CN112300986B

CN112300986B - Method for preparing adipose-derived mesenchymal stem cells by serum-free medium

Info

Publication number: CN112300986B
Application number: CN202011381205.0A
Authority: CN
Inventors: 王正; 肖海蓉
Original assignee: BOYALIFE Inc
Current assignee: BOYALIFE Inc
Priority date: 2020-11-30
Filing date: 2020-11-30
Publication date: 2022-07-26
Anticipated expiration: 2040-11-30
Also published as: CN112300986A

Abstract

The invention relates to a method for preparing adipose-derived mesenchymal stem cells by a serum-free culture medium. In particular to a method for separating and culturing primary adipose-derived mesenchymal stem cells in a serum-free manner, which comprises the following steps: processing a fat sample; centrifuging to obtain the upper layerCleaning adipose tissue with D-Hanks solution, centrifuging to obtain adipose tissue, and digesting with collagenase II; diluting with D-hanks liquid, centrifuging, taking cell precipitate, adding primary complete culture medium for resuspension, sampling and counting, and inoculating to a culture bottle according to a specified cell amount; placing CO ₂ Culturing in an incubator; and (3) after culturing until the cell fusion degree reaches more than 80%, removing the old culture medium, cleaning the cells by using D-hanks liquid, adding the recombinant pancreatin solution to digest the cells to make the cells fall off, adding the D-hanks liquid to dilute, centrifuging, and resuspending the cell sediment by using a primary complete culture medium to obtain the primary adipose mesenchymal stem cells. The method of the present invention exhibits excellent technical effects as described in the specification.

Description

Method for preparing adipose-derived mesenchymal stem cells by serum-free medium

Technical Field

The invention belongs to the technical field of biology, and relates to a method for separating adipose mesenchymal stem cells from fat. The invention also relates to a culture medium and a related test solution used in the culture of the adipose-derived mesenchymal stem cells. When the adipose-derived mesenchymal stem cells are isolated and cultured by using the method, the excellent technical effect can be shown. In particular, the present invention relates to methods of isolating cultured mesenchymal stem cells from adipose using serum-free media.

Background

Mesenchymal Stem Cells (MSCs), such as human mesenchymal stem cells, were first isolated from bone marrow and a class of tissue stem cells derived from the mesoderm, with multipotent differentiation potential and self-renewal capacity, have the ability to differentiate into a variety of adult cells, such as osteoblasts, chondrocytes, adipocytes, endothelial cells, neurons, myocytes, hepatocytes, etc., under specific conditions in vivo and in vitro (Cap AI. Mesenchyl stem cells. J Orthop Res.1991,9:641-650.Pittenger MF, Mackay AM, Beck, et al. multilineagent stem cell of derived human mesenchymal stem cells. science.1999; 284: 143. 147). The latest research shows that the mesenchymal stem cells have the functions of immunoregulation and hematopoiesis support, and are easy to introduce and express exogenous genes. Therefore, the mesenchymal stem cells are not only seed cells in the construction of tissue engineering bone, cartilage and cardiac muscle and important carrier cells in gene therapy, but also have wide application prospect in hematopoietic stem cell transplantation and organ transplantation because the mesenchymal stem cells promote hematopoietic reconstruction and inhibit graft-versus-host reaction. Mesenchymal stem cells have the characteristic of adherent growth in vitro, and by utilizing the characteristic, the mesenchymal stem cells are successfully separated and cultured from various tissues such as liver, kidney, pancreas, muscle, cartilage, skin, peripheral blood and the like.

Stem cells are progenitors of human cells, and all cells in our body are derived from stem cells. When cells in the body age, die or damage denatures, stem cells grow and transform out of the cells that can replace them. As seed cells, the traditional Chinese medicine composition is mainly used for clinically treating various intractable diseases caused by tissue cell and organ injuries which cannot be naturally repaired by an organism; as an immunoregulatory cell, in the treatment of immune rejection and autoimmune diseases. Human mesenchymal stem cells are important members of a stem cell family, are derived from mesoderm in early development and belong to pluripotent stem cells, and are discovered in bone marrow initially, so that the human mesenchymal stem cells are increasingly concerned because of the characteristics of multidirectional differentiation potential, hematopoietic support, stem cell implantation promotion, immune regulation, self-replication and the like. Initial clinical studies were conducted in 1995 by Lazarus et al, who collected autologous MSCs of patients with hematological tumors in remission, cultured for 4-7 weeks in vitro for expansion, and then injected intravenously into patients, who were divided into 3 groups, administered different doses of MSCs, respectively, and no toxic side effects were observed after injection, suggesting that MSCs are safe and reliable for transplantation therapy. Then, clinical reports of autologous MSCs are gradually increased, and the disease types comprise hematopoietic reconstruction after radiotherapy and chemotherapy, graft-versus-host disease (GVHD), heart system diseases and the like, and clinical intravenous infusion is proved to be safe and reliable in the reports.

The isolation culture and subculture process of mesenchymal stem cells are key steps related to the use safety of stem cells as therapeutic drugs. The composition of the cultivation process, in particular of the culture medium, is a major influencing parameter. In the methods described in the prior documents, when mesenchymal stem cells are isolated and subcultured, the culture medium mostly needs to be supplemented with serum, such as fetal bovine serum, and particularly, 10% fetal bovine serum is usually required to be supplemented, and for example, the mesenchymal stem cells are isolated and subcultured usually by using MSC complete medium (which is DMEM-F12 medium containing 10% fetal bovine serum).

On the one hand, however, the cost of fetal bovine serum is rather high, which is disadvantageous for the culture of mesenchymal stem cells; on the other hand, the presence of fetal bovine serum as an exogenous material of animal origin in stem cells poses a potential risk to the safety of clinical use of the cells. Therefore, serum-free isolation and subculture of mesenchymal stem cells are of interest.

In addition, stem cells with multipotential differentiation potential have been isolated from adipose tissue and successfully differentiated into adipocytes, osteoblasts, chondrocytes, myocytes, etc. in a special system cultured in vitro, and such differentiated stem cells are recognized as adipose-derived mesenchymal cells, i.e., adipose-derived stem cells or adipose-derived stem cells (ADSCs), which are stem cells with multipotential differentiation potential isolated from adipose tissue in recent years, and adipose stem cells have totipotent characteristics of differentiated polyembryony, including the capability of mesodermal, endodermal and ectodermal differentiated cells, and can secrete many potential favorable growth factors and cytokines to rapidly develop a cell-based therapeutic method for promoting wound healing. The adipose-derived stem cells are a group of multifunctional mesenchymal stem cells, can be differentiated into non-adipose (mesenchymal) fragments of other cell lines, have a large number of adipose-derived cells, and are easy to separate and obtain.

As an adjuvant treatment method for various tissue defects, adipose-derived stem cells are recommended. In the wound healing process, angiogenesis is an important step, and the formation of new blood vessels is a necessary condition for supporting the formation of granulation tissues and the survival and the expansion of keratinocytes to heal wounds. Therefore, it is considered that the adipose-derived stem cells promote the healing of the wound surface by promoting the rapid angiogenesis. In addition, adipose-derived stem cells have also been shown to promote wound healing through 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 of animals in combination with extracellular matrix scaffold components, and have been found to promote the vascularization ability of the scaffold. Research results show that the promotion effect of the adipose-derived cells on wound healing is related to the promotion of epithelization and granulation tissue formation of the adipose-derived stem cells, and the research results of potential epidermal cell differentiation capacity of the adipose-derived stem cells also indicate that the distant differentiation capacity of the adipose-derived stem cells is a reaction of the multifunctional adipose-derived stem cells on 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, which are beneficial for regeneration of blood vessels. That is, the adipose-derived stem cells promote the healing of the wound surface through cell differentiation and angiogenesis. In addition, adipose-derived stem cells have been used in the treatment of wounds in combination with platelet-derived growth factor, a well-known factor involved in the normal wound healing process. The research result shows that the combined application of the two has a synergistic effect, namely, the wound healing can be promoted by increasing the level of the growth factors.

Theoretically, the cell therapy has a promising prospect because the multifunctional stem cells or progenitor cells promote the healing of the wound by regenerating and continuously providing a plurality of beneficial factors for the lost tissues of the wound. Nevertheless, the research on the mechanism of wound healing by adipose-derived stem cells remains 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, 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 intervening and treating diabetes, liver injury repair, muscle reconstruction, type 2 diabetes positive eruption, myocardial infarction, cerebral infarction, cognitive dysfunction, stroke, intervertebral disc repair, glioblastoma treatment, bone defect, blood vessel neogenesis after trauma, rheumatoid arthritis, labyrinthine lip, heart failure, colitis, urinary incontinence and the like. Researchers have transformed adipose-derived mesenchymal stem cells into cardiomyocytes, which not only reprograms mature stem cells, but also improve the treatment of heart disease. Also, the adipose-derived mesenchymal stem cells and fibrin glue are compounded and then injected into the left ventricular wall of a myocardial infarction rat, and the ADSCs have great prospects in the clinical tissue engineering treatment of myocardial infarction. The adipose-derived mesenchymal stem cells are put in the reticular scaffold as seed cells, so that the skull injury of the dog is successfully repaired, and the skull injury is clinically treated. The research also applies that after the adipose-derived mesenchymal stem cells of abdominal fat of a heart patient are injected into the heart of the patient, the damage to the heart is reduced, and the blood flow is also increased, after 6 months after the stem cells are injected into the heart, the capacity of the heart of the patient receiving oxygenated blood is improved, the blood sent out by the left ventricle of the heart is also increased by 3.5 times, SPELT development proves that the capacity of the heart pump of the patient receiving the adipose-derived mesenchymal stem cells is increased by 5.7%, nuclear magnetic scanning also shows that the average myocardial scar area of the patient is reduced to 15.4% from 31.6%, and the research result is released in the scientific society of American Heart Association in 2010. Experiments prove that the adipose-derived mesenchymal stem cells can secrete various skin growth factors such as fibroblast growth factor (bFGF) to promote the propagation of fibroblasts, and have the effects of resisting photoaging, oxidation, wrinkle, ultraviolet radiation and the like. Research on the treatment of complicated anus of patients with adipose-derived mesenchymal stem cells has often progressed to phase II clinical trials. And the human adipose derived mesenchymal stem cells are transplanted into the breasts of patients with atrophy and different sizes of breasts or requiring breast augmentation, the left and right breasts are balanced and symmetrical in size, and the repaired breasts are naturally soft, completely calcified, cyst-free and free of obvious injection scars by the X-ray detection of the breasts. Recent clinical research shows that intramuscular injection of adipose-derived mesenchymal stem cells has certain curative effects on diabetic foot and arteriosclerosis obliterans. After 6 months of injecting the adipose-derived mesenchymal stem cells, clinically, the rest pain of the patient is relieved, the distance of painless walking is obviously prolonged, and no complication is found. Tests show that the adipose-derived mesenchymal stem cells induce the islet-like cells to be returned to the diabetic patient, and after 23 months of postoperative follow-up, the exogenous insulin dosage of the patient is reduced, the average weight of the patient is increased, and the patient has no discomfort or side reaction.

Researchers find that adipose tissues participate in wound repair, when adipocytes are stimulated by wound, the gene expression of inflammatory substrates such as TNF-a, IL-1, IL-6, intercellular adhesion factors, chemotactic factors, acute response proteins and receptors and the like is subjected to stress secretion, the regulation of the factors is an intrinsic treatment mechanism for understanding the participation of adipocytes in repair, and the regulation system is precise regulation worthy of thinking. The obtained scientific research result is the theoretical basis for enriching the wound repair. Clinical application traumas and deep skin injury wounds of a hypodermis adipose tissue fat layer and fat infusion treatment can achieve an ideal treatment effect of accelerating wound repair, foreign researchers also apply adipose cells, collagen and elastin apply tissue engineering technology to prepare artificial skin containing the adipose cells, clinical application finds that wound contraction caused by tissue repair of full-layer skin defects can be reduced, and the phenomenon indicates that factors and cells with repair effects exist in adipose tissues.

The fat mesenchymal stem cells have strong adhesion and rapid characteristics to extracellular matrix, and the mesenchymal stem cells with human fat sources screened and cultured in vitro can be specifically developed in a domestic and foreign method. The biological performance of the stem cells can improve functions, avoid potential harmful genes, control differentiation time and control different differentiation directions of the stem cells and the like, and has irreplaceable guiding significance in historical periods in basic scientific research, clinical treatment, stem cell treatment research directions, skin regeneration injury repair, construction of highly simulated tissue engineering skin and the like.

Adipose-derived mesenchymal stem cells (ADSCs) are used as mesenchymal stem cells derived from fat, the material is convenient to obtain, the extraction efficiency of the ADSCs is 40 times higher than that of bone marrow mesenchymal stem cells, and the proliferation speed of the ADSCs under the in vitro culture condition is higher; has multipotential differentiation ability, and can be differentiated into fat cell, osteoblast, chondrocyte, cardiomyocyte, and even nerve cell. At present, clinical phase I and phase II experiments prove that the application of the ADSCs in the injury repair of various organs such as heart, rectum, mammary gland and the like is safe and effective, and researches prove that the ADSCs have the effect of promoting the regeneration and repair of skin.

ADSCs present their inherent advantages in several respects. The ADSCs belong to the monocyte family and are one of adult stem cells. It is derived from treated adipose tissues, grows in a cell culture dish in an adherent and adherent manner during culture, has the capability of being variously differentiated, not only into adipocytes, but also into myocytes, chondrocytes, nerve cells, vascular endothelial cells, osteocytes and the like, and thus is also widely used in tissue engineering. For example, ADSCs can differentiate into adipocytes by self-replication, playing a role in tissue regeneration; the ADSCs can be differentiated into vascular endothelial cells or peripheral cells; the ADSCs can secrete blood vessel growth factors under the condition of hypoxia or other conditions; when the ADSCs and the fat cells are transplanted together, the ADSCs can be differentiated into endothelial cells, so that the occurrence of fibrosis and fat necrosis is prevented, and the fat survival rate is improved; according to the results of many recent specific studies, the ADSCs can express various cytokines such as VEGF, IGF, TGF- β 1, bFGF, EGF, etc., and also prevent the occurrence of apoptosis and maintain the survival rate of adipocytes by expressing vascular growth factors such as VEGF, IGF-1, etc.

The prior art discloses many methods related to the culture of adipose-derived mesenchymal stem cells. For example, CN106479970A (201611050980.1) discloses a method for large-scale culturing human adipose mesenchymal stem cells. The method for culturing the human adipose-derived mesenchymal stem cells in a large scale is characterized in that an adipose-derived mesenchymal stem cell culture medium is added into a glass culture bottle, and the mixture is incubated and balanced for 30 minutes at 35 ℃ in an incubator with the humidity of 95% and the humidity of 5% CO 2; adjusting the temperature of the adipose-derived mesenchymal stem cell culture medium to 35 ℃, adjusting the pH to 7.05, adding the pretreated microcarrier and the adipose-derived mesenchymal stem cells, and stirring for 2 hours at 5-20 RPM; and (4) raising the temperature to 37 ℃, and culturing for 96 hours under the conditions of 15RPM-40RPM and 5% CO2, wherein the adipose mesenchymal stem cell culture medium is supplemented. Compared with the prior art, 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 shape and good stem cell characteristics of the adipose-derived mesenchymal stem cells.

CN104762260A (201510197879.8) relates to a preparation method of adipose-derived mesenchymal stem cells, which is characterized by comprising the following steps: step 1: obtaining fat, pretreating, performing enzymolysis, centrifuging, and collecting precipitate to obtain adipose mesenchymal stem cells; and 2, step: culturing the adipose-derived mesenchymal stem cells, when the adipose-derived mesenchymal stem cells grow to 80% fusion, discarding the culture solution, adding PBS (phosphate buffer solution) for cleaning, adding EDTA-pancreatin solution for digestion, terminating digestion, centrifuging and subculturing. The invention is believed to have the following advantages: 1. the stem cells obtained by culture have high purity; 2. various factors secreted in the culture process of the adipose-derived mesenchymal stem cells are recovered, so that the proliferation of epidermal cells can be remarkably promoted, and the regeneration and replacement of epidermal cells can be accelerated, thereby achieving the remarkable anti-aging effect.

CN104974984A (application No. 201510173453.9) discloses an amplification culture method of adipose tissue-derived mesenchymal stem cells, comprising the following steps: carrying out heavy suspension treatment on the separated mesenchymal stem cells from the adipose tissues by using an adipose-derived stem cell culture medium to obtain a heavy suspension containing the mesenchymal stem cells; inoculating the resuspension into a stem cell culture vessel for amplification culture; replacing the adipose-derived stem cell culture medium after 24 hours of amplification culture, replacing the adipose-derived stem cell culture medium every two days, digesting with pancreatin when the growth is fused to 80-90%, and then carrying out subculture; wherein the adipose-derived stem cell culture medium contains 0.1-10 v/v% of fetal calf serum, 1-10 v/v% of gentamicin and 80-98.9 v/v% of high-sugar DMEM culture medium; the stem cell culture device is a stem cell culture device which is coated with fibronectin. According to the method for amplifying and culturing the adipose tissue-derived mesenchymal stem cells, the fibrous adhesion protein is adopted to coat a solid support to which the adipose tissue-derived mesenchymal stem cells are attached, and the synergistic screening culture of the low-concentration fetal calf serum and the high-concentration gentamicin is utilized, so that the adipose tissue-derived mesenchymal stem cells can be amplified by 400-fold and 500-fold within two weeks, have high purity and can be directionally induced and differentiated into the adipose tissues.

CN106520686A (application No. 201610887111.8) provides a method for culturing adipose-derived mesenchymal stem cells. The adipose tissue-derived mesenchymal stem cell culture method comprises the following culture steps: 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): (5-20 ug/ml). The culture method can improve the amplification 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,the method is characterized by comprising the following steps: (1) obtaining of adipose tissue: adipose tissue is waste obtained by fat sucking operation of swelling method in professional hospital or beauty salon, and is stored at 4-20 deg.C under aseptic condition for less than 48 hr; (2) preliminary removal of erythrocytes: standing the fat extract for layering, carefully removing the lower layer liquid, and washing with D-Hanks solution for several times until the eluate is clear; (3) digestion of fat extracts: adding collagenase I prepared by D-Hanks solution to 0.01-2g/100ml, and digesting for 15-120 minutes at 37 ℃ by shaking at 20-400 r/min; (4) obtaining of adipose-derived stem cells: centrifuging the digested product for 5min at 4 ℃ under the centrifugal force of 450g, resuspending the adipose-derived stem cell culture medium, and filtering with a filter membrane with the aperture of 100 mu m to remove impurities to finally obtain adipose-derived stem cells; (5) removing the red blood cells again, and removing the red blood cells again by using a mode of repeated washing and red blood cell lysis; (6) counting adipose-derived stem cells, detecting activity and culturing: taking 100 μ l of the separated cells, and counting the cells with a cell counting plate; at the same time, 100. mu.l of cells were removed and viable cells were counted by trypan blue staining, based on the results of both counts in 3X 10 ⁴ /cm ² The density of (2) was inoculated in a T-75 flask and cultured at 37 ℃ under the conditions of a CO2 concentration of 5% and a humidity of 100%. The method is believed to be capable of obtaining a large number of adipose-derived stem cells with good states and good multi-directional differentiation capacity by using a small amount of adipose tissues, and has the advantages of simple and easy operation and strong repeatability. However, the method of this document is directed to mesenchymal stem cells at the same time as the cells are lysed, which is disadvantageous for the survival of the stem cells.

CN106222134A (application No. 201610605996.8) discloses a culture method for efficiently obtaining adipose-derived mesenchymal stem cells, wherein only the adipose grain part in the middle layer after centrifugation is aspirated when the adipose is separated, and the cell sediment at the bottom of centrifugation is not obtained.

CN102329783A (application No. 201110310461.5) discloses a method for separating adipose mesenchymal stem cells from a distending fluid in a surgical liposuction operation, which comprises the following steps: (1) before the collection and liposuction of the swelling liquid, pouring excessive hypotonic swelling liquid into a part to be extracted, then placing the fat and the swelling liquid obtained in the liposuction process at room temperature for 10min, and after the fat and the swelling liquid are completely separated, sucking red liquid below the yellow fat by using a liquid transfer gun, namely the swelling liquid containing adipose stem cells; (2) processing the expansion liquid, namely subpackaging the collected expansion liquid into 50ml centrifuge tubes, and placing the centrifuge tubes into a centrifuge to centrifuge for 5min at 1500 rpm; centrifuging to remove the liquid on the upper part of the centrifuge tube, collecting the precipitate, suspending each tube by using PBS (phosphate buffer solution), adding erythrocyte lysate, standing for 5 minutes at room temperature, centrifuging at 1200rpm for 3min, and removing the supernatant; (3) washing the cells, adding 5ml of DMEM (serum-free) to blow away the precipitate, filtering by using a cell filter, centrifuging the filtered liquid at 1200rpm for 3min to obtain the precipitate, namely the adipose-derived mesenchymal stem cells, and adding DMEM (serum-free) to repeat the above operation and washing for 3 times; (4) and (3) cell culture: inoculating the obtained cells into a 75cm2 cell culture bottle, inoculating, adding a mesenchymal stem cell culture medium, and culturing at 37 ℃ under 5% CO 2. However, CN102329783A only separated stem cells from the fraction of the swollenin, not from the fat fluid that was contaminated with the swollenin.

Although the prior art discloses some culture methods such as those described above in relation to adipose mesenchymal stem cells, these methods often require the use of a medium containing relatively high concentrations of fetal bovine serum.

It is still expected to provide a new method for isolated culture or even subculture of mesenchymal stem cells, and in particular to provide a new method without fetal calf serum for isolated culture or even subculture of mesenchymal stem cells.

Disclosure of Invention

The invention aims to provide a novel method for preparing adipose-derived mesenchymal stem cells, in particular to a method suitable for separating and obtaining adipose-derived mesenchymal stem cells from fat, and the method is expected to show the characteristics of high cell yield, high cell viability and the like and/or other excellent performances. The present inventors have surprisingly found that the technical effects of one or more aspects as described herein can be obtained using the process of the present invention. The present invention has been completed based on this finding.

To this end, the present invention provides in a first aspect a method for isolated culture of primary adipose mesenchymal stem cells, comprising the steps of:

(1) processing a fat sample transported to a laboratory through a cold chain at 2-8 ℃ in a biological safety cabinet;

(2) centrifuging the fat sample, removing upper-layer adipose tissue, cleaning with D-Hanks solution, centrifuging again, removing adipose tissue, adding 1% type II collagenase, and performing oscillation digestion;

(3) after digestion, adding one-time volume of D-hanks liquid for dilution, centrifuging, and reserving the cell sediment at the bottom layer for the next operation;

(4) taking the cell sediment obtained in the step (3), adding a primary complete culture medium for resuspension, sampling and counting, and inoculating the cell sediment into a culture bottle according to the specified cell quantity; placing CO ₂ Culturing in an incubator;

(5) and (3) after culturing for 3D, completely replacing the culture medium once until the cell fusion degree reaches more than 80%, removing the old culture medium, cleaning the cells by using D-hanks liquid, adding a recombinant pancreatin solution to digest the cells to make the cells fall off, adding the D-hanks liquid to dilute, centrifuging, and re-suspending the cell precipitate by using a primary complete culture medium to obtain the primary adipose-derived mesenchymal stem cells (namely P0 generation).

The method according to the first aspect of the present invention, wherein in the step (2), both centrifugations are performed under the condition of 100Xg centrifugation for 5 min.

The method according to the first aspect of the present invention, wherein in the step (2), 1% collagenase type II is added in an amount of 2 times the volume thereof for digestion with shaking for 30 min.

The method according to the first aspect of the present invention, wherein in the step (3), the centrifugation is performed under a condition of centrifugation at 100Xg for 5 min.

The method according to the first aspect of the present invention, wherein the inoculation into the culture flask in the prescribed cell amount in the step (4) means that the cell amount is 1 to 5X 10 ⁴ /cm ² Inoculation into T75 flask means, for example, 2X 10 in terms of cell amount ⁴ /cm ² Inoculated into a T75 flask.

The process according to the first aspect of the present invention, wherein in the step (4), CO ₂ The conditions for the culture in the incubator were: 5% CO ₂ 37 ℃ and saturated humidity.

The method according to the first aspect of the present invention, wherein in the step (5), 2ml of the recombinant pancreatin solution is added per vial for digesting the cells for 2 min.

The method according to the first aspect of the present invention, wherein in step (5), 10ml of D-hanks solution is added per bottle for dilution.

The method according to the first aspect of the present invention, wherein in the step (5), the centrifugation is carried out at 100Xg for 10 min.

The method according to the first aspect of the present invention, wherein the formulation of the D-Hanks liquid is as follows: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4.12H2O, 0.35g NaHCO3, water to 1000 ml. For example, the preparation method is as follows: dissolving the materials in 1000ml, and filtering with 0.22 μm microporous membrane for sterilization.

The method according to the first aspect of the invention, wherein said primary complete medium is prepared with DMEM-F12 medium as a matrix and comprises: 1% platelet lysate, 1% human serum albumin, 2. mu.g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF.

The method according to the first aspect of the invention, wherein said primary complete medium is replaced by primary supplemented medium. The method according to the first aspect of the invention, wherein said primary supplementary medium is formulated in DMEM-F12 medium as a medium and comprises: 1% platelet lysate, 1% human serum albumin, 2. mu.g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF, 0.12% thioglycerol, 1% fructose.

The method according to the first aspect of the present invention, wherein the DMEM-F12 medium formulation consists of: 116.6mg of anhydrous calcium chloride, 6.65mg of L-aspartic acid, 2.65mg of L-leucine, 0.042mg of linoleic acid, 0.0013mg of copper sulfate pentahydrate, 91.25mg of L-lysine hydrochloride, 0.105mg of lipoic acid, 0.05mg of ferric nitrate nonahydrate, 17.24mg of L-methionine, 8.1mg of phenol red, 0.417mg of ferrous sulfate heptahydrate, 35.48mg of L-phenylalanine, 0.081mg of 1, 4-butanediamine dihydrochloride, 311.8mg of potassium chloride, 26.25mg of L-serine, 55mg of sodium pyruvate, 28.64mg of magnesium chloride, 53.45mg of L-threonine, 0.0035mg of vitamin H, 48.84mg of anhydrous magnesium sulfate, 4.45mg of L-alanine, 2.24mg of D-calcium pantothenate, 7000mg of sodium chloride, 7.5mg of L-asparagine, 8.98mg of choline chloride, 54.35mg of anhydrous sodium dihydrogen phosphate, 6.65mg of L-aspartic acid, 2.65mg of L-24.24 mg of L-methionine, 17.17 mg of L-disodium hydrogen phosphate, 17.17 mg of L-asparagine, 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 a proper amount of water added to 1000 mL; preparation: dissolving the materials in 1000ml, and filtering and sterilizing with 0.22 μm microporous membrane.

The method according to the first aspect of the present invention, further comprising detecting the primary adipose mesenchymal stem cells obtained by isolated culture. For example, detection of cell morphology and/or immunophenotypic identification. In one embodiment, said immunophenotypic identification refers to detection of CD73, CD90, CD105 and CD19, CD11b, CD31, CD45, HLADR, CD 34. The primary adipose-derived mesenchymal stem cells obtained by the invention are positive in CD73, CD90 and CD105 (all more than 98%), and negative in CD19, CD11b, CD31, CD45, HLADR and CD34 (all less than 2%).

Further, the second aspect of the present invention provides a mesenchymal stem cell culture medium, which may also be referred to as primary complete medium, prepared by taking DMEM-F12 medium as a matrix and comprising: 1% platelet lysate, 1% human serum albumin, 2. mu.g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF.

The mesenchymal stem cell culture medium according to the second aspect of the present invention is prepared with DMEM-F12 medium as a matrix and comprises: 1% platelet lysate, 1% human serum albumin, 2. mu.g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF, 0.12% thioglycerol, 1% fructose.

The mesenchymal stem cell culture medium according to the second aspect of the present invention is used for isolation and culture of primary adipose mesenchymal stem cells.

The mesenchymal stem cell culture medium according to the second aspect of the present invention, which is used for the method for isolating and culturing primary adipose mesenchymal stem cells, comprises the following steps:

(2) centrifuging the fat sample, removing the upper layer adipose tissue, cleaning with D-Hanks solution, centrifuging again, removing adipose tissue, adding 1% type II collagenase, and performing oscillatory digestion;

(3) after digestion is finished, adding one-time volume of D-hanks liquid for dilution, centrifuging, and reserving the cell sediment at the bottom layer for the next operation;

(4) taking the cell sediment obtained in the step (3), adding a primary complete culture medium for resuspension, sampling and counting, and inoculating the cell sediment into a culture bottle according to a specified cell amount; placing CO ₂ Culturing in an incubator;

In the present invention, "10 ^ 6" indicates the power of 6 of 10 when indicating the number of cells; in the present invention, "cm ^ 2" represents a square centimeter when representing a culture area; other cases involving the "^" symbol have similar meanings; the meaning of this symbol is also well known in the art.

According to the method of any one of the embodiments of the first aspect of the present invention, the fat is fat obtained from any part of the human body.

Of the various process steps described above, although specific steps are described in some detail or in language specific to the process steps described in the examples of the following detailed description, those skilled in the art will be able to fully appreciate the above-described process steps from the detailed disclosure of the invention as a whole.

Any embodiment of any aspect of the invention may be combined with other embodiments, as long as they do not contradict. Furthermore, in any embodiment of any aspect of the invention, any feature may be applicable to that feature in other embodiments, so long as they do not contradict. The invention is further described below.

All documents cited herein are incorporated herein by reference in their entirety and to the extent they do not conform to the teachings of the present invention, the statements made therein shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.

In the present invention, the term "adipose-derived mesenchymal stem cell" refers to a mesenchymal stem cell derived from adipose. Thus in the present invention, and in particular in the context relating to the present invention, the term "adipose mesenchymal stem cell" may be used interchangeably with "adipose stem cell", "mesenchymal stem cell", unless explicitly indicated otherwise.

In the present invention, the term "PBS buffer" or "PBS" refers to phosphate buffer. The general formulation and formulation of the PBS used in the context of the present invention, as well as their general properties such as pH value or pH range, are well known to those skilled in the art and are typically commercially available pre-formulations (or powders), e.g. the PBS used in the field of the present invention is typically a commercial buffer at pH7.4(± 0.1), e.g. HyClone brand PBS buffer; in the present invention, the composition of PBS buffer solution in the classical application of the art includes 137mM sodium chloride, 2.7nM potassium chloride and 10mM phosphate, and PBS used in the present invention has the same composition as that in the present invention, unless otherwise specified.

The adipose-derived mesenchymal stem cells are isolated and cultured by the method, and have very high survival rate and very high yield. The present methods exhibit superior technical effects in one or more aspects as described herein.

Drawings

FIG. 1: microscopic cell morphology of mesenchymal stem cells (100 ×).

FIG. 2 is a schematic diagram: cell directed differentiation potential, A) adipogenic control, B) adipogenic induction; C) osteogenic control, D) osteogenic induction; E) chondrogenic control, F) chondrogenic induction.

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. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention generally and/or specifically describes the materials used in the tests, as well as the test methods. Although many materials and methods of operation are known in the art for the purposes of this invention, the invention is nevertheless described herein in as detail as possible.

In the present invention, the DMEM-F12 medium formulation used in the experiments, unless otherwise specified, consisted of: 116.6mg of anhydrous calcium chloride, 6.65mg of L-aspartic acid, 2.65mg of L-leucine, 0.042mg of linoleic acid, 0.0013mg of copper sulfate pentahydrate, 91.25mg of L-lysine hydrochloride, 0.105mg of lipoic acid, 0.05mg of ferric nitrate nonahydrate, 17.24mg of L-methionine, 8.1mg of phenol red, 0.417mg of ferrous sulfate heptahydrate, 35.48mg of L-phenylalanine, 0.081mg of 1, 4-butanediamine dihydrochloride, 311.8mg of potassium chloride, 26.25mg of L-serine, 55mg of sodium pyruvate, 28.64mg of magnesium chloride, 53.45mg of L-threonine, 0.0035mg of vitamin H, 48.84mg of anhydrous magnesium sulfate, 4.45mg of L-alanine, 2.24mg of D-calcium pantothenate, 7000mg of sodium chloride, 7.5mg of L-asparagine, 8.98mg of choline chloride, 54.35mg of anhydrous sodium dihydrogen phosphate, 6.65mg of L-aspartic acid, 2.65mg of L-24.24 mg of L-methionine, 17.17 mg of L-disodium hydrogen phosphate, 17.17 mg of L-asparagine, 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 a proper amount of water added to 1000 mL; preparation: dissolving the materials in 1000ml, and filtering and sterilizing with 0.22 μm microporous membrane.

In the present invention, Platelet lysates used in the experiments were readily available commercially, and as used in the experiments herein, no more than specifically indicated, were obtained from PLTGold Human Platelet Lysate, available from Sigma-Aldrich, having the designation SCM 151.

In the present invention, collagenase type II used in the experiments can be easily obtained from the market, and as not specifically mentioned, used in the experiments herein is from Gibco.

In the present invention, bFGF (basic fibroblast growth factor) used in the experiment can be easily purchased from the market, and as not specifically mentioned, it is purchased from Sigma-Aldrich under the trade name GF003 in the experiment herein.

In the present invention, EGF (epidermal growth factor) used in the test is readily commercially available, and as not specifically mentioned, it is used in the test herein that it is available from Gibco under the trade name PHG 0311L.

In the present invention, recombinant insulin used in the test can be easily obtained from the market, and as not specifically mentioned, it is used in the test herein that it is obtained from Solarbio and its product number is I8830.

In the present invention, the recombinant pancreatin solution used in the test can be readily commercially available, and as used in the test herein, if not specifically mentioned, is a 2000u/ml concentration of recombinant pancreatin solution available from Rambox corporation, cat # RT2S 01.

In the present invention, the D-Hanks solution used in the test, unless otherwise specified, was formulated and prepared as follows: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4.12H2O, 0.35g NaHCO3 and water to 1000 ml; preparation: dissolving the materials in 1000ml, and filtering and sterilizing with 0.22 μm microporous membrane.

In the present invention, the primary complete medium used in the experiments was prepared with DMEM-F12 medium as a medium and contained, unless otherwise specified: 1% platelet lysate, 1% human serum albumin, 2. mu.g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF.

In the present invention, the primary supplement medium used in the experiments was prepared with DMEM-F12 medium as a medium and contained, if not specifically stated: 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 a specific experiment of the present invention, the prepared stem cells of a certain generation were sampled, nucleated cells, i.e., MSC cells were counted using a sysmex hemocytometer, cell viability was detected by trypan blue staining, and the samples were taken for microbial detection.

Example 1: isolation culture of Primary adipose mesenchymal Stem cells

(1) Fat donated by volunteers transported to the laboratory via the cold chain at 2-8 ℃ was processed in a biosafety cabinet (sample a);

(2) centrifuging at 100Xg for 5min, removing upper adipose tissue, cleaning with D-Hanks solution, centrifuging at 100Xg for 5min, removing adipose tissue, adding 1% type II collagenase with 2 times volume, and performing oscillatory digestion for 30 min;

(3) after digestion, adding one-time volume of D-hanks liquid for dilution, centrifuging at 100Xg for 5min, and leaving the cell sediment at the bottom layer for the next operation;

(4) taking the cell sediment obtained in the step (3), adding primary complete culture medium for heavy suspension, sampling and counting, and performing 2X 10 ⁴ /cm ² Inoculating into a T75 bottle; placing CO ₂ Incubator (5% CO) ₂ At 37 ℃, saturated humidity);

(5) and (3) after culturing for 3D, completely replacing the culture medium once until the cell fusion degree reaches more than 80% (about 5D), removing the old culture medium, cleaning the cells by using D-hanks liquid, adding 2ml of recombinant pancreatin solution into each bottle to digest the cells for 2min so as to enable the cells to fall off, adding 10ml of D-hanks liquid into each bottle for dilution, centrifuging for 10min at 100Xg, and re-suspending the cell precipitate by using a primary complete culture medium to obtain the primary adipose mesenchymal stem cells (namely P0 generation).

The above steps (1) to (5) are performed at 2X 10 in step (4) ⁴ /cm ² After the cells were quantitatively inoculated into T75 flasks, the number of nucleated cells obtained per flask was: (Mean of 5 replicates) was 1.13 × 10^6(n ═ 5) (this data can be referred to herein as cell harvest) and cell viability 91.3% (n ═ 5).

Example 1 a: isolation culture of Primary adipose mesenchymal Stem cells

The operations and materials from step (1) to step (3) are continued in example 1;

(4) the cell pellet obtained in step (3) of example 1 was taken, added to primary supplement medium for resuspension, sampled and counted in 2X 10 ⁴ /cm ² Inoculating into a T75 bottle; placing CO ₂ Incubator (5% CO) ₂ 37 ℃, saturated humidity);

(5) and (3) after culturing for 3D, completely replacing the culture medium once until the cell fusion degree reaches more than 80% (about 5D), removing the old culture medium, cleaning the cells by using D-hanks liquid, adding 2ml of recombinant pancreatin solution into each bottle to digest the cells for 2min so as to make the cells fall off, adding 10ml of D-hanks liquid into each bottle for dilution, centrifuging at 100Xg for 10min, and re-suspending the cell precipitate by using a primary supplement culture medium to obtain the primary adipose mesenchymal stem cells (namely P0 generation). Example 1a Steps (1) to (5) described above were carried out at 2X 10 in step (4) ⁴ /cm ² After inoculation into T75 flasks, a nucleated cell number (mean of 5 replicates) of 5.45 × 10^6(n ═ 5) was obtained per flask (this data can be referred to herein as cell harvest), and a cell viability of 92.7% (n ═ 5).

Example 1 b: primary adipose mesenchymal stem cells were obtained by isolated culture as in example 1a, except that primary supplemented medium was supplemented with thioglycerol. The cell harvest of the primary adipose mesenchymal stem cells of example 1b was 1.26 × 10^6(n ═ 5) and the cell viability was 93.3% (n ═ 5).

Example 1 c: primary adipose mesenchymal stem cells were obtained by performing the same procedure as in example 1a, except that fructose was not added to the primary supplemented medium, and isolated and cultured. The cell harvest of the primary adipose mesenchymal stem cells of example 1b was 0.87 x 10^6(n ═ 5) and the cell viability was 90.4% (n ═ 5).

In this document, the embodiment 1a, the embodiment 1b and the embodiment 1c can also be referred to as an auxiliary example of the embodiment 1, while the embodiment 1 can be referred to as a main example, and they can be referred to as an example family.

Compared with the example 1, the cell viability rates of the example 1a, the example 1b and the example 1c are basically the same and are all in the range of 90-94%; however, the cell harvest was about 4.82 times that of example 1a, about 1.12 times that of example 1b, and about 0.77 times that of example 1c for example 1a for example 1 b; these unexpected findings indicate that adding a small amount of cheap thioglycerol and fructose in the primary complete culture medium can not only obtain primary stem cells with substantially equivalent cell viability rate, but also promote a significant increase in cell yield of up to 4 times, and a significant increase in cell yield with substantially unchanged production cost. However, when only thioglycerol or only fructose is added, although the cell viability rate is not changed, the cell yield is not increased, and even when only thioglycerol is additionally added, the cell yield is obviously reduced.

In terms of cell viability, the main examples 2-8 and their respective subsidiary a, b, c examples also show substantially the same results as those of the above example 1 and its subsidiary examples (example 1a, example 1b, example 1c), and the cell viability is in the range of 90-95%, for example, the cell viability of the primary mesenchymal stem cells obtained in example 2 and the fourth of example 2a, example 2b, example 2c is 93.3%, 92.4%, 94.1%, 90.7%.

In terms of cell harvest, the main examples 2-8 and their respective auxiliary examples a, b, c also show substantially the same trend and even result as the above-mentioned example 1 and its auxiliary examples (example 1a, example 1b, example 1c), the cell harvest of the main examples 2-8 is in the range of (0.93-1.27) × 10^6, the cell harvest of the group a auxiliary examples is 4.16-5.23 times of their respective main examples, the cell harvest of the group b auxiliary examples is 0.96-1.24 times of their respective main examples, and the cell harvest of the group c auxiliary examples is 0.69-0.88 times of their respective main examples; for example, the cell yields (n ═ 5) in examples 2, 2a, 2b and 2c were 1.18X 10^6, 5.14X 10^6(4.36 times), 1.23X 10^6(1.04 times) and 0.93X 10^6(0.79 times), respectively.

The primary placental mesenchymal stem cells obtained from examples 1-8 and their respective appendixes a, b and c were tested and the morphology was normal, and immunophenotyping showed that each primary placental mesenchymal stem cell was positive for CD73, CD90 and CD105 (all greater than 98%, for example, CD73 obtained from example 1 was greater than 98.7%), negative for CD19, CD11b, CD31, CD45, HLADR and CD34 (all less than 2%, for example, CD19 obtained from example 1 was less than 0.35%).

Example 2: isolation culture of primary adipose-derived mesenchymal stem cells

(1) Fat donated by volunteers transported to the laboratory via the cold chain at 2-8 ℃ was processed in a biosafety cabinet (sample B);

(2) centrifuging at 100Xg for 5min, removing upper adipose tissue, cleaning with D-Hanks solution, centrifuging at 100Xg for 5min, removing adipose tissue, adding 1% collagenase II with 2 times volume, and performing shake digestion for 30 min;

(4) taking the cell sediment obtained in the step (3), adding primary complete culture medium for resuspension, sampling and counting according to 2 multiplied by 10 ⁴ /cm ² Inoculating into a T75 bottle; placing CO ₂ Incubator (5% CO) ₂ At 37 ℃, saturated humidity);

(5) and (3) after 3D culture, 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 by using D-hanks liquid, adding 2ml of recombinant pancreatin solution into each bottle to digest the cells for 2min so as to make the cells fall off, adding 10ml of D-hanks liquid into each bottle for dilution, centrifuging at 100Xg for 10min, and re-suspending the cell precipitate by using a primary complete culture medium to obtain the primary adipose-derived mesenchymal stem cells (namely P0 generation).

Example 2 a: isolation culture of primary adipose-derived mesenchymal stem cells

The operations and materials from step (1) to step (3) were continued in example 2;

(4) the cell pellet obtained in step (3) of example 2 was taken, added to primary supplement medium for resuspension, sampled and counted in 2X 10 ⁴ /cm ² Inoculating into a T75 bottle; placing CO ₂ Incubator (5% CO) ₂ 37 ℃, saturated humidity);

(5) and (3) after culturing for 3D, completely replacing the culture medium once until the cell fusion degree reaches more than 80% (about 5D), removing the old culture medium, cleaning the cells by using D-hanks liquid, adding 2ml of recombinant pancreatin solution into each bottle to digest the cells for 2min so as to make the cells fall off, adding 10ml of D-hanks liquid into each bottle for dilution, centrifuging at 100Xg for 10min, and re-suspending the cell precipitate by using a primary supplement culture medium to obtain the primary adipose mesenchymal stem cells (namely P0 generation).

Example 2 b: the procedure of example 2a was followed, except that thioglycerol was not added to the primary supplement medium, and isolated culture was performed to obtain primary adipose mesenchymal stem cells.

Example 2 c: the procedure of example 2a was followed, except that fructose was not added to the primary supplemented medium, and isolated to obtain primary adipose mesenchymal stem cells.

Example 3: isolation culture of primary adipose-derived mesenchymal stem cells

(1) Fat donated by volunteers transported to the laboratory via the cold chain at 2-8 ℃ was processed in a biosafety cabinet (sample C);

(3) after digestion, adding one-time volume of D-hanks liquid for dilution, centrifuging for 5min at 100Xg, and reserving the cell sediment at the bottom layer for next operation;

Example 3 a: isolation culture of primary adipose-derived mesenchymal stem cells

The operations and materials from the step (1) to the step (3) are continued in the example 3;

(4) taking the cell pellet obtained in step (3) of example 3, adding primary supplement medium for resuspension, sampling and counting according to 2X 10 ⁴ /cm ² Inoculating into a T75 bottle; placing CO ₂ Incubator (5% CO) ₂ 37 ℃, saturated humidity);

(5) and (3) after culturing for 3D, completely replacing the culture medium once until the cell fusion degree reaches more than 80% (about 5D), removing the old culture medium, cleaning the cells by using D-hanks liquid, adding 2ml of recombinant pancreatin solution into each bottle to digest the cells for 2min so as to enable the cells to fall off, adding 10ml of D-hanks liquid into each bottle for dilution, centrifuging for 10min at 100Xg, and re-suspending the cell precipitate by using a primary supplement culture medium to obtain the primary adipose mesenchymal stem cells (namely P0 generation).

Example 3 b: primary adipose mesenchymal stem cells were obtained by isolated culture as in example 3a, except that the primary supplemented medium was supplemented with thioglycerol.

Example 3 c: primary adipose mesenchymal stem cells were obtained by performing the same procedure as in example 3a, except that fructose was not added to the primary supplemented medium and isolated and cultured.

Example 4: isolation culture of primary adipose-derived mesenchymal stem cells

(1) Fat donated by volunteers transported to the laboratory via the cold chain at 2-8 ℃ was processed in a biosafety cabinet (sample D);

Example 4 a: isolation culture of Primary adipose mesenchymal Stem cells

The operations and materials from step (1) to step (3) were continued in example 4;

(4) the cell pellet obtained in step (3) of example 4 was taken, added to primary supplement medium for resuspension, sampled and counted in 2X 10 ⁴ /cm ² Inoculating into a T75 bottle; placing CO ₂ Incubator (5% CO) ₂ At 37 ℃, saturated humidity);

Example 4 b: the procedure of example 4a was followed, except that thioglycerol was not added to the primary supplement medium, and isolated culture was performed to obtain primary adipose mesenchymal stem cells.

Example 4 c: primary adipose mesenchymal stem cells were obtained by performing the same procedure as in example 4a, except that fructose was not added to the primary supplemented medium and isolated and cultured.

Example 5: isolation culture of Primary adipose mesenchymal Stem cells

(1) Fat donated by volunteers transported to the laboratory via the cold chain at 2-8 ℃ was processed in a biosafety cabinet (sample E);

(4) taking the cell sediment obtained in the step (3), adding primary complete culture medium for resuspension, sampling and counting according to 2 multiplied by 10 ⁴ /cm ² Inoculating into a T75 bottle; placing CO ₂ Incubator (5% CO) ₂ 37 ℃, saturated humidity);

Example 5 a: isolation culture of Primary adipose mesenchymal Stem cells

The operations and materials from step (1) to step (3) were continued in example 5;

(4) taking the cell pellet obtained in step (3) of example 5, adding primary supplement medium to resuspend, sampling and counting according to 2X 10 ⁴ /cm ² Inoculating into a T75 bottle; placing CO ₂ Incubator (5% CO) ₂ 37 ℃, saturated humidity);

Example 5 b: primary adipose mesenchymal stem cells were obtained by isolated culture as in example 5a, except that the primary supplemented medium was supplemented with thioglycerol.

Example 5 c: the procedure of example 5a was followed, except that fructose was not added to the primary supplemented medium, and isolated to obtain primary adipose mesenchymal stem cells.

Example 6: isolation culture of Primary adipose mesenchymal Stem cells

(1) Fat donated by volunteers transported to the laboratory via the cold chain at 2-8 ℃ was processed in a biosafety cabinet (sample F);

Example 6 a: isolation culture of Primary adipose mesenchymal Stem cells

The operations and materials from step (1) to step (3) were continued in example 6;

(4) the cell pellet obtained in step (3) of example 6 was added to a primary supplement cultureResuspending the medium, sampling and counting according to 2X 10 ⁴ /cm ² Inoculating into a T75 bottle; placing CO ₂ Incubator (5% CO) ₂ 37 ℃, saturated humidity);

Example 6 b: the procedure of example 6a was followed, except that thioglycerol was not added to the primary supplement medium, and isolated culture was performed to obtain primary adipose mesenchymal stem cells.

Example 6 c: primary adipose mesenchymal stem cells were obtained by performing the same procedure as in example 6a, except that fructose was not added to the primary supplemented medium and isolated and cultured.

Example 7: isolation culture of Primary adipose mesenchymal Stem cells

(1) Fat donated by volunteers transported to the laboratory via the cold chain at 2-8 ℃ (sample G) was processed in a biosafety cabinet;

(4) taking the cell sediment obtained in the step (3), adding primary complete culture medium for heavy suspension, sampling and counting, and performing 2X 10 ⁴ /cm ² Inoculating into a T75 bottle; placing CO ₂ Incubator (5% CO) ₂ 37 ℃, saturated humidity);

Example 7 a: isolation culture of primary adipose-derived mesenchymal stem cells

The operations and materials of the steps (1) to (3) are continued in example 7;

(4) the cell pellet obtained in step (3) of example 7 was taken, added to primary supplement medium for resuspension, sampled and counted in 2X 10 ⁴ /cm ² Inoculating into a T75 bottle; placing CO ₂ Incubator (5% CO) ₂ 37 ℃, saturated humidity);

Example 7 b: the procedure of example 7a was followed, except that thioglycerol was not added to the primary supplement medium, and isolated culture was performed to obtain primary adipose mesenchymal stem cells.

Example 7 c: the procedure of example 7a was followed, except that fructose was not added to the primary supplemented medium, and isolated to obtain primary adipose mesenchymal stem cells.

Example 8: isolation culture of primary adipose-derived mesenchymal stem cells

(1) Fat donated by volunteers transported to the laboratory via the cold chain at 2-8 ℃ (sample H) was processed in a biosafety cabinet;

Example 8 a: isolation culture of primary adipose-derived mesenchymal stem cells

The operations and materials from the step (1) to the step (3) are continued in the example 8;

(4) taking the cell pellet obtained in step (3) of example 8, adding primary supplement medium to resuspend, sampling and counting according to 2X 10 ⁴ /cm ² Inoculating into a T75 bottle; placing CO ₂ Incubator (5% CO) ₂ At 37 ℃, saturated humidity);

Example 8 b: the procedure of example 8a was followed, except that thioglycerol was not added to the primary supplement medium, and isolated culture was performed to obtain primary adipose mesenchymal stem cells.

Example 8 c: the procedure of example 8a was followed, except that fructose was not added to the primary supplemented medium, and isolated to obtain primary adipose mesenchymal stem cells.

The subculture of the mesenchymal stem cells of generation P0 prepared in examples 1 to 8 and their respective subsidiary a, b and c shows that all the cells can be continuously passaged to more than P10 generations, the cells can maintain stable continuous proliferation capacity, and the cell viability of each generation is stable to more than 87% during continuous passage, for example, the cell viability of a batch of mesenchymal stem cells of generation P0 obtained in example 1a is stable to 93.2% when the batch of mesenchymal stem cells of generation P0 is continuously passaged to P10 generations.

Test example 1: detection of mesenchymal stem cells

Typical characteristics of mesenchymal stem cells include: the cells are fusiform and grow adherently under a microscope, flow cytometry identifies that CD73, CD90 and CD105 are positive and CD19, CD11b, CD31, CD45, HLADR and CD34 are negative, and a directional differentiation potential test shows that the cells have the differentiation potential of osteogenesis, chondrogenesis and adipogenesis.

The mesenchymal stem cells prepared in the embodiments 1 to 8 and the auxiliary a, b and c examples thereof are detected by using a method known in the field, and the results are as follows: all mesenchymal stem cells exhibited spindle-shaped and adherent growth (e.g., microscopic cell morphology of the mesenchymal stem cells of generation P0 obtained in example 1a is shown in fig. 1), CD73, CD90 and CD105 of all mesenchymal stem cells were greater than 98% (e.g., CD73 ═ 99.2%, CD90 ═ 99.7%, CD105 ═ 98.8% for one batch of the mesenchymal stem cells of generation P0 obtained in example 1 a), CD19, CD11b, CD31, CD45 of all mesenchymal stem cells, HLADR and CD34 were less than 2% (for example, CD19 ═ 0.25%, CD11b ═ 0.22%, CD31 ═ 0.06%, CD45 ═ 0.36%, HLADR ═ 0.2%, and CD34 ═ 0.12% for the batch of mesenchymal stem cells of P0 obtained in example 1 a), and the directed differentiation potential test showed that all mesenchymal stem cells had osteogenic, chondrogenic and adipogenic differentiation potential (for example, the differentiation potential of osteogenic, chondrogenic and adipogenic for the batch of mesenchymal stem cells of P0 obtained in example 1a was shown in fig. 2). The above-described embodiments are merely preferred embodiments for fully illustrating the present application, and the scope of the present application is not limited thereto. The equivalent substitution or change made by the person skilled in the art on the basis of the present application is within the protection scope of the present application. The protection scope of this application is subject to the claims.

Claims

1. The method for separating and culturing the primary adipose-derived mesenchymal stem cells comprises the following steps:

(4) taking the cell sediment obtained in the step (3), adding a primary supplement culture medium for resuspension, sampling and counting, and inoculating the cell sediment to a culture bottle according to the specified cell quantity; placing CO ₂ Culturing in an incubator;

(5) after culturing for 3D, completely replacing the culture medium, removing the old culture medium until the cell fusion degree reaches more than 80%, cleaning the cells by using D-hanks liquid, adding a recombinant pancreatin solution to digest the cells to enable the cells to fall off, adding the D-hanks liquid to dilute, centrifuging, and re-suspending the cell precipitate by using a primary supplement culture medium to obtain primary adipose mesenchymal stem cells;

wherein 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.

2. The method according to claim 1, wherein in step (2), both centrifugations are performed at 100Xg for 5 min.

3. The method according to claim 1, wherein in the step (2), 1% collagenase type II is added in an amount of 2 times the volume of the mixture for digestion with shaking for 30 min.

4. The method according to claim 1, wherein in step (3), the centrifugation is performed at 100Xg for 5 min.

5. The method according to claim 1, wherein the step (4) of inoculating the culture flask with the predetermined amount of cells is performed in an amount of 1 to 5X 10 cells ⁴ /cm ² Inoculated into a T75 flask.

6. The process according to claim 1, step (4), wherein CO ₂ The conditions for the culture in the incubator were: 5% CO ₂ 37 ℃ and saturated humidity.

7. The method according to claim 1, wherein the cells are digested in step (5) by adding 2ml of the recombinant pancreatin solution per vial for 2 min.

8. The method according to claim 1, wherein the centrifugation is carried out at 100Xg for 10min in step (5).

9. The process according to claim 1, wherein 10ml of D-hanks liquid is added per bottle for dilution in step (5).

10. The method according to claim 1 wherein the formulation of the D-Hanks liquid consists of: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4.12H2O, 0.35g NaHCO3, water to 1000 ml.

11. The method according to claim 10, wherein the D-Hanks liquid is formulated as follows: dissolving each material by 1000ml, and filtering and sterilizing by a microporous filter membrane of 0.22 mu m to obtain the product.

12. The method according to claim 1, wherein said DMEM-F12 medium formula consists of: 116.6mg of anhydrous calcium chloride, 6.65mg of L-aspartic acid, 2.65mg of L-leucine, 0.042mg of linoleic acid, 0.0013mg of copper sulfate pentahydrate, 91.25mg of L-lysine hydrochloride, 0.105mg of lipoic acid, 0.05mg of ferric nitrate nonahydrate, 17.24mg of L-methionine, 8.1mg of phenol red, 0.417mg of ferrous sulfate heptahydrate, 35.48mg of L-phenylalanine, 0.081mg of 1, 4-butanediamine dihydrochloride, 311.8mg of potassium chloride, 26.25mg of L-serine, 55mg of sodium pyruvate, 28.64mg of magnesium chloride, 53.45mg of L-threonine, 0.0035mg of vitamin H, 48.84mg of anhydrous magnesium sulfate, 4.45mg of L-alanine, 2.24mg of D-calcium pantothenate, 7000mg of sodium chloride, 7.5mg of L-asparagine, 8.98mg of choline chloride, 54.35mg of anhydrous sodium dihydrogen phosphate, 6.65mg of L-aspartic acid, 2.65mg of L-24.24 mg of L-methionine, 17.17 mg of L-disodium hydrogen phosphate, 17.17 mg of L-asparagine, 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 a proper amount of water added to 1000 mL.

13. The mesenchymal stem cell culture medium is prepared by taking a DMEM-F12 culture medium as a matrix and adding the following components in parts by weight: 1% platelet lysate, 0.12% thioglycerol, 1% fructose, 1% human serum albumin, 2 μ g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF.