote vascularization capacity of the scaffold. The research results show that the promotion effect of the adipose-derived stem cells on wound healing is related to the promotion of epithelialization and granulation tissue formation of the adipose-derived stem cells, and the research results of the potential epidermal cell differentiation capability of the adipose-derived stem cells also suggest that the remote differentiation capability of the adipose-derived stem cells is a reaction of the multifunctional adipose-derived stem cells to the microenvironment of the wound. Remodeling of local blood vessels by adipose-derived stem cells involves direct differentiation into vascular endothelial cells and secretion of vascular growth factors, both of which are beneficial for regeneration of blood vessels. That is, adipose-derived stem cells promote healing of wound surfaces through cell differentiation and angiogenesis. In addition, adipose-derived stem cells and platelet-derived growth factors, a well-known factor involved in the normal wound healing process, are used in combination for wound treatment. The research results show that the combined application of the two has a synergistic effect, namely, the healing of the wound surface can be promoted by improving the level of the growth factors.
In theory, cell therapy has a broad prospect because the multifunctional stem or progenitor cells promote wound healing by providing many beneficial factors to the regeneration and persistence of the wound's missing tissue. Nevertheless, studies on the mechanisms of adipose-derived stem cells on wound healing remain to be explored further. Remodeling of local blood vessels by adipose-derived stem cells involves direct differentiation into vascular endothelial cells and secretion of angiogenic growth factors, both of which are beneficial for regeneration of blood vessels.
The application prospect of the adipose-derived mesenchymal stem cells is verified by application to animal models of various diseases, and the adipose-derived mesenchymal stem cells are used for the intervention treatment of diabetes, liver injury repair, muscle reconstruction of the shoulder, type 2 diabetes yang eruption, myocardial infarction, cerebral infarction, cognitive dysfunction, stroke, intervertebral disc repair, glioblastoma treatment, bone defect, post-traumatic angiogenesis, rheumatoid arthritis, lip fissure, heart failure, colonitis, urinary incontinence and the like. Researchers have transformed adipose-derived mesenchymal stem cells into cardiomyocytes, both reprogramming mature stem cells and improving treatment of heart disease. Also, adipose-derived mesenchymal stem cells and fibrin glue are compounded and then injected to the left ventricle wall of a rat with myocardial infarction, and ADSCs has great prospect in clinical tissue engineering for treating myocardial infarction. The adipose-derived mesenchymal stem cells are used as seed cells to be placed in the reticular stent, so that the skull injury of the dog is successfully repaired, and the skull injury is clinically treated. After the adipose-derived mesenchymal stem cells of abdominal fat of heart patients are injected into the heart, the damage of the heart is reduced, the blood flow is increased, the capacity of the heart to receive oxygenated blood is improved after the stem cells are injected into the heart for 6 months, the capacity of the left ventricle of the heart to send out blood is increased by 3.5 times, SPELT imaging proves that the capacity of the heart pump of the patient receiving the adipose-derived mesenchymal stem cells is increased by 5.7%, and nuclear magnetic scanning also shows that the average myocardial scar area of the patient is reduced from 31.6% to 15.4%, and the research result is published in the American heart Association of the year of science of 2010. The adipose-derived mesenchymal stem cells can regulate growth hormone, cytokine and the like in surrounding tissues to prevent and treat apoptosis in the process of tissue repair, and experiments prove that the adipose-derived mesenchymal stem cells can secrete various skin growth factors such as fibroblast growth factors (Basic fibroblastgrowth factor, bFGF) and the like to promote fibroblast proliferation, and have the effects of resisting photoaging, resisting oxidization, resisting wrinkles, resisting ultraviolet radiation and the like. There are studies on adipose tissue-derived stem cells to treat patient complications anorectal frequency that have entered phase II clinical trials. And the human adipose-derived mesenchymal stem cells are transplanted into breasts of patients with breast atrophy, different sizes or breast augmentation, the sizes of the left and right breasts are balanced and symmetrical, and the breasts are detected by the X-ray, so that the repaired breasts are naturally soft, completely free from calcification, free from cyst and free from obvious injection scars. Recent clinical studies have shown that intramuscular injection of adipose-derived mesenchymal stem cells also has a therapeutic effect on diabetic foot and occlusive arteriosclerosis. After 6 months of injection of adipose-derived mesenchymal stem cells, the resting pain of the patient was clinically alleviated, the distance of painless walking was significantly prolonged, and no complications were found. Experiments show that adipose tissue-derived stem cells induce islet-like cells to reinfusion diabetics, and the patients are found to have reduced exogenous insulin dosage after 23 months of postoperative follow-up, the average weight of the patients is increased, and the patients have no discomfort or side effects.
The characteristic of the adhesion strength of the adipose-derived mesenchymal stem cells to the extracellular matrix is rapid, and the mesenchymal stem cells derived from human adipose which are screened and cultured in vitro can be specifically developed in domestic and foreign methods. The biological performance of the stem cells can achieve the purposes of improving functions, avoiding potential harmful genes, controlling differentiation time and different differentiation directions of the stem cells, and the like, and has irreplaceable guiding significance in the historic period in basic scientific research, clinical treatment, stem cell treatment research direction, skin regeneration injury repair, high tissue engineering skin construction and the like.
Adipose-derived mesenchymal stem cells (ADSCs) are used as adipose-derived mesenchymal stem cells, the materials are convenient to obtain, the extraction efficiency is 40 times higher than that of bone marrow mesenchymal stem cells, and the proliferation speed is faster under the in vitro culture condition; has multi-directional differentiation ability, and can differentiate into adipocytes, osteoblasts, chondrocytes, cardiomyocytes, and even nerve cells. The clinical experiments in phase I and phase II at present prove that the ADSCs are safe and effective in repairing injuries of various organs such as heart, rectum, mammary gland and the like, and the ADSCs have the effect of promoting skin regeneration and repair.
ADSCs present their inherent advantages in several respects. ADSCs belong to the monocyte family and are one of the adult stem cells. It is derived from treated adipose tissue, grows in a cell culture dish in an adherent and adherent manner during culture, and has various differentiation abilities, not only into adipocytes, but also into myocytes, chondrocytes, nerve cells, vascular endothelial cells, bone cells, etc., so that they are also widely used in tissue engineering. For example, ADSCs can differentiate into adipocytes by self-replication, playing a role in tissue regeneration; ADSCs can differentiate into vascular endothelial cells or peripheral cells; ADSCs can secrete vascular growth factors under hypoxic or other conditions; when ADSCs and fat cells are transplanted together, the ADSCs can differentiate into endothelial cells, prevent fibrosis and fat necrosis, and improve the survival rate of fat; according to the recent results of many specific researches, ADSCs can express various cytokines VEGF, IGF, TGF-beta 1, bFGF, EGF and the like, and can prevent apoptosis and maintain the survival rate of adipocytes by expressing vascular growth factors such as VEGF, IGF-1 and the like.
The prior art discloses a number of methods for culturing adipose-derived mesenchymal stem cells. For example, CN106479970a (201611050980.1) discloses a method for large scale culturing of human adipose mesenchymal stem cells. The method for culturing human adipose-derived mesenchymal stem cells on a large scale comprises the steps of adding a adipose-derived mesenchymal stem cell culture medium into a glass culture flask, and incubating and balancing the culture medium for 30 minutes at 35 ℃ in an incubator with 95% of CO2 and 5% of humidity; regulating the temperature of the adipose-derived mesenchymal stem cell culture medium to 35 ℃, and adding the pretreated microcarrier and the adipose-derived mesenchymal stem cells to the culture medium at pH7.05, and stirring for 2 hours at 5-20 RPM; the temperature is raised to 37 ℃ and the culture is carried out for 96 hours under the conditions of 15RPM-40RPM and 5% CO2, and the adipose-derived mesenchymal stem cell culture medium is supplemented. Compared with the existing method, the method for culturing the human adipose-derived mesenchymal stem cells on a large scale is believed to obtain more cells after proliferation, has strong cell activity and proliferation capacity, and can well maintain the morphology and good stem cell characteristics of the adipose-derived mesenchymal stem cells.
CN104762260a (201510197879.8) relates to a method for preparing adipose-derived mesenchymal stem cells, which is characterized by comprising the following steps: step 1: obtaining fat, pretreating, carrying out enzymolysis, centrifuging, and collecting precipitate to obtain adipose-derived mesenchymal stem cells; step 2: culturing the adipose-derived mesenchymal stem cells, when the adipose-derived mesenchymal stem cells grow to 80% and are fused, discarding the culture solution, adding PBS for cleaning, adding EDTA-pancreatin solution for digestion, stopping digestion, centrifuging, and subculturing. The invention is believed to have the following advantages: 1. the purity of stem cells obtained by culture is high; 2. various factors secreted in the culture process of the adipose-derived mesenchymal stem cells are recovered, so that proliferation of the epidermal cells can be obviously promoted, and the regeneration and replacement of the epidermal cells can be quickened, and an obvious anti-aging effect is achieved.
CN104974984a (application No. 201510173453.9) discloses an expansion culture method of adipose tissue-derived mesenchymal stem cells, comprising the following steps: carrying out resuspension treatment on the isolated adipose tissue-derived mesenchymal stem cells by using an adipose-derived stem cell culture medium to obtain a heavy suspension containing the mesenchymal stem cells; inoculating the heavy suspension into a stem cell incubator for expansion culture; after the expansion culture is carried out for 24 hours, the adipose-derived stem cell culture medium is replaced, then the adipose-derived stem cell culture medium is replaced every two days, and when the adipose-derived stem cell culture medium grows to be fused to 80-90%, the adipose-derived stem cell culture medium is subjected to the pancreatin digestion treatment and then is subjected to the subculture; the adipose-derived stem cell culture medium comprises 0.1-10 v/v% of fetal bovine serum, 1-10 v/v% of gentamicin and 80-98.9 v/v% of high-sugar DMEM culture medium; the stem cell incubator is a stem cell incubator treated with a fibronectin coating. The amplification culture method of adipose tissue-derived mesenchymal stem cells is believed to adopt a solid support to which the fibronectin is coated and is attached for the growth of adipose-derived mesenchymal stem cells, and the adipose-derived mesenchymal stem cells can be amplified 400-500 times in two weeks by utilizing the synergistic screening culture of low-concentration fetal calf serum and high-concentration gentamicin, and have high purity and can be directionally induced and differentiated into adipose tissues.
CN106520686a (application No. 201610887111.8) provides a method for culturing adipose-derived mesenchymal stem cells. The adipose-derived mesenchymal stem cell culture method comprises the following culture steps: and inoculating the obtained primary adipose-derived mesenchymal stem cells into a stem cell culture medium for culture treatment, wherein the stem cell culture medium contains stem cell factors and interleukin 3 factors, and the content ratio of the stem cell factors to the interleukin 3 factors is (5-20 mu mol/L) to (5-20 ug/ml). The culture method can improve the expansion capacity of the adipose-derived mesenchymal stem cells and obtain higher migration capacity.
CN101984049a (application No. 201010580537.1) discloses a method for isolating mesenchymal stem cells from adipose tissue, which is characterized by comprising the steps of: (1) obtaining adipose tissue: adipose tissues are waste obtained by swelling liposuction operation in professional hospitals or beauty parlors, and are stored for less than 48 hours at 4-20 ℃ under the aseptic condition; (2) preliminary removal of erythrocytes: after resting the fat extract for stratification, carefully removing the underlying liquid, washing with D-Hanks liquid multiple times until the eluate is clear; (3) digesting the fat extract: adding a type I collagenase prepared by D-Hanks liquid to 0.01-2g/100ml, and shaking and digesting for 15-120 minutes at 37 ℃ at 20-400 rpm; (4) obtaining adipose-derived stem cells: centrifuging the digested product at 4deg.C under centrifugal force 450g for 5min, re-suspending the fat stem cell culture medium, and filtering with 100 μm pore size filter membrane to remove impurities to obtain fat stem cells; (5) Removing red blood cells again by means of repeated washing and red blood cell lysis; (6) adipose-derived stem cell count, activity detection and culture: taking 100 μl of the isolated cells and performing cell counting by a cell counting plate; simultaneously taking 100 μl of cells, performing viable cell count by trypan blue staining, and performing 3×10 based on the results of both counts 4 /cm 2 Is inoculated in a T-75 flask at 37℃and a CO2 concentration of 5% and a humidity of 100%. The method of the invention is believed to be capable of obtaining a large number of adipose-derived stem cells in good condition and maintaining good multi-directional differentiation ability by using a small amount of adipose tissues, and the operation method is simple and easy to implement and has strong repeatability. However, the method of this document is directed to mesenchymal stem cells simultaneously when the cells are lysed, which is detrimental to the survival of the stem cells.
In the culture method for efficiently obtaining adipose-derived mesenchymal stem cells disclosed in CN106222134a (application No. 201610605996.8), only the fat pellet part of the intermediate layer after centrifugation was sucked up, and the cell pellet at the bottom of centrifugation was not obtained when fat was separated.
CN102329783a (application No. 201110310461.5) discloses a method for separating adipose mesenchymal stem cells from the inflation fluid in a surgical liposuction procedure, consisting of the steps of: (1) The method comprises the steps of (1) pouring excessive hypotonic expansion liquid into a part to be extracted before extracting fat from the expansion liquid, then placing fat and the expansion liquid obtained in the fat extracting process at room temperature for 10min, and sucking red liquid at the lower part of yellow fat by a pipetting gun after the fat and the expansion liquid are completely separated, namely the expansion liquid containing fat stem cells; (2) Treating the expansion liquid, packaging the collected expansion liquid into a 50ml centrifuge tube, and centrifuging in a centrifuge at 1500rpm for 5min; centrifuging to remove the liquid above the centrifuge tube, collecting precipitate, suspending with PBS liquid for each tube, adding erythrocyte lysate, standing at room temperature for 5min, centrifuging at 1200rpm for 3min, and discarding supernatant; (3) Washing cells, adding 5ml of DMEM (without serum), blowing off the precipitate, filtering by a cell filter, centrifuging the filtered liquid at 1200rpm for 3min, precipitating to obtain adipose-derived mesenchymal stem cells, adding DMEM (without serum), and repeating the above operation for 3 times; (4) cell culture: the obtained cells were inoculated into 75cm2 cell culture flasks, inoculated, and cultured in 5% CO2 at 37℃with the addition of mesenchymal stem cell medium. However, the CN102329783a only performs the separation of stem cells in the swelled liquid portion, not the fat liquid doped with the swelled liquid.
As described in CN112522193B (chinese patent application No. CN 2020115689421), a general method for obtaining human adipose-derived mesenchymal stem cells comprises (a) two stages of isolated culture of primary adipose-derived mesenchymal stem cells and (B) subculture of adipose-derived mesenchymal stem cells, wherein the (B) stage subculture is typically performed in an incubator atmosphere of normal oxygen concentration of 5% carbon dioxide and the balance of air (about 21% oxygen: about 78% nitrogen).
MSCs for clinical research and application require in vitro culture and expansion, and different culture environments affect characteristics of MSCs, such as immunoregulatory function, secretion level of active factors, cell differentiation ability, and the like. How to select a proper culture environment for the application direction of MSC, thereby generating cells which are more beneficial to clinic is a problem which needs to be solved at present. At present, MSC is usually cultured in vitro in ambient normoxic (20% oxygen) but in reality the O2 concentration in the physiological microenvironment of MSC is much lower [ Nekanti U, et al Increased proliferation and analysis of differential gene expression in human Wharton's jelly-derived mesenchymal stromal cells under hypoxia, int J Biol Sci, 2010, 6 (5): 499]. In vivo, the O2 level of the tissue is maintained within a small range called "physiological normoxic", typically 2% -9%.
Currently, many studies have been made to select an intermediate 5% oxygen environment (i.e., an atmosphere of 5% carbon dioxide, 5% oxygen and the balance nitrogen) of "physiological normoxic" as a hypoxic culture environment, which achieves some desired effects, in particular, a cell doubling time (also referred to as population doubling time, PDT, in hours) can be significantly shortened by, for example, decreasing the oxygen concentration. However, it is well known that a reduction in cell doubling time means an increase in cell senescence, which is undesirable in some cases for clinical studies and the use of mesenchymal stem cells. It would be highly valuable for clinical applications and would be highly desirable by those skilled in the art if the culture conditions could be adjusted by simple means to further adjust the cell doubling time of MSCs, and thus improve the proliferation efficiency of adipose-derived mesenchymal stem cells.
Disclosure of Invention
An object of the present invention is to provide a method of separately culturing adipose-derived mesenchymal stem cells, or to provide a method of improving proliferation efficiency of adipose-derived mesenchymal stem cells, or to provide a method of culturing adipose-derived mesenchymal stem cells using a low-oxygen environment to improve proliferation efficiency thereof; alternatively, it is an object of the present invention to use the adipose mesenchymal stem cells obtained by the method of the present invention for the treatment of premature ovarian failure. It has been unexpectedly found that the use of the stem cell preparation of the present invention for the treatment of premature ovarian failure exhibits satisfactory results. The present invention has been completed based on such findings.
To this end, the present invention provides in a first aspect a method for isolating and subculturing adipose-derived mesenchymal stem cells comprising (a) two stages of isolating and culturing primary adipose-derived mesenchymal stem cells and (b) subculturing adipose-derived mesenchymal stem cells, wherein:
(a) The stage of isolated culture of primary adipose-derived mesenchymal stem cells comprises the following steps:
(a1) Processing fat samples transported to a laboratory via a cold chain at 2-8 ℃ in a biosafety cabinet;
(a2) Centrifuging a fat sample, removing upper adipose tissue, washing the upper adipose tissue once again by using D-Hanks liquid, centrifuging again, removing adipose tissue, and adding 1% type II collagenase for shake digestion;
(a3) After digestion, adding one volume of D-hanks liquid for dilution, centrifuging, and reserving cell sediment at the bottom layer for subsequent operation;
(a4) Taking the cell sediment obtained in the step (3), adding a primary supplementary culture medium for resuspension, sampling and counting, and inoculating the cell sediment into a culture bottle according to the specified cell quantity; CO placement 2 Culturing in an incubator;
(a5) Culturing for 3D, completely replacing the culture medium once until the cell fusion degree reaches more than 80%, removing the old culture medium, cleaning cells by using D-hanks solution, adding recombinant pancreatin solution to digest the cells, allowing the cells to fall off, adding D-hanks solution for dilution, centrifuging, and re-suspending cell precipitation by using a primary supplementary culture medium to obtain primary adipose-derived mesenchymal stem cells, namely P0 generation;
(b) The stage of subculturing the adipose-derived mesenchymal stem cells comprises the following steps: taking P0 generation cells, washing with PBS, adding a pancreatin solution for digestion until most of the cells fall off, adding a complete culture medium for stopping digestion, transferring the cells into a centrifuge tube, centrifuging, discarding the supernatant, adding a subculture medium for resuspension, counting, inoculating to a culture bottle, and placing 5% oxygen and 5% CO 2 Subculturing in a three-gas incubator at 37 ℃ until the cell fusion rate reaches more than 80%, and digesting by using digestive enzyme to obtain P1 generation cells; and then carrying out passage for the next generation according to the method of the step, and sequentially obtaining the cell suspension of the adipose-derived mesenchymal stem cells for each generation.
According to the method of the first aspect of the invention, the subculture medium comprises: 12% of fetal bovine serum, 1% of L-glutamine, 12 mu g/mL of Epidermal Growth Factor (EGF),Zinc monomethionine15-120. Mu.g/mL, e.g., 15-100. Mu.g/mL, e.g., 15-75. Mu.g/mL, e.g., 50. Mu.g/mL, DMEM-F12 is added to 100%.
According to the method of the first aspect of the invention, the PBS is a disodium hydrogen phosphate/sodium dihydrogen phosphate buffer having a phosphate ion concentration of 0.025M and a pH of 6.8.
According to the method of the first aspect of the invention, the complete medium is DMEM-F12 medium comprising 12% fetal bovine serum.
According to the method of the first aspect of the invention, in step (b) a cell suspension of adipose mesenchymal stem cells is obtained by passing through the step (b) for P1-P15 passages, for example, P1-P9 passages.
According to the method of the first aspect of the invention, the formula of the D-Hanks liquid comprises the following components: 8.0g NaCl, 0.4g KCl, 0.06g KH 2 PO 4 Na of 0.08g 2 HPO 4 .12H 2 O, 0.35g NaHCO 3 Water to 1000ml.
According to the method of the first aspect of the invention, the primary supplemental medium is formulated with DMEM-F12 medium as a substrate and added: 1% platelet lysate, 1% human serum albumin, 2. Mu.g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF, 0.12% thioglycerol, 1% fructose.
According to the method of the first aspect of the present invention, in step (a 2), both the centrifugation is performed at 100Xg for 5 min.
According to the method of the first aspect of the present invention, in the step (a 2), 2 times the volume of 1% type II collagenase is added for shaking digestion for 30min.
According to the method of the first aspect of the present invention, in step (a 3), the centrifugation is performed at 100Xg for 5 min.
According to the method of the first aspect of the present invention, in the step (a 4), the step of inoculating the culture flask with a predetermined cell amount means that the cell amount is 1 to 5X 10 4 /cm 2 Inoculate to T75 flask.
According to the method of the first aspect of the present invention, in the step (a 4), the inoculation into the culture flask in accordance with the prescribed cell amount means that the cell amount is 2X 10 4 /cm 2 Inoculate to T75 flask.
In the method according to the first aspect of the invention, in step (a 4), CO 2 The conditions for culturing in the incubator are: 5% CO 2 Saturated humidity at 37 ℃.
According to the method of the first aspect of the present invention, in step (a 5), 2ml of the recombinant pancreatin solution is added to each flask to digest the cells for 2min.
According to the method of the first aspect of the present invention, in the step (a 5), 10ml of D-hanks liquid is added to each bottle for dilution.
According to the method of the first aspect of the present invention, in step (a 5), centrifugation is performed at 100Xg for 10min.
The method according to the first aspect of the invention, wherein the DMEM-F12 medium formulation consists of: anhydrous calcium chloride 116.6mg, L-leucine 59.05mg, linoleic acid 0.042mg, cupric sulfate pentahydrate 0.0013mg, L-lysine hydrochloride 91.25mg, lipoic acid 0.105mg, ferric nitrate nonahydrate 0.05mg, L-methionine 17.24mg, phenol red 8.1mg, ferrous sulfate heptahydrate 0.417mg, L-phenylalanine 35.48mg, 1, 4-butanediamine dihydrochloride 0.081mg, potassium chloride 311.8mg, L-serine 26.25mg, sodium pyruvate 55mg, magnesium chloride 28.64mg, L-threonine 53.45mg, vitamin H0.0035mg, anhydrous magnesium sulfate 48.84mg, L-alanine 4.45mg, calcium D-pantothenate 2.24mg, sodium chloride 7000mg, L-asparagine 7.5mg, choline chloride 8.98mg, anhydrous sodium dihydrogen phosphate 54.35mg, L-aspartic acid 6.65mg folic acid 2.65mg, disodium hydrogen phosphate 71.02mg, L-cysteine hydrochloride 17.56mg, i-inositol 12.6mg, zinc sulfate heptahydrate 0.432mg, L-glutamic acid 7.35mg, nicotinamide 2.02mg, L-arginine hydrochloride 147.5mg, L-proline 17.25mg, pyridoxal hydrochloride 2mg, L-cystine hydrochloride 31.29mg, L-tryptophan 9.02mg, pyridoxine hydrochloride 0.031mg, L-glutamine 365mg, L-tyrosine 38.4mg, riboflavin 0.219mg, glycine 18.75mg, L-valine 52.85mg, thiamine hydrochloride 2.17mg, L-histidine hydrochloride 31.48mg, D-glucose 3151mg, thymidine 0.365mg, L-isoleucine 54.47mg, hypoxanthine 2mg, vitamin B12 0.68mg, and water in an appropriate amount to 1000mL; preparing: dissolving each material with 1000ml, and filtering and sterilizing by a microporous filter membrane with the size of 0.22 mu m.
The method according to the first aspect of the present invention, further comprising detecting primary adipose mesenchymal stem cells obtained by isolated culture, detecting cell morphology and/or immunophenotyping.
According to the method of the first aspect of the invention, the immunophenotyping is performed by detecting CD73, CD90, CD105 and CD19, CD11b, CD31, CD45, HLADR, CD 34.
The method according to the first aspect of the invention, wherein in step (b), the cells are subjected to 5% oxygen, 5% CO 2 Subculturing in a three-gas incubator at 37 ℃; it is well known that this three-gas incubator is a saturated humidity condition. The substantial meaning of the gas composition in the three-gas incubator is 5% oxygen, 5% carbon dioxide, and the balance nitrogen. In addition, as shown in step (a), the cells are placed in CO 2 In the incubator at 37℃with 5% CO 2 The gas atmosphere for the culture under saturated humidity conditions was 5% carbon dioxide and the remaining air (about 21% oxygen and about 78% nitrogen), and the gas composition in the incubator, if only the carbon dioxide concentration was indicated and not the oxygen concentration, was the atmosphere of 5% carbon dioxide and the remaining air, to be distinguished from the atmosphere of 5% oxygen, 5% CO of step (5) 2 Is a three-gas culture environment. In short, the three-gas incubator refers to three of nitrogen, oxygen and carbon dioxide, and the total amount thereof is 100%.
According to the method of the first aspect of the invention, in the step (b), the cells are subjected to subculture in an incubator until the cell fusion rate reaches 80% -90%.
The method according to the first aspect of the present invention, wherein in step (b), the digestive enzyme is a TrypLE Express digestive enzyme.
The method according to the first aspect of the present invention, wherein in the step (b), the inoculation density of the cells in the culture flask is 6000 to 10000 cells/cm 2 For example 8000 cells/cm 2 。
The method according to the first aspect of the invention, wherein step (b) is operated as follows: taking P0 generation cells, washing with PBS, adding 2ml of recombinant pancreatin solution for digestion for 2-5min until most of the cells fall off, adding 5ml of complete culture medium for stopping digestion, transferring the cells into a centrifuge tube, centrifuging at 1400rpm for 5min, discarding the supernatant, adding 5ml of passage culture medium for resuspension, counting, inoculating to a culture flask, and obtaining the cells with the density of 8000 cells/cm 2 Placing 5% oxygen and 5% CO 2 Subculturing in a three-gas incubator at 37 ℃ until the cell fusion rate reaches 80% -90%, and using digestive enzyme TrypLE expressss is digested for 3min to obtain P1 generation cells; and then carrying out passage for the next generation according to the method of the step, and sequentially obtaining the cell suspension of the adipose-derived mesenchymal stem cells for each generation.
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 more than 90%.
Further, the second aspect of the present invention provides a adipose-derived mesenchymal stem cell isolated and subcultured by the method of any one of the embodiments of the first aspect of the present invention.
Further, a third aspect of the present invention provides a cell preparation for use in an injection mode, comprising the following components in percentage by weight: adipose-derived mesenchymal stem cells 2×10 6 Sodium chloride 9.0mg, magnesium citrate (the concentration of magnesium ions reaches 5 mmol/L), soybean lecithin for injection 0.2mg and proper amount of water to 1ml; the adipose-derived mesenchymal stem cells are P1-to P9-generation cells and are isolated and subcultured by the method of any one embodiment of the first aspect of the present invention.
The cell preparation according to the third aspect of the invention, which is prepared according to a method comprising the steps of: dissolving sodium chloride, magnesium citrate and phospholipid with proper amount of water to obtain saline solution; transferring the mesenchymal stem cells obtained by subculture into a centrifuge tube, centrifuging (for example, centrifuging at 2000rpm for 5 min), discarding the supernatant, and adding saline solution to resuspend the cells to obtain a cell preparation.
Further, the fourth aspect of the present invention provides the use of the adipose-derived mesenchymal stem cells of any aspect of the present invention, or the adipose-derived mesenchymal stem cells isolated and subcultured by the method of any aspect of the present invention, or the cell preparation of any aspect of the present invention, in the preparation of a medicament for treating premature ovarian failure.
In describing the method steps of the various aspects of the present invention, the specific steps described therein may be distinguished in some details or in language description from the steps described in the examples of the detailed description section below, however, those skilled in the art will be able to summarize the above described method steps in light of the detailed disclosure of the invention throughout.
Any of the embodiments of any of the aspects of the invention may be combined with other embodiments, provided that they do not contradict. Furthermore, in any of the embodiments of any of the aspects of the present invention, any technical feature may be applied to the technical feature in other embodiments as long as they do not contradict. The present invention is further described below.
All documents cited herein are incorporated by reference in their entirety and are incorporated by reference herein to the extent they are not inconsistent with this invention. Furthermore, various terms and phrases used herein have a common meaning known to those skilled in the art, and even though they are still intended to be described and explained in greater detail herein, the terms and phrases used herein should not be construed to be inconsistent with the ordinary meaning in the sense of the present invention.
In the present invention, the term "adipose mesenchymal stem cells" refers to adipose-derived mesenchymal stem cells. Thus, in the context of the present invention, and in particular in relation to the present invention, the term "adipose mesenchymal stem cells" may be used interchangeably with "adipose stem cells", "mesenchymal stem cells", unless explicitly indicated otherwise.
Mesenchymal stem cells have regenerative potential and immunoregulatory capacity, and are found in bone marrow, adipose tissue, umbilical cord blood, amniotic fluid, placenta, dental pulp, tendon, synovial membrane, skeletal muscle, and the like [ Widowati W, et al.Hypoxia in mesenchymal stem cell [M/OL]. Licensee InTech, 2017. Doi:10.5772/62960；Yin J Q, et al. Manufacturing of primed mesenchymal stromal cells for therapy [J]. Nat Biomed Eng, 2019, 3(2): 90-104]. Umbilical cords, which have been used as waste, are favored by researchers and applications because of their wide sources, unexplained disputes, easy separation, low immunogenicity, etc., and a large number of researchers are evaluating the effectiveness and safety of their use in clinical therapy. The international cell therapy association updated the minimum standard of mesenchymal stem cells in 2006: (1) anchorage growth in a standard culture environment; (2) CD73, CD90. CD105 positive expression, the expression rate is more than or equal to 95%, CD34, CD45, CD14 or CD11b, CD79 alpha or CD19, HLA-DR negative expression, the expression rate is less than or equal to 2%; (3) Can differentiate into osteoblasts, adipocytes and chondroblasts in vitro [ Dominici M, et al Minimal criteria for defining multipotent mesenchymal stromal cells, the International Society for Cellular Therapy position statement [ J ] ]. Cytotherapy, 2006, 8(4): 315-317]. Nevertheless, the mesenchymal stem cells prepared by researchers in different laboratories still have great differences in the in vitro proliferation capacity, the immunoregulation capacity, the expression level and the secretion capacity of cytokines and growth factors, and the like, and a more suitable in vitro culture scheme of the mesenchymal stem cells is required to be continuously explored [ Yin J Q, et al Manufacturing of primed mesenchymal stromal cells for therapy [ J ]]. Nat Biomed Eng, 2019, 3(2): 90-104]。
The methods of isolating and subculturing adipose-derived mesenchymal stem cells of the present invention exhibit excellent effects in one or more aspects as described in the context herein. For example, the method of the invention uses 5% oxygen to separate and subculture the adipose mesenchymal stem cells, so that the proliferation capacity of the adipose mesenchymal stem cells can be enhanced, and the phenotype, differentiation capacity and lymphocyte proliferation inhibition capacity of the original adipose mesenchymal stem cells can be maintained.
Drawings
FIG. 1 is a typical microscopic cell morphology of adipose mesenchymal stem cells.
Fig. 2 is a typical osteogenic, adipogenic, chondrogenic differentiation staining pattern of adipose mesenchymal stem cells, fig. 2A, 2B, 2C, respectively.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof. The present invention generally and/or specifically describes the materials used in the test as well as the test methods. Although many materials and methods of operation are known in the art for accomplishing the objectives of the present invention, the present invention will be described in as much detail herein. The following examples further illustrate the invention, but do not limit it.
As not otherwise illustrated, DMEM/F12 used in the present invention was added as a basal medium, and epidermal growth factor, fetal bovine serum and DMEM/F12 were purchased from the brand of GIBCO of ThermoFisher technologies, EGF was purchased from PeproTech, L-glutamine, gentamicin, zinc monomethionine, and the digestive enzyme TrypLE Express were all purchased from Sigma-aldrich.
The complete medium used in the present invention is DMEM-F12 medium containing 12% fetal bovine serum, unless otherwise specified.
The Hank's balanced salt solution used in the present invention, unless otherwise specified, is composed of: the pH was adjusted to 7.4 with 8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO4.7H2O, 0.1g/L MgSO2.6H2O, 0.06g/L Na2HPO4.2H2O, 0.06g/L KH2PO4, 1.0g/L glucose, 0.14g/L CaCl2, 0.35g/L NaHCO3, 0.2g/L phenol red, hydrochloric acid or sodium hydroxide.
Some steps of the method used in the examples of isolation and passaging of adipose-derived mesenchymal stem cells of the present invention were carried out with reference to the relevant procedures described in chinese patent application CN112522193a (CN 2020115689421) of the applicant's team.
In the present invention, the DMEM-F12 medium formulation used in the test was composed as follows, unless otherwise specified: anhydrous calcium chloride 116.6mg, L-leucine 59.05mg, linoleic acid 0.042mg, cupric sulfate pentahydrate 0.0013mg, L-lysine hydrochloride 91.25mg, lipoic acid 0.105mg, ferric nitrate nonahydrate 0.05mg, L-methionine 17.24mg, phenol red 8.1mg, ferrous sulfate heptahydrate 0.417mg, L-phenylalanine 35.48mg, 1, 4-butanediamine dihydrochloride 0.081mg, potassium chloride 311.8mg, L-serine 26.25mg, sodium pyruvate 55mg, magnesium chloride 28.64mg, L-threonine 53.45mg, vitamin H0.0035mg, anhydrous magnesium sulfate 48.84mg, L-alanine 4.45mg, calcium D-pantothenate 2.24mg, sodium chloride 7000mg, L-asparagine 7.5mg, choline chloride 8.98mg, anhydrous sodium dihydrogen phosphate 54.35mg, L-aspartic acid 6.65mg folic acid 2.65mg, disodium hydrogen phosphate 71.02mg, L-cysteine hydrochloride 17.56mg, i-inositol 12.6mg, zinc sulfate heptahydrate 0.432mg, L-glutamic acid 7.35mg, nicotinamide 2.02mg, L-arginine hydrochloride 147.5mg, L-proline 17.25mg, pyridoxal hydrochloride 2mg, L-cystine hydrochloride 31.29mg, L-tryptophan 9.02mg, pyridoxine hydrochloride 0.031mg, L-glutamine 365mg, L-tyrosine 38.4mg, riboflavin 0.219mg, glycine 18.75mg, L-valine 52.85mg, thiamine hydrochloride 2.17mg, L-histidine hydrochloride 31.48mg, D-glucose 3151mg, thymidine 0.365mg, L-isoleucine 54.47mg, hypoxanthine 2mg, vitamin B12 0.68mg, and water in an appropriate amount to 1000mL; preparing: dissolving each material with 1000ml, and filtering and sterilizing by a microporous filter membrane with the size of 0.22 mu m.
In the present invention, the platelet lysate used in the test is readily available from the market, as not specifically described, and as used in the test herein is PLTGold Human Platelet Lysate from Sigma-Aldrich under the designation SCM151.
In the present invention, the type II collagenase used in the test is readily available from the market, as not specifically described, and used in the test herein is available from Gibco.
In the present invention, bFGF (basic fibroblast growth factor) used in the test is readily available from the market, as not specifically described herein, and is available from Sigma-Aldrich under the designation GF003.
In the present invention, EGF (epidermal growth factor) used in the test is readily available from the market, and as not specifically described, gibco, product number PHG0311L, was used in the test herein.
In the present invention, recombinant insulin used in the test is readily available commercially, as not specifically described, and is used in the test herein as available from Solarbio under the designation I8830.
In the present invention, the recombinant pancreatin solution used in the test is readily commercially available, and as not specifically described, the recombinant pancreatin solution of 2000u/ml concentration available from lanbo Kang Si company, cat No. RT2S01 was used in the test herein.
In the present invention, the D-Hanks solution used in the test had the following composition and preparation method, unless otherwise specified: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4.12H2O, 0.35g NaHCO3, water to 1000ml; preparing: dissolving each material with 1000ml, and filtering and sterilizing by a microporous filter membrane with the size of 0.22 mu m.
In the present invention, the primary supplemental medium used in the test was formulated with DMEM-F12 medium as a substrate and included, unless specified otherwise: 1% platelet lysate, 1% human serum albumin, 2. Mu.g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF, 0.12% thioglycerol, 1% fructose.
In the present invention, PBS is disodium hydrogen phosphate/sodium dihydrogen phosphate buffer having a phosphate ion concentration of 0.025M and a pH of 6.8, unless otherwise specified.
In the specific experiments of the present invention, the obtained generation of stem cells were sampled, nucleated cells, i.e., MSC cells were counted using a sysmex hemocytometer, and cell viability was measured by trypan blue staining and sampled for microbial detection.
Example 1: isolated culture of primary adipose-derived mesenchymal stem cells
(1) Processing in a biosafety cabinet the fat donated by volunteers transported to the laboratory via a cold chain at 2-8 ℃;
(2) Centrifuging at 100xg for 5min, removing upper adipose tissue, washing with D-Hanks solution for one time, centrifuging at 100xg for 5min, removing adipose tissue, adding 2 times of 1% type II collagenase, and performing shake digestion for 30min;
(3) After digestion, adding one volume of D-hanks liquid for dilution, centrifuging for 5min at 100xg, and reserving cell sediment at the bottom layer for subsequent operation;
(4) Taking the cell sediment obtained in the step (3), adding a primary supplementary culture medium for resuspension, sampling and counting, and obtaining the cell sediment according to the ratio of 2 multiplied by 10 4 /cm 2 Inoculating to a T75 bottle; CO placement 2 Incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) After culturing for 3D, completely changing the culture medium once until the cell fusion degree reaches more than 80% (about 5D), removing the old culture medium, cleaning the cells with D-hanks solution, adding 2ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 10ml of D-hanks solution into each bottle to dilute, centrifuging for 10min at 100Xg, and re-suspending the cell sediment with the primary supplementary culture medium to obtain the primary adipose mesenchymalStem cells (i.e., P0 generation), which can be used for the next passage; in the embodiment 1, the steps (1) to (5) are performed in the step (4) at a rate of 2X 10 4 /cm 2 After quantitative inoculation into T75 flasks, the number of nucleated cells obtained per flask (average of 5 replicates) was 5.72X10A 6 (n=5) (this data may be referred to herein as cell harvest), and the cell viability was 92.2% (n=5) ]
(6) Cell passage: taking P0 generation cells, washing with PBS, adding 2ml of recombinant pancreatin solution for digestion for 2-5min until most of the cells fall off, adding 5ml of complete culture medium for stopping digestion, transferring the cells into a centrifuge tube, centrifuging at 1400rpm for 5min, discarding the supernatant, adding 5ml of passage culture medium for resuspension, counting, inoculating to a culture flask, and obtaining the cells with the density of 8000 cells/cm 2 Placing 5% oxygen and 5% CO 2 Subculturing in a three-gas incubator at 37 ℃, and when the cell fusion rate reaches 80% -90%, using digestive enzyme TrypLE Express to digest for 3min to obtain P1 generation cells; and then carrying out passage for the next generation according to the method of the step, and sequentially obtaining the cell suspension of the adipose-derived mesenchymal stem cells for the subsequent P2-P9 generation. The subculture medium used in this example comprises: 12% of fetal bovine serum, 1% of L-glutamine, 12 mu g/mL of Epidermal Growth Factor (EGF),Zinc monomethionine50. Mu.g/mL, DMEM-F12 was added to 100%.Zinc monomethionineCAS number 56329-42-1, available fromSigma-Aldrich。
Test example 1: identification of biological characteristics of adipose-derived mesenchymal stem cells
The biological characteristics of the adipose-derived mesenchymal stem cells of the generation P1 to P9 obtained by the method of the example 1 are identified, and the results are as follows:
each generation of cells is in the form of typical stem cells, namely spindle-shaped, full and adherent growth, and the cell morphology of typical P3 generation mesenchymal stem cells under a microscope is shown in fig. 1.
The results of the flow phenotype identification of the generation P1-P9 show that the positive expression of CD73, CD90 and CD105 is more than 98 percent, for example, the positive expression of CD73, CD90 and CD105 of the generation P6 cells is 99.72 percent, 99.33 percent and 99.58 percent respectively; the results of the flow phenotype identification show that all the passages of adipose mesenchymal stem cells have the differentiation potential of osteogenesis, chondrogenesis and adipogenesis, and the microscopic image results of the differentiation potential of osteogenesis, chondrogenesis and adipogenesis of all the passages of adipose mesenchymal stem cells, such as CD 19=0.33, CD 11b=0.18, CD 31=0.12, CD 45=0.43, HLADR=0.36 and CD 34=0.24 of all the passages of adipose mesenchymal stem cells, for example, the differentiation potential of osteogenesis, chondrogenesis and adipogenesis of one batch of adipose mesenchymal stem cells of the passage P6 obtained in example 1 is shown in fig. 2; these results indicate that the cells isolated and cultured from fat using the method of this example are mesenchymal stem cells and have high purity.
In addition, the growth cycle was measured for the P1-P9 generation cells, and the results showed that all the generation times of the G2 phase cells were <1%, the S phase cells were >10%, for example, the G2 phase cells of the P5 generation cells were 0.63% and the S phase cells were 12.48%, and these results showed that the cells obtained in example 1 were strong in proliferation capacity and did not enter into the division stage.
The P1-P9 generation cells obtained in other examples of the present invention also substantially exhibit the above-described cell characteristics.
Test example 2: cell proliferation assay
The test uses cell population doubling time (population doubling time, PDT) as a parameter to evaluate proliferation of adipose-derived mesenchymal stem cells. Specifically, the P1 generation or other generation cell suspensions obtained in example 1 were each treated with 5X10 4 The density of each ml was inoculated into 30 dishes of 35mm diameter, randomly divided into 10 groups of 3 dishes each, and 5% oxygen and 5% CO were placed using the subculture medium described in example 1 2 Culturing in a three-gas incubator at 37 ℃, taking one group (3 dishes) at the same time every day, digesting for 3min by using digestive enzyme TrypLE Express to obtain cell suspension, counting cells (counting by using a cell counting plate), taking the average value of the 3 dishes as the total number of cells per day, taking the number of cells as an ordinate to the end of the 10 th group (10 days), drawing a cell growth curve by taking the number of cells as an abscissa, and calculating the cell population doubling time (PDT, h) according to the following formula:
PDT=Δt×Lg2/(LgNt-LgN0)
in the method, in the process of the invention,
Δt is the culture time (h) for cell proliferation to reach the plateau,
n0 is the number of cells initially inoculated in culture,
nt is the number of cells that proliferate up to the plateau.
Using the above method, the cell population doubling times (PDT values) of the adipose mesenchymal stem cells of the generation P1, the generation P3, the generation P5, the generation P7 and the generation P9 obtained in example 1 were respectively measured, and the results were: 27.86h, 28.16 h, 28.82h, 27.23h, 28.35h, mean.+ -. SD is 28.08.+ -. 0.53. This indicates that the population doubling time of the different generation sub-cells is substantially the same.
The adipose-derived mesenchymal stem cells obtained using the method of example 1 were subjected to cell proliferation test using the method of test example 2, and the present inventors have unexpectedly found that when the amounts of zinc monomethionine added to the subculture medium were varied, the cell population doubling time exhibited a certain tendency to vary, and specific tests and results were as follows.
EXAMPLE 2 isolation and passage of adipose-derived mesenchymal Stem cells
This embodiment performs a related operation with reference to embodiment 1.
(1) Processing in a biosafety cabinet the fat donated by volunteers transported to the laboratory via a cold chain at 2-8 ℃;
(2) Centrifuging at 100xg for 5min, removing upper adipose tissue, washing with D-Hanks solution for one time, centrifuging at 100xg for 5min, removing adipose tissue, adding 2 times of 1% type II collagenase, and performing shake digestion for 30min;
(3) After digestion, adding one volume of D-hanks liquid for dilution, centrifuging for 5min at 100xg, and reserving cell sediment at the bottom layer for subsequent operation;
(4) Taking the cell sediment obtained in the step (3), adding a primary supplementary culture medium for resuspension, sampling and counting, and obtaining the cell sediment according to the ratio of 2 multiplied by 10 4 /cm 2 Inoculating to a T75 bottle; CO placement 2 Incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) After culturing for 3D, the culture medium is completely changed for one time until the cell fusion degree reaches more than 80 percent (about 5D), the old culture medium is removed, the cells are washed by using D-hanks liquid, 2ml of recombinant pancreatin solution is added into each bottle to digest the cells for 2min so as to enable the cells to fall off, 10ml of D-hanks liquid is added into each bottle to dilute, 100xg is centrifuged for 10min, and the cell sediment is resuspended by using a primary supplementary culture medium, so that primary adipose mesenchymal stem cells (namely P0 generation) can be obtained and can be used for the next passage;
(6) Cell passage: taking P0 generation cells, washing with PBS, adding 2ml of recombinant pancreatin solution for digestion for 2-5min until most of the cells fall off, adding 5ml of complete culture medium for stopping digestion, transferring the cells into a centrifuge tube, centrifuging at 1400rpm for 5min, discarding the supernatant, adding 5ml of passage culture medium for resuspension, counting, inoculating to a culture flask, and obtaining the cells with the density of 8000 cells/cm 2 Placing 5% oxygen and 5% CO 2 Subculturing in a three-gas incubator at 37 ℃, and when the cell fusion rate reaches 80% -90%, using digestive enzyme TrypLE Express to digest for 3min to obtain P1 generation cells; and then carrying out passage for the next generation according to the method of the step, and sequentially obtaining the cell suspension of the adipose-derived mesenchymal stem cells for the subsequent P2-P9 generation. The subculture medium used in this example comprises: fetal bovine serum 12%, L-glutamine 1%, epidermal Growth Factor (EGF) 12. Mu.g/mL, zinc monomethionine 80. Mu.g/mL, DMEM-F12 to 100%.
EXAMPLE 3 isolation and passage of adipose-derived mesenchymal Stem cells
This embodiment performs a related operation with reference to embodiment 1.
(1) Processing in a biosafety cabinet the fat donated by volunteers transported to the laboratory via a cold chain at 2-8 ℃;
(2) Centrifuging at 100xg for 5min, removing upper adipose tissue, washing with D-Hanks solution for one time, centrifuging at 100xg for 5min, removing adipose tissue, adding 2 times of 1% type II collagenase, and performing shake digestion for 30min;
(3) After digestion, adding one volume of D-hanks liquid for dilution, centrifuging for 5min at 100xg, and reserving cell sediment at the bottom layer for subsequent operation;
(4) Taking the cell sediment obtained in the step (3), adding a primary supplementary culture medium for resuspension, sampling and counting, and obtaining the cell sediment according to the ratio of 2 multiplied by 10 4 /cm 2 Inoculating to a T75 bottle; CO placement 2 Incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) After culturing for 3D, the culture medium is completely changed for one time until the cell fusion degree reaches more than 80 percent (about 5D), the old culture medium is removed, the cells are washed by using D-hanks liquid, 2ml of recombinant pancreatin solution is added into each bottle to digest the cells for 2min so as to enable the cells to fall off, 10ml of D-hanks liquid is added into each bottle to dilute, 100xg is centrifuged for 10min, and the cell sediment is resuspended by using a primary supplementary culture medium, so that primary adipose mesenchymal stem cells (namely P0 generation) can be obtained and can be used for the next passage;
(6) Cell passage: taking P0 generation cells, washing with PBS, adding 2ml of recombinant pancreatin solution for digestion for 2-5min until most of the cells fall off, adding 5ml of complete culture medium for stopping digestion, transferring the cells into a centrifuge tube, centrifuging at 1400rpm for 5min, discarding the supernatant, adding 5ml of passage culture medium for resuspension, counting, inoculating to a culture flask, and obtaining the cells with the density of 8000 cells/cm 2 Placing 5% oxygen and 5% CO 2 Subculturing in a three-gas incubator at 37 ℃, and when the cell fusion rate reaches 80% -90%, using digestive enzyme TrypLE Express to digest for 3min to obtain P1 generation cells; and then carrying out passage for the next generation according to the method of the step, and sequentially obtaining the cell suspension of the adipose-derived mesenchymal stem cells for the subsequent P2-P9 generation. The subculture medium used in this example comprises: fetal bovine serum 12%, L-glutamine 1%, epidermal Growth Factor (EGF) 12 μg/mL, zinc monomethionine 120 μg/mL, DMEM-F12 to 100%.
EXAMPLE 4 isolation and passage of adipose-derived mesenchymal Stem cells
This embodiment performs a related operation with reference to embodiment 1.
(1) Processing in a biosafety cabinet the fat donated by volunteers transported to the laboratory via a cold chain at 2-8 ℃;
(2) Centrifuging at 100xg for 5min, removing upper adipose tissue, washing with D-Hanks solution for one time, centrifuging at 100xg for 5min, removing adipose tissue, adding 2 times of 1% type II collagenase, and performing shake digestion for 30min;
(3) After digestion, adding one volume of D-hanks liquid for dilution, centrifuging for 5min at 100xg, and reserving cell sediment at the bottom layer for subsequent operation;
(4) Taking the cell sediment obtained in the step (3), adding a primary supplementary culture medium for resuspension, sampling and counting, and obtaining the cell sediment according to the ratio of 2 multiplied by 10 4 /cm 2 Inoculating to a T75 bottle; CO placement 2 Incubator (5% CO) 2 ，37 ℃, saturated humidity);
(5) After culturing for 3D, the culture medium is completely changed for one time until the cell fusion degree reaches more than 80 percent (about 5D), the old culture medium is removed, the cells are washed by using D-hanks liquid, 2ml of recombinant pancreatin solution is added into each bottle to digest the cells for 2min so as to enable the cells to fall off, 10ml of D-hanks liquid is added into each bottle to dilute, 100xg is centrifuged for 10min, and the cell sediment is resuspended by using a primary supplementary culture medium, so that primary adipose mesenchymal stem cells (namely P0 generation) can be obtained and can be used for the next passage;
(6) Cell passage: taking P0 generation cells, washing with PBS, adding 2ml of recombinant pancreatin solution for digestion for 2-5min until most of the cells fall off, adding 5ml of complete culture medium for stopping digestion, transferring the cells into a centrifuge tube, centrifuging at 1400rpm for 5min, discarding the supernatant, adding 5ml of passage culture medium for resuspension, counting, inoculating to a culture flask, and obtaining the cells with the density of 8000 cells/cm 2 Placing 5% oxygen and 5% CO 2 Subculturing in a three-gas incubator at 37 ℃, and when the cell fusion rate reaches 80% -90%, using digestive enzyme TrypLE Express to digest for 3min to obtain P1 generation cells; and then carrying out passage for the next generation according to the method of the step, and sequentially obtaining the cell suspension of the adipose-derived mesenchymal stem cells for the subsequent P2-P9 generation. The subculture medium used in this example comprises: fetal bovine serum 12%, L-glutamine 1%, epidermal Growth Factor (EGF) 12 μg/mL, zinc monomethionine 30 μg/mL, DMEM-F12 to 100%.
EXAMPLE 5 isolation and passage of adipose-derived mesenchymal Stem cells
This embodiment performs a related operation with reference to embodiment 1.
(1) Processing in a biosafety cabinet the fat donated by volunteers transported to the laboratory via a cold chain at 2-8 ℃;
(2) Centrifuging at 100xg for 5min, removing upper adipose tissue, washing with D-Hanks solution for one time, centrifuging at 100xg for 5min, removing adipose tissue, adding 2 times of 1% type II collagenase, and performing shake digestion for 30min;
(3) After digestion, adding one volume of D-hanks liquid for dilution, centrifuging for 5min at 100xg, and reserving cell sediment at the bottom layer for subsequent operation;
(4) Taking the cell sediment obtained in the step (3), addingPrimary supplemented medium was resuspended, sampled and counted according to 2X 10 4 /cm 2 Inoculating to a T75 bottle; CO placement 2 Incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) After culturing for 3D, the culture medium is completely changed for one time until the cell fusion degree reaches more than 80 percent (about 5D), the old culture medium is removed, the cells are washed by using D-hanks liquid, 2ml of recombinant pancreatin solution is added into each bottle to digest the cells for 2min so as to enable the cells to fall off, 10ml of D-hanks liquid is added into each bottle to dilute, 100xg is centrifuged for 10min, and the cell sediment is resuspended by using a primary supplementary culture medium, so that primary adipose mesenchymal stem cells (namely P0 generation) can be obtained and can be used for the next passage;
(6) Cell passage: taking P0 generation cells, washing with PBS, adding 2ml of recombinant pancreatin solution for digestion for 2-5min until most of the cells fall off, adding 5ml of complete culture medium for stopping digestion, transferring the cells into a centrifuge tube, centrifuging at 1400rpm for 5min, discarding the supernatant, adding 5ml of passage culture medium for resuspension, counting, inoculating to a culture flask, and obtaining the cells with the density of 8000 cells/cm 2 Placing 5% oxygen and 5% CO 2 Subculturing in a three-gas incubator at 37 ℃, and when the cell fusion rate reaches 80% -90%, using digestive enzyme TrypLE Express to digest for 3min to obtain P1 generation cells; and then carrying out passage for the next generation according to the method of the step, and sequentially obtaining the cell suspension of the adipose-derived mesenchymal stem cells for the subsequent P2-P9 generation. The subculture medium used in this example comprises: fetal bovine serum 12%, L-glutamine 1%, epidermal Growth Factor (EGF) 12 μg/mL, zinc monomethionine 15 μg/mL, DMEM-F12 to 100%.
EXAMPLE 6 isolation and passage of adipose-derived mesenchymal Stem cells
This embodiment performs a related operation with reference to embodiment 1.
(1) Processing in a biosafety cabinet the fat donated by volunteers transported to the laboratory via a cold chain at 2-8 ℃;
(2) Centrifuging at 100xg for 5min, removing upper adipose tissue, washing with D-Hanks solution for one time, centrifuging at 100xg for 5min, removing adipose tissue, adding 2 times of 1% type II collagenase, and performing shake digestion for 30min;
(3) After digestion, adding one volume of D-hanks liquid for dilution, centrifuging for 5min at 100xg, and reserving cell sediment at the bottom layer for subsequent operation;
(4) Taking the cell sediment obtained in the step (3), adding a primary supplementary culture medium for resuspension, sampling and counting, and obtaining the cell sediment according to the ratio of 2 multiplied by 10 4 /cm 2 Inoculating to a T75 bottle; CO placement 2 Incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) After culturing for 3D, the culture medium is completely changed for one time until the cell fusion degree reaches more than 80 percent (about 5D), the old culture medium is removed, the cells are washed by using D-hanks liquid, 2ml of recombinant pancreatin solution is added into each bottle to digest the cells for 2min so as to enable the cells to fall off, 10ml of D-hanks liquid is added into each bottle to dilute, 100xg is centrifuged for 10min, and the cell sediment is resuspended by using a primary supplementary culture medium, so that primary adipose mesenchymal stem cells (namely P0 generation) can be obtained and can be used for the next passage;
(6) Cell passage: taking P0 generation cells, washing with PBS, adding 2ml of recombinant pancreatin solution for digestion for 2-5min until most of the cells fall off, adding 5ml of complete culture medium for stopping digestion, transferring the cells into a centrifuge tube, centrifuging at 1400rpm for 5min, discarding the supernatant, adding 5ml of passage culture medium for resuspension, counting, inoculating to a culture flask, and obtaining the cells with the density of 8000 cells/cm 2 Placing 5% oxygen and 5% CO 2 Subculturing in a three-gas incubator at 37 ℃, and when the cell fusion rate reaches 80% -90%, using digestive enzyme TrypLE Express to digest for 3min to obtain P1 generation cells; and then carrying out passage for the next generation according to the method of the step, and sequentially obtaining the cell suspension of the adipose-derived mesenchymal stem cells for the subsequent P2-P9 generation. The subculture medium used in this example comprises: fetal bovine serum 12%, L-glutamine 1%, epidermal Growth Factor (EGF) 12. Mu.g/mL, zinc monomethionine 0. Mu.g/mL, DMEM-F12 to 100%.
EXAMPLE 7 isolation and passage of adipose-derived mesenchymal Stem cells
This embodiment performs a related operation with reference to embodiment 1.
(1) Processing in a biosafety cabinet the fat donated by volunteers transported to the laboratory via a cold chain at 2-8 ℃;
(2) Centrifuging at 100xg for 5min, removing upper adipose tissue, washing with D-Hanks solution for one time, centrifuging at 100xg for 5min, removing adipose tissue, adding 2 times of 1% type II collagenase, and performing shake digestion for 30min;
(3) After digestion, adding one volume of D-hanks liquid for dilution, centrifuging for 5min at 100xg, and reserving cell sediment at the bottom layer for subsequent operation;
(4) Taking the cell sediment obtained in the step (3), adding a primary supplementary culture medium for resuspension, sampling and counting, and obtaining the cell sediment according to the ratio of 2 multiplied by 10 4 /cm 2 Inoculating to a T75 bottle; CO placement 2 Incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) After culturing for 3D, the culture medium is completely changed for one time until the cell fusion degree reaches more than 80 percent (about 5D), the old culture medium is removed, the cells are washed by using D-hanks liquid, 2ml of recombinant pancreatin solution is added into each bottle to digest the cells for 2min so as to enable the cells to fall off, 10ml of D-hanks liquid is added into each bottle to dilute, 100xg is centrifuged for 10min, and the cell sediment is resuspended by using a primary supplementary culture medium, so that primary adipose mesenchymal stem cells (namely P0 generation) can be obtained and can be used for the next passage;
(6) Cell passage: taking P0 generation cells, washing with PBS, adding 2ml of recombinant pancreatin solution for digestion for 2-5min until most of the cells fall off, adding 5ml of complete culture medium for stopping digestion, transferring the cells into a centrifuge tube, centrifuging at 1400rpm for 5min, discarding the supernatant, adding 5ml of passage culture medium for resuspension, counting, inoculating to a culture flask, and obtaining the cells with the density of 8000 cells/cm 2 Placing 20% oxygen and 5% CO 2 Subculturing in a three-gas incubator at 37 ℃, and when the cell fusion rate reaches 80% -90%, using digestive enzyme TrypLE Express to digest for 3min to obtain P1 generation cells; and then carrying out passage for the next generation according to the method of the step, and sequentially obtaining the cell suspension of the adipose-derived mesenchymal stem cells for the subsequent P2-P9 generation. The subculture medium used in this example comprises: fetal bovine serum 12%, L-glutamine 1%, epidermal Growth Factor (EGF) 12. Mu.g/mL, zinc monomethionine 120. Mu.g/mL or 80. Mu.g/mL or 50. Mu.g/mL or 15. Mu.g/mL or 0. Mu.g/mL, DMEM-F12 to 100%.
Test example 3: cell proliferation assay
Cell proliferation experiments were performed according to the method of test example 2. Specifically, for each of the P1, P3, P5, P7, and P9 generation cells obtained in examples 2 to 6, the cell population doubling time was tested and calculated, and the cell population doubling time (PDT value, hour, mean±sd, n=5, sd value small indicates that the difference between the results of the 5 generations is very small, and the following is true) of each of the 5 generation adipose-derived mesenchymal stem cells obtained in examples: (PDT value=28.08±0.53 for example 1), PDT value= 25.27 ±0.84 for example 2, PDT value=21.48±1.32 for example 3, PDT value=33.17±1.06 for example 4, PDT value=38.32±0.95 for example 5, PDT value= 44.58 ±1.16 for example 6. The above results show that the cell population doubling time (PDT value) and the single methionine zinc concentration show typical trend relationship, and the cell population doubling time is shortened with an increase in the single methionine zinc concentration within a range of 0 to 80 μg/mL, but is substantially unchanged after the concentration is increased to 120 μg/mL (in other test examinations, the PDT values obtained by using the average values of 100 μg/mL, 150 μg/mL and 200 μg/mL are 23.18h, 21.75h, 35 h and 23h, respectively, and 23h are all unchanged within a range of 23 to 20 h). This finding is of considerable practical value, for example, the concentration of zinc monomethionine added can be appropriately adjusted according to specific practical requirements, particularly different requirements of cell growth rates, preferably the concentration of zinc monomethionine is in the range of 15-100 μg/mL, more preferably the concentration of zinc monomethionine is in the range of 15-80 μg/mL.
In another test, the method of test example 3 was used for measurement,
example 7 subcultured with normoxic cells of the P1, P3, P5, P7, P9 generation obtained with a subculture medium containing zinc monomethionate 120 μg/mL had a cell population doubling time PDT value=43.41±1.58,
example 7 subcultured with normoxic cells of the P1, P3, P5, P7, P9 generation obtained with a subculture medium containing 80 μg/mL zinc monomethionate had a cell population doubling time PDT value=42.17±1.01,
example 7 subcultured with normoxic cells of the P1, P3, P5, P7, P9 generation obtained with a subculture medium containing zinc monomethionate at 50 μg/mL had a cell population doubling time PDT value=43.82±1.73,
example 7 subcultured with normoxic cells of the P1, P3, P5, P9 generation obtained with a subculture medium containing 15 μg/mL of zinc monomethionate had a cell population doubling time PDT value= 41.57 ±1.45,
example 7 subcultured with normoxic cells of the P1, P3, P5, P9 generation obtained with a subculture medium containing 0 μg/mL of zinc monomethionate had a cell population doubling time PDT value= 42.82 ±1.26,
the cell population doubling time of the cells obtained by the normal oxygen condition passage is long, and the zinc methionine concentration has no influence on the cell population doubling time.
In addition, although the inventors found that zinc glycinate also exhibited the above-described rule when the inventors studied the similar experiments using umbilical cord mesenchymal stem cells and placenta mesenchymal stem cells, the rule of the above-described cell population doubling time related to the concentration of zinc monomethionine could not be obtained when zinc glycinate was used for the adipose mesenchymal stem cells of the present invention, for example, when zinc monomethionine was replaced with equal amount of zinc glycinate in examples 1 to 6 of the present invention, the cell population doubling time PDT values of P1, P3, P5, and P9 generation cells obtained by the subculture medium were all in a small range of 27 to 33 h.
Test example 4: detection of surface markers
The adipose-derived mesenchymal stem cells of the generation P2, the generation P5 and the generation P8 obtained in examples 1 to 6 were treated with 500XgCentrifugation was performed for 5min, and 2mL of 1 XPBS was added to resuspend, and the mixture was labeled 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 detection was performed by a flow analyzer. Each antibody used for the assay was purchased from BioVision, usa. The expression of CD73, CD90 and CD105 of the three-generation fat mesenchymal stem cells obtained in 6 examples is positive >96 percent) and the expression of CD34, CD45 and HLA-DR of the adipose-derived mesenchymal stem cells obtained by 3 generations of 6 examples is negative<2%). For example, the expression of the P5 generation adipose tissue stem cell surface markers CD73, CD90 and CD105 obtained in example 4 are 99.62%, 99.37% and 99.58% respectively, and the P5 generation adipose tissue stem cell surface markers obtained in example 4The expression levels of CD34, CD45 and HLA-DR were 0.226%, 0.173% and 0.292%, respectively.
Test example 5: induced differentiation and staining
The P4-generation adipose-derived mesenchymal stem cells obtained in each of examples 1 to 6 were plated into 24-well plates at a certain cell density, 3 wells each. Osteogenic/adipogenic induced differentiation was all 3X 10 4 Cell/square centimeter density cells were plated in 24 well plates and the chondrogenic induced differentiation cell plating density was halved. After culturing with DMEM/F12 containing 10% FBS for 24 hours, the culture medium was replaced with osteogenic differentiation medium, adipogenic differentiation medium and chondrogenic differentiation medium (all from Biological Industriers), respectively. After 21d of induction culture, calcium-containing bone cells were stained with alizarin red-S to detect osteogenic differentiation, lipid droplets were stained with oil red O to detect adipogenic differentiation, and proteoglycans were stained with alcian blue 8GX to detect chondrogenic differentiation (each reagent was purchased from Sigma-Aldrich). The results show that the P4-generation adipose-derived mesenchymal stem cells obtained in 6 examples have excellent osteogenic, adipogenic and chondrogenic three-line differentiation capacity. Typically, the osteogenic, adipogenic, chondrogenic differentiation staining patterns of the P6-generation adipose mesenchymal stem cells of example 1 are shown in FIG. 2A, FIG. 2B, FIG. 2C, respectively.
Test example 6: adipose-derived mesenchymal stem cells inhibit lymphocyte proliferation
The P5-generation adipose-derived mesenchymal stem cells prepared in examples 1 to 6 were cultured in 24-well plates at a ratio of 1X 10 per well 5 Inoculating individual cells, placing 5% oxygen and 5% CO 2 Incubated in a three-gas incubator at 37℃for 2h, and treated with 10. Mu.g/mL mitomycin C (purchased from Sigma-Aldrich) for 3h.
Freshly isolated peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMC, prepared by the method of the example with reference to CN 110551686A) were stained with 2.0. Mu. Mol/L carboxyfluorescein diacetate succinimidyl ester (CFSE, available from Beijing Zhijacking Co., ltd.) for 10min at 37℃and the excess CFSE was washed off. CFSE-stained PBMCs were co-cultured with mitomycin C-de-proliferated adipose-derived mesenchymal stem cells in a ratio of 10:1 for 5d, and examined by flow cytometry.
The test is divided into: negative control group, PBMC group, positive stimulation group, pbmc+pha-P group, experimental group, adipose mesenchymal stem cell inhibition group [ pbmc+pha-p+mscs group ], wherein the remaining groups except PBMC group were stimulated with 10 μg/mL phytohemagglutinin P (PHA-P, purchased from invitogen), the daughter cell ratio was analyzed, and the inhibition ratio was calculated by the following formula:
The ratio of daughter cells of the negative control group (PBMC group) was measured to be 2.27%, the ratio of daughter cells of the positive stimulation group (PBMC+PHA-P group) was measured to be 85.47%, and the inhibition rates of the P5-generation adipose-derived mesenchymal stem cell experimental groups prepared in examples 1 to 6 were calculated to be respectively: 53.25%, 57.41%, 60.36%, 48.47%, 43.62%, 38.64%. In addition, the inhibition rates of the P8-generation adipose-derived mesenchymal stem cells prepared in examples 1 to 6 were measured by the above method, and the results were respectively: 52.27%, 56.74%, 61.52%, 47.36%, 42.84%, 39.76%. These results indicate that the higher the inhibition of adipose mesenchymal stem cells obtained using a subculture medium containing zinc monomethionate at a higher concentration, this finding is completely unexpected. In addition, referring to the test of the method of test example 6, the results of the inhibition rates of the P5-generation and P8-generation adipose mesenchymal stem cells obtained in the passaging medium containing 50. Mu.g/mL of zinc monomethionine in example 7 were 40.35% and 41.18%, respectively, the results of the inhibition rates of the P4-generation and P7-generation adipose mesenchymal stem cells obtained in the passaging medium containing 15. Mu.g/mL of zinc monomethionine in example 7 were 39.53% and 38.84%, respectively, and the results of the inhibition rates of the P3-generation and P6-generation adipose mesenchymal stem cells obtained in the passaging medium containing 0. Mu.g/mL of zinc monomethionine in example 7 were 39.23% and 40.52%, respectively, and the results showed that the inhibition rates of the mesenchymal stem cells obtained in the presence or absence of zinc monomethionine in the case of normal oxygen were significantly weaker than those in the passaging conditions using 5% oxygen in example 1 and the like, and that the zinc monomethionine concentration was independent of the inhibition rates in the normal oxygen conditions.
Preparation example 1 preparation of mesenchymal Stem cells
Prescription: mesenchymal stem cells (generation P5, example 1) 2X 10 6 Sodium chloride 9.0mg, magnesium citrate (to make the concentration of magnesium ion reach 5 mmol/L), soybean lecithin 0.2mg for injection, and water with proper amount to 1ml.
The preparation method comprises the following steps: dissolving sodium chloride, magnesium citrate and phospholipid with proper amount of water to obtain saline solution; the P5-generation mesenchymal stem cells obtained in example 1 were transferred into a centrifuge tube, centrifuged at 2000rpm for 5min, and the supernatant was discarded, and the cells were resuspended in saline solution to prepare a cell preparation.
Test example 7 effectiveness of mesenchymal Stem cells in treating Premature Ovarian Failure (POF)
This test example refers to the method described in CN109652366a (chinese patent application No. 2018115685527) by the inventor team for the efficacy study of mesenchymal stem cells for the treatment of Premature Ovarian Failure (POF).
(1) Female C57BL/6 mice of 6 weeks old were intraperitoneally injected with 50 mg/kg/day Cyclophosphamide (CTX) for 15 days, and injected at the same time every day to establish a POF mouse model. Ovarian reserve function is assessed by indicators of follicular number, hormone levels, estrus cycle, and fertility tests.
(2) Female C57BL/6 mice of 6 weeks of age were randomly assigned to: control group (n=20), POF model group (n=20), adipose-derived mesenchymal stem cell-treated group (PD-MSC group, n=20). Treatment groups of mice, each 2×10 mice, were given 2 injections of adipose-derived mesenchymal stem cell preparation on days 1 and 14, respectively, after POF modeling 5 And tail vein injection of adipose-derived mesenchymal stem cells. The POF model group was injected with an equal volume of physiological saline.
In this experiment, the adipose-derived mesenchymal stem cell injection preparation was the cell preparation obtained in preparation example 1.
(3) Hormone levels
After 14 days and 28 days after the second transplantation of the adipose-derived mesenchymal stem cells, 10 mice were taken from each group, the eyebox was sampled, serum was isolated, and the temperature was kept at-20 ℃. Enzyme-linked immunosorbent assay (ELISA) analysis of the levels of estradiol (E2), follicle Stimulating Hormone (FSH), anti-Miaole hormone (AMH) (specific methods are carried out with reference to the methods of the Philippies). The results show that: compared with the POF model group, the serum level of E2 in mice of the adipose-derived mesenchymal stem cell group was increased and the FSH level was decreased at each time point, and the difference was remarkable (p < 0.05), and the specific results are shown in table 1 below.
Table 1:
p <0.05, p <0.01 compared to the POF model group for the same day of detection.
(4) Follicular count of mouse ovarian tissue
After 28 days of the second transplantation of the adipose-derived mesenchymal stem cells, 10 mice are respectively taken from each group, sacrificed, left ovarian tissues of the mice are fixed in 4% paraformaldehyde, the fixed tissues are subjected to serial alcohol dehydration, xylene transparency, paraffin embedding, serial slicing, slice thickness of 5um, HE staining and microscopic observation.
The results show that: compared with a control group, the number of primary follicles, secondary follicles and mature follicles of mice in the POF model group is obviously reduced, and the number of closed follicles is obviously increased; the number of follicles at each stage is recovered to different degrees 28 days after adipose-derived mesenchymal stem cells are treated, so that the growth of granulosa cells is increased, the apoptosis is reduced, the morphology of ovarian epithelial cells is stable, the number of primary follicles, secondary follicles and mature follicles is obviously increased, and the number of atresia follicles is obviously reduced. The follicular counts at each stage 28 days after adipose-derived mesenchymal stem cell transplantation were significantly different from those of POF group, P <0.01, and the specific results are shown in table 2 below.
Table 2:
p <0.05, < p <0.01 compared to the POF model group.
(5) Mice fertility observations:
on day 28 after adipose-derived mesenchymal stem cell transplantation, male and female mice were treated with the following 2:1 proportion cage feeding, counting the fertility rate of mice, comparing the litter size of the mice, observing the repair effect of adipose-derived mesenchymal stem cell transplantation on the ovary function of the mice, wherein the adipose-derived mesenchymal stem cell group is obviously different from the POF group, and the P is less than 0.01. The specific results are shown in Table 3 below.
Table 3:
p <0.05, < p <0.01 compared to the POF model group.
According to the results, the adipose-derived mesenchymal stem cell transplantation treatment can obviously improve the reserve function of the damaged ovary of the POF mouse, the follicular number is increased, the oestrogen is increased, and part of the fertility of the mouse is recovered, so that the adipose-derived mesenchymal stem cell can be applied to the clinical treatment of the POF.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (10)

1. A method of isolating and subculturing adipose-derived mesenchymal stem cells comprising two stages of (a) isolating and culturing primary adipose-derived mesenchymal stem cells and (b) subculturing adipose-derived mesenchymal stem cells, wherein:
(a) The stage of isolated culture of primary adipose-derived mesenchymal stem cells comprises the following steps:
(a1) Processing fat samples transported to a laboratory via a cold chain at 2-8 ℃ in a biosafety cabinet;
(a2) Centrifuging a fat sample, removing upper adipose tissue, washing the upper adipose tissue once again by using D-Hanks liquid, centrifuging again, removing adipose tissue, and adding 1% type II collagenase for shake digestion;
(a3) After digestion, adding one volume of D-hanks liquid for dilution, centrifuging, and reserving cell sediment at the bottom layer for subsequent operation;
(a4) Taking the cell sediment obtained in the step (3), adding a primary supplementary culture medium for resuspension, sampling and counting, and inoculating the cell sediment into a culture bottle according to the specified cell quantity; CO placement 2 Culturing in an incubator;
(a5) Culturing for 3D, completely replacing the culture medium once until the cell fusion degree reaches more than 80%, removing the old culture medium, cleaning cells by using D-hanks solution, adding recombinant pancreatin solution to digest the cells, allowing the cells to fall off, adding D-hanks solution for dilution, centrifuging, and re-suspending cell precipitation by using a primary supplementary culture medium to obtain primary adipose-derived mesenchymal stem cells, namely P0 generation;
(b) The stage of subculturing the adipose-derived mesenchymal stem cells comprises the following steps: taking P0 generation cells, washing with PBS, adding a pancreatin solution for digestion until most of the cells fall off, adding a complete culture medium for stopping digestion, transferring the cells into a centrifuge tube, centrifuging, discarding the supernatant, adding a subculture medium for resuspension, counting, inoculating to a culture bottle, and placing 5% oxygen and 5% CO 2 Subculturing in a three-gas incubator at 37 ℃ until the cell fusion rate reaches more than 80%, and digesting by using digestive enzyme to obtain P1 generation cells; and then carrying out passage for the next generation according to the method of the step, and sequentially obtaining the cell suspension of the adipose-derived mesenchymal stem cells for each generation.
2. The method according to claim 1, wherein the subculture medium comprises: 12% of fetal bovine serum, 1% of L-glutamine, 12 mu g/mL of Epidermal Growth Factor (EGF),Zinc monomethionine15-120. Mu.g/mL, e.g., 15-100. Mu.g/mL, e.g., 15-75. Mu.g/mL, e.g., 50. Mu.g/mL, DMEM-F12 is added to 100%.
3. The method according to claim 1, wherein:
the PBS is disodium hydrogen phosphate/sodium dihydrogen phosphate buffer solution with the concentration of phosphate ions of 0.025M and the pH value of 6.8;
the complete medium is DMEM-F12 medium comprising 12% fetal bovine serum;
in the step (b), cell suspension of the adipose tissue stem cells is obtained by carrying out the generation of P1-P15 generation times such as P1-P9 generation times;
the formula of the D-Hanks liquid comprises the following components: 8.0g NaCl, 0.4g KCl, 0.06g KH 2 PO 4 Na of 0.08g 2 HPO 4 .12H 2 O, 0.35g NaHCO 3 Water to 1000ml; and/or
The primary supplementary culture medium is prepared by taking DMEM-F12 culture medium as a matrix and adding: 1% platelet lysate, 1% human serum albumin, 2. Mu.g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF, 0.12% thioglycerol, 1% fructose.
4. The method according to claim 1, wherein:
in step (a 2), both centrifuges are operated at 100Xg for 5 min;
In the step (a 2), adding 2 times of 1% type II collagenase by volume for shaking digestion for 30min;
in step (a 3), the centrifugation is performed under a condition of centrifugation at 100Xg for 5 min;
in the step (a 4), the cells are inoculated into a culture flask in a predetermined cell amount of 1 to 5X 10 cells 4 /cm 2 Inoculating to a T75 bottle;
in the step (a 4), the cells are inoculated into the culture flask in a predetermined cell amount of 2X 10 cells 4 /cm 2 Inoculating to a T75 bottle; and/or
In step (a 4), CO 2 The conditions for culturing in the incubator are: 5% CO 2 Saturated humidity at 37 ℃.
5. The method according to claim 1, wherein: in the step (a 5), 2ml of recombinant pancreatin solution is added to each bottle to digest the cells for 2min;
in the step (a 5), 10ml of D-hanks liquid is added into each bottle to dilute;
in step (a 5), centrifuging at 100Xg for 10min; and/or the number of the groups of groups,
the DMEM-F12 culture medium comprises the following components: anhydrous calcium chloride 116.6mg, L-leucine 59.05mg, linoleic acid 0.042mg, cupric sulfate pentahydrate 0.0013mg, L-lysine hydrochloride 91.25mg, lipoic acid 0.105mg, ferric nitrate nonahydrate 0.05mg, L-methionine 17.24mg, phenol red 8.1mg, ferrous sulfate heptahydrate 0.417mg, L-phenylalanine 35.48mg, 1, 4-butanediamine dihydrochloride 0.081mg, potassium chloride 311.8mg, L-serine 26.25mg, sodium pyruvate 55mg, magnesium chloride 28.64mg, L-threonine 53.45mg, vitamin H0.0035mg, anhydrous magnesium sulfate 48.84mg, L-alanine 4.45mg, calcium D-pantothenate 2.24mg, sodium chloride 7000mg, L-asparagine 7.5mg, choline chloride 8.98mg, anhydrous sodium dihydrogen phosphate 54.35mg, L-aspartic acid 6.65mg folic acid 2.65mg, disodium hydrogen phosphate 71.02mg, L-cysteine hydrochloride 17.56mg, i-inositol 12.6mg, zinc sulfate heptahydrate 0.432mg, L-glutamic acid 7.35mg, nicotinamide 2.02mg, L-arginine hydrochloride 147.5mg, L-proline 17.25mg, pyridoxal hydrochloride 2mg, L-cystine hydrochloride 31.29mg, L-tryptophan 9.02mg, pyridoxine hydrochloride 0.031mg, L-glutamine 365mg, L-tyrosine 38.4mg, riboflavin 0.219mg, glycine 18.75mg, L-valine 52.85mg, thiamine hydrochloride 2.17mg, L-histidine hydrochloride 31.48mg, D-glucose 3151mg, thymidine 0.365mg, L-isoleucine 54.47mg, hypoxanthine 2mg, vitamin B12 0.68mg, and water in an appropriate amount to 1000mL; preparing: dissolving each material with 1000ml, and filtering and sterilizing by a microporous filter membrane with the size of 0.22 mu m.
6. The method according to claim 1, wherein:
the method also comprises the steps of detecting primary adipose-derived mesenchymal stem cells obtained by isolated culture, detecting cell morphology and/or identifying immunophenotype;
the immunophenotype identification refers to detection of CD73, CD90, CD105 and CD19, CD11b, CD31, CD45, HLADR and CD 34.
7. The method according to claim 1, wherein:
in the step (b), the cells are subjected to subculture in an incubator until the cell fusion rate reaches 80% -90%;
in step (b), the digestive enzyme is a TrypLE Express digestive enzyme;
in the step (b), the inoculation density of the cells in the culture flask is 6000-10000 cells/cm 2 For example 8000 cells/cm 2 ；
Step (b) is performed as follows: taking P0 generation cells, washing with PBS, adding 2ml of recombinant pancreatin solution for digestion for 2-5min until most of the cells fall off, adding 5ml of complete culture medium for stopping digestion, transferring the cells into a centrifuge tube, centrifuging at 1400rpm for 5min, discarding the supernatant, adding 5ml of passage culture medium for resuspension, counting, inoculating to a culture flask, and obtaining the cells with the density of 8000 cells/cm 2 Placing 5% oxygen and 5% CO 2 Subculturing in a three-gas incubator at 37 ℃, and when the cell fusion rate reaches 80% -90%, using digestive enzyme TrypLE Express to digest for 3min to obtain P1 generation cells; then carrying out passage for the next generation according to the method of the step, and sequentially obtaining cell suspension of adipose-derived mesenchymal stem cells of each generation; and/or the number of the groups of groups,
The cell purity of each generation of placenta mesenchymal stem cells obtained in the step (b) is more than 90%.
8. A adipose-derived mesenchymal stem cell isolated and subcultured by the method of any one of claims 1 to 7.
9. A cell preparation for use in an injection method, comprising the following components in percentage by weight: adipose-derived mesenchymal stem cells 2×10 6 Sodium chloride 9.0mg, magnesium citrate (the concentration of magnesium ions reaches 5 mmol/L), soybean lecithin for injection 0.2mg and proper amount of water to 1ml; the adipose-derived mesenchymal stem cells are cells of the generation P1 to generation P9, and are obtained by the method of any one of claims 1 to 7, isolated and subcultured; for example, the cell preparation is prepared according to a method comprising the steps of: dissolving sodium chloride, magnesium citrate and phospholipid with proper amount of water to obtain saline solution; transferring the mesenchymal stem cells obtained by subculture into a centrifuge tube, centrifuging (for example, centrifuging at 2000rpm for 5 min), discarding the supernatant, and adding saline solution to resuspend the cells to obtain a cell preparation.
10. Use of the adipose-derived mesenchymal stem cells isolated and subcultured by the method of any one of claims 1 to 7, or the adipose-derived mesenchymal stem cells of claim 8, or the cell preparation of claim 9, for the preparation of a medicament for treating premature ovarian failure.
CN202311275612.7A 2023-09-28 2023-09-28 Adipose-derived mesenchymal stem cell low-oxygen atmosphere culture and application thereof in treating premature ovarian failure Pending CN117305236A (en)

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CN115737681A (en) * 2022-08-25 2023-03-07 博品(上海)生物医药科技有限公司 Application of human adipose-derived mesenchymal stem cells in preparation of medicine for treating premature ovarian failure

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115737681A (en) * 2022-08-25 2023-03-07 博品(上海)生物医药科技有限公司 Application of human adipose-derived mesenchymal stem cells in preparation of medicine for treating premature ovarian failure

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