阅读说明：本技术 一种间充质干细胞体外筛选、激活、扩增、冻存及其细胞库建立的方法 (Method for in vitro screening, activating, amplifying and cryopreserving of mesenchymal stem cells and establishing cell bank of mesenchymal stem cells ) 是由 张炳强 陈梦梦 于 2020-05-25 设计创作，主要内容包括：本发明公开了一种间充质干细胞体外筛选、激活、扩增、冻存及其细胞库建立的方法,该方法包括：利用间充质干细胞专用原代筛选培养基进行第一阶段筛选培养,得到纯化的间充质干细胞；利用间充质干细胞专用激活扩增培养基将纯化的间充质干细胞进行第二阶段激活和大规模扩增培养,获得大量激活功能的间充质干细胞；利用间充质干细胞专用冻存液冻存干细胞,并按ABO/RH分型和HLA分型进行保存,建立可供检索的信息档案,构建间充质干细胞库。(The invention discloses a method for in vitro screening, activating, amplifying and cryopreserving of mesenchymal stem cells and establishing a cell bank of the mesenchymal stem cells, which comprises the following steps: carrying out first-stage screening culture by using a primary screening culture medium special for the mesenchymal stem cells to obtain purified mesenchymal stem cells; performing second-stage activation and large-scale amplification culture on the purified mesenchymal stem cells by using the mesenchymal stem cell special activation amplification culture medium to obtain a large amount of mesenchymal stem cells with activation functions; and (3) freezing and storing the stem cells by using the freezing storage solution special for the mesenchymal stem cells according to ABO/RH typing and HLA typing, establishing an information file for retrieval, and constructing a mesenchymal stem cell bank.)

1. A mesenchymal stem cell in-vitro screening, activating, amplifying, freezing and storing method and a cell bank establishing method thereof are characterized by comprising the following steps: carrying out first-stage screening culture by using a primary screening culture medium special for the mesenchymal stem cells to obtain purified mesenchymal stem cells; performing second-stage activation and large-scale amplification culture on the purified mesenchymal stem cells by using the mesenchymal stem cell special activation amplification culture medium to obtain a large amount of mesenchymal stem cells with activation functions; and (3) freezing and storing the stem cells by using the freezing storage solution special for the mesenchymal stem cells according to ABO/RH typing and HLA typing, establishing an information file for retrieval, and constructing a mesenchymal stem cell bank.

2. The method for in vitro screening, activating, amplifying, freezing and establishing of mesenchymal stem cells and cell bank thereof according to claim 1, wherein the primary screening culture medium dedicated for mesenchymal stem cells is a mesenchymal stem cell serum-free complete culture medium supplemented with 2-8ng/ml SCF, 2-4ng/ml BMP-4, 10-30IU/ml IL-10 and 1-4ng/ml LIF, 1-4ng/ml TGF-beta, 2-8ng/ml rapamycin, 2-12ng/ml trametinib, 10-20ng/ml acetaminophen, 1-3ng/ml 5-HMF, 10-20ng/ml chloroquine phosphate.

3. The method for in vitro screening, activating, amplifying, freezing and establishing the mesenchymal stem cells and the cell bank thereof according to claim 1, wherein the mesenchymal stem cell specific activating and amplifying culture medium is a mesenchymal stem cell serum-free complete culture medium added with 2-8ng/ml SCF, 1-4ng/ml bFGF, 10-20ng/ml paeoniflorin, 20-30ng/ml metformin hydrochloride, 1-4ng/ml hydrocortisone, 2-4ng/ml CXCL10, 1-2ng/ml Forskolin, 1-3ng/ml 5-HMF and 10-20ng/ml chloroquine phosphate.

4. The method for in vitro screening, activating, amplifying, freezing and establishing the mesenchymal stem cells and the cell bank thereof according to claim 1, wherein the freezing medium special for the mesenchymal stem cells comprises 5-10 vol% of DMSO, 5-10 vol% of compound electrolyte injection, 5-10 vol% of hydroxyethyl starch 200/0.5 sodium chloride injection, 1-2 vol% of albumin, 1-2 vol% of hydroxycamptothecin injection and 66-83 vol% of serum-free complete medium for the mesenchymal stem cells.

5. The method for in vitro screening, activating, expanding, freezing and establishing of mesenchymal stem cells and cell bank thereof according to any one of claims 2, 3 and 4, wherein the primary screening Medium dedicated to mesenchymal stem cells, the activating and expanding Medium dedicated to mesenchymal stem cells and the freezing and storing solution dedicated to mesenchymal stem cells are selected from the group consisting of TheraPEAK MSCGM-CD Medium, MesenPRO RS Medium, Corning MSC Xeno-Free SFM and other commercially available serum-Free media.

Technical Field

The invention relates to the technical field of biology, in particular to a method for in-vitro screening, activation, amplification, cryopreservation and cell bank establishment of mesenchymal stem cells.

Background

The development of stem cells, which is the most hot spot in the biological world in recent years, will provide revolutionary technical means for the medical field. The stem cell is one kind of primitive cell with self replicating capacity and multidirectional differentiation potential, and may be differentiated into several kinds of functional cells under certain condition, and may be used in treating leukemia, congenital metabolic disease, some solid tumors, diabetes, heart disease, cerebral palsy and other diseases. The human body has over 220 kinds of cells, which form complex tissues and organs by organic integration, each having its specific functions such as a contraction function of cardiac muscle cells, an information transmission function of nerve cells, etc. Stem cells are the progenitors of these cells, also known in the medical community as "universal cells".

Mesenchymal Stem Cells (MSCs) are derived from mesoderm and ectoderm in early development, belong to pluripotent stem cells, have multidirectional differentiation potential, and can be differentiated into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium and the like. In 2006, the international cell therapy association (ISCT) regulated the definition of MSCs. Only cells that meet the following three criteria at the same time can be called MSCs: firstly, growing adherent; ② some specific antigens (markers) are expressed on the cell surface; ③ the ability to differentiate into adipocytes, osteoblasts and chondrocytes. MSC is the adult stem cell which is currently the most safe, effective and widely used in therapy, and mainly comes from bone marrow, fat, umbilical cord, placenta, amnion, etc. Compared with other stem cells, the MSC has the advantages of easy acquisition, easy in-vitro culture, long-term passage stability, low immunogenicity, strong tissue repair capacity and the like. In addition, MSCs are derived from adult cells, and can even be obtained from the patient himself, rather than embryonic or fetal stem cells, and thus do not involve ethical and ethical issues. MSCs have the potential to differentiate divergently and to replicate themselves after serial passage and cryopreservation. Studies have shown that human bone marrow MSCs can maintain stem cell characteristics for more than 40 passages in vitro.

MSC has multiple functions and wide application, has the main function of cell transplantation treatment, can also be used as an ideal target cell for gene therapy, and has certain application in biological tissue engineering and immunotherapy. In recent years, MSC has been extensively used in experimental and clinical studies, and a great deal of research has revealed its application value in diagnosis and treatment of cardiovascular, nervous, motor, digestive, autoimmune, blood, urinary, ophthalmic, and orthopedic system diseases.

In 2016, China promulgated a first 30 clinical research institutions for stem cells, from 2019, the clinical research institutions for stem cells and project records will carry out dynamic management, and by 9 months in 2019, the number of national approved clinical treatment research hospitals for stem cells is increased to 106, and the number of hospital approved hospitals of a military system is 12 and 118; the number of recorded projects is increased to 62, and the patent application of literature research is continuously increased. By 3 months 2020, the clinical studies related to stem cells registered on clinical trial. gov have reached 5432, of which 469 in china and the most developed cities are guangzhou, beijing and shanghai.

However, obtaining higher quality cells is critical for MSC therapy. The challenge of stem cell therapy is that the stem cell product is very complex, and the difference of cell sources and production processes has great influence on the quality and therapeutic effect of stem cells, which is also the main reason why the results of the previous clinical trials of stem cell therapy are not ideal. Therefore, how to obtain a large amount of standardized, high-quality and high-activity MSCs becomes the most important factor for restricting the development of the stem cell industry, and methods for screening, activating, amplifying, freezing and establishing MSCs in vitro need to be improved.

Disclosure of Invention

The invention aims to solve the technical problems in the prior art. Therefore, the invention provides a method for in vitro screening, activating, amplifying and cryopreservation of mesenchymal stem cell bodies (MSC) and establishing a cell bank, the method has the advantages of high efficiency, high speed, high safety and low cost of screening, activating and amplifying the MSC, and a large amount of function-activated MSC obtained by screening and amplifying can establish the cell bank for long-term storage and still maintain good cell activity after recovery.

The invention provides a method for in vitro screening, activating, amplifying and freezing mesenchymal stem cells and establishing a cell bank of the mesenchymal stem cells. According to an embodiment of the invention, the method comprises:

carrying out first-stage screening culture by using a primary screening culture medium special for mesenchymal stem cells to obtain purified MSCs; performing second-stage activation and large-scale amplification culture on the purified MSC by using the special activation amplification culture medium for the mesenchymal stem cells to obtain a large amount of activated functional MSC; and (3) freezing and storing the stem cells by using the freezing storage solution special for the mesenchymal stem cells according to ABO/RH typing and HLA typing, establishing an information file for retrieval, and constructing a mesenchymal stem cell bank.

The primary screening culture medium special for the mesenchymal stem cells is a serum-free complete culture medium for the mesenchymal stem cells, which is added with 2-8ng/ml SCF, 2-4ng/ml BMP-4, 10-30IU/ml IL-10 and 1-4ng/ml LIF, 1-4ng/ml TGF-beta, 2-8ng/ml rapamycin, 2-12ng/ml trametinib, 10-20ng/ml acetaminophen, 1-3ng/ml 5-HMF and 10-20ng/ml chloroquine phosphate.

The special activation and amplification culture medium for the mesenchymal stem cells is a mesenchymal stem cell serum-free complete culture medium added with 2-8ng/ml SCF, 1-4ng/ml bFGF, 10-20ng/ml paeoniflorin, 20-30ng/ml metformin hydrochloride, 1-4ng/ml hydrocortisone, 2-4ng/ml CXCL10, 1-2ng/ml Forskolin, 1-3ng/ml 5-HMF and 10-20ng/ml chloroquine phosphate.

The special cryopreservation solution for the mesenchymal stem cells comprises 5-10 vol% of DMSO, 5-10 vol% of compound electrolyte injection, 5-10 vol% of hydroxyethyl starch 200/0.5 sodium chloride injection, 1-2 vol% of albumin, 1-2 vol% of hydroxycamptothecin injection and 66-83 vol% of a mesenchymal stem cell serum-free complete culture medium.

The serum-Free complete culture Medium is a TheraPEAK (therapeutic growth hormone receptor) MSCGM-CD (multiple growth hormone-binding domain.

The method of the invention divides the mesenchymal stem cell culture into a first stage screening culture and a second stage activation and large-scale amplification culture, and the two stages utilize different culture conditions.

The first stage of screening culture is mainly to eliminate primary medium impurity cells. MSCs, whether derived from bone marrow, umbilical cord, placenta, or the like, are more or less contaminated with blood cells, endothelial cells, and other foreign cells during the primary preparation. The primary screening culture medium special for the mesenchymal stem cells is particularly added with cell growth promoting factors and screening factors. SCF (Recombinant Human StemCell factor) can stimulate cell proliferation and migration in vitro. BMP-4 (Recombinant Human BoneMorphogenic Protein 4) is a potent bone morphogenetic Protein and is part of the transforming growth factor (TGF-. beta.) superfamily and plays a role in the formation of mesenchymal cells and the development of multiple organs. The invention discloses a method for preparing a non-MSC (non-mesenchymal stem cell) analgesic drug, which is characterized in that acetaminophen is a common antipyretic analgesic drug, and the acetaminophen with a proper concentration is added, so that non-MSC in primary culture is obviously inhibited, the apoptosis speed of mixed cells is accelerated, and no influence is caused on MSC. Rapamycin is a specific mTOR inhibitor with an IC50 of 0.1nM, and exerts an antitumor effect by inducing autophagy, inhibiting tumor cell viability in a dose-dependent manner. The low-dose rapamycin can induce autophagy and apoptosis of non-MSC in primary culture. The invention discovers that the chloroquine phosphate (the conventional concentration is about 5ug/ml and can be used as an autophagy inhibitor and a lysosome inhibitor) with extremely low dose (not higher than 20 ng/ml), can remarkably promote the proliferation of MSC, and can be used as an autophagy inhibitor to resist the autophagy induced by rapamycin. Trametinib (Trametinib) is a reversible inhibitor of mitogen-activated extracellular signal-regulated kinase 1(MEK1) and MEK2 activation and MEK1 and MEK2 kinase activity. MEK proteins are upstream regulators of the extracellular signal-related kinase (ERK) pathway, which promote cell proliferation. 5-HMF (5-hydroxymethylfurfural) has an antioxidant effect and can resist oxidative damage caused by hydrogen peroxide, and the action mechanism of the 5-HMF is probably related to the reduction of nuclear factor kB protein expression and the increase of Bcl-2 protein expression.

The second stage is activation and large scale amplification culture. SCF and bFGF (basic fibroblast growth factor) can stimulate cell proliferation and migration in vitro. Metformin has been known from the advent to the present, has been used for 50 years, and has been widely used for the treatment of metabolic diseases such as type 2 diabetes, polycystic ovary syndrome, obesity and the like, because it has various pharmacological actions such as inhibition of hepatic glucose output, increase of the sensitivity of peripheral tissues to insulin and the like. In recent years, the research finds that metformin has more and more effects. The invention discovers for the first time that metformin hydrochloride can activate MSC, promote the proliferation and activation of stem cells and obviously improve the secretion capacity of MSC factors. Paeoniflorin (PF) is the main effective component of common Chinese medicine peony, and is a monoterpene glycoside compound. In recent years, scholars at home and abroad carry out more intensive research on the pharmacological action of paeoniflorin, and the paeoniflorin is found to have the activities of resisting free radical damage, inhibiting intracellular calcium overload, resisting neurotoxicity and the like, and in vivo experiments prove that the paeoniflorin has various biological effects of reducing blood viscosity, resisting platelet aggregation, expanding blood vessels, improving microcirculation, resisting oxidation, resisting convulsion and the like, and has small toxic and side effects. The invention discovers for the first time that paeoniflorin can activate MSC, promote the proliferation and activation of stem cells, inhibit the apoptosis of stem cells and obviously improve the secretion capacity of MSC factors. CXCL10 (CXC chemokine ligand 10), namely IP-10 (interferon-lymphocyte protein-10), can inhibit the formation of hematopoietic cell colonies, chemotaxis monocytes, activate T cells and natural killer cells, stimulate the fusion of T cell adhesion endothelial cells and natural killer cells mediated cells, inhibit angiogenesis and the like, but the invention firstly discovers that CXCL10 can activate MSC, promote the proliferation and activation of stem cells, inhibit the apoptosis of stem cells and obviously improve the secretion capacity of MSC factors. Forskolin, a ubiquitous activator of eukaryotic Adenylate Cyclase (AC), is commonly used to elevate cAMP levels in cell physiology studies. Adenylate Cyclase (AC) can be directly activated by its catalytic subunit to increase intracellular cyclic adenosine monophosphate (cAMP) levels. Literature research finds that Forskolin can promote the proliferation of olfactory ensheathing cells cultured in vitro and also induce the differentiation of stem cells. The invention finds that Forskolin with proper concentration can promote the proliferation and activation of MSC, inhibit the apoptosis of stem cells and promote the expression of MSC surface markers.

The compound electrolyte injection with the volume of 5-10% is specially added into the freezing storage solution special for the mesenchymal stem cells, so that the osmotic pressure of the crystals can be better maintained. The colloid osmotic pressure can be better maintained by adding 5-10 vol% hydroxyethyl starch 200/0.5 sodium chloride injection and 1-2 vol% albumin. The addition of 1-2 vol% hydroxycamptothecin injection can greatly improve the cell survival rate after resuscitation.

The invention can be used for screening, activating, amplifying and culturing the MSC to obtain a large amount of standardized, high-quality and high-activity MSC. The invention has the advantages of high screening efficiency, high amplification speed, high safety, low cost and the like. The invention establishes a corresponding cell bank, classifies and stores the stem cells in large scale, has long effective preservation time, still keeps good cell activity after recovery and high cell recovery rate, thereby meeting the requirements of a large number of stem cells in clinical treatment.

Drawings

FIG. 1 is a flow chart of the method of the present invention;

FIG. 2 is an adipose MSC (200X) obtained by culturing 14d according to the method of the present invention;

FIG. 3 is a comparison of the proliferation rate of stem cells in the method of the present invention and in the conventional culture method.

Detailed Description

The invention provides a method for in vitro screening, activating, amplifying and cryopreserving of mesenchymal stem cells and establishing a cell bank of the mesenchymal stem cells, which comprises the following steps: carrying out first-stage screening culture by using a primary screening culture medium special for mesenchymal stem cells to obtain purified MSCs; performing second-stage activation and large-scale amplification culture on the purified mesenchymal stem cells by using the mesenchymal stem cell special activation amplification culture medium to obtain a large amount of MSCs with activation functions; and (3) freezing and storing the stem cells by using the freezing storage solution special for the mesenchymal stem cells according to ABO/RH typing and HLA typing, establishing an information file for retrieval, and constructing a mesenchymal stem cell bank.

According to the embodiment of the invention, the primary screening culture medium special for the mesenchymal stem cells is a mesenchymal stem cell serum-free complete culture medium added with 2-8ng/ml SCF, 2-4ng/ml BMP-4, 10-30IU/ml IL-10 and 1-4ng/ml LIF, 1-4ng/ml TGF-beta, 2-8ng/ml rapamycin, 2-12ng/ml trametinib, 10-20ng/ml acetaminophen, 1-3ng/ml 5-HMF and 10-20ng/ml chloroquine phosphate.

According to the embodiment of the invention, the mesenchymal stem cell special activation and amplification culture medium is a mesenchymal stem cell serum-free complete culture medium added with 2-8ng/ml SCF, 1-4ng/ml bFGF, 10-20ng/ml paeoniflorin, 20-30ng/ml metformin hydrochloride, 1-4ng/ml hydrocortisone, 2-4ng/ml CXCL10, 1-2ng/ml Forskolin, 1-3ng/ml 5-HMF and 10-20ng/ml chloroquine phosphate.

According to the embodiment of the invention, the serum-Free complete culture Medium is TheraPEAK (mesenchymal Stem cell) -MSCGM-CD (multiple Mass Messaging culture Medium), MesenPRO RS (Medium), Corning MSC Xeno-Free SFM or other commercially available types of serum-Free culture Medium.

According to the embodiment of the invention, in the first stage of primary screening culture of the mesenchymal stem cells, the cells are passaged for 2 times every 2-3 d; and in the second mesenchymal stem cell activation and large-scale amplification culture process, the passage is carried out once every 2-3d for a plurality of times. Therefore, MSCs can be expanded in a high purity and large scale in a short time, and sufficient function-activated MSCs are obtained for possible clinical treatment.

According to the embodiment of the invention, the special cryopreservation solution for the mesenchymal stem cells is a serum-free complete culture medium containing 5-10 vol% of DMSO, 5-10 vol% of compound electrolyte injection, 5-10 vol% of hydroxyethyl starch 200/0.5 sodium chloride injection, 1-2 vol% of albumin, 1-2 vol% of hydroxycamptothecin injection and 66-83 vol% of the mesenchymal stem cells, wherein the cryopreservation concentration of the mesenchymal stem cells is (1 × 10)⁷-5×10⁸⁾And/ml. Therefore, the frozen cells have high concentration, are suitable for freezing and storing stem cells on a large scale, have low freezing and storing cost, good freezing and storing effect, and high cell survival rate and cell yield after recovery.

According to the embodiment of the invention, the activated and amplified mesenchymal stem cells are stored according to ABO/RH typing and HLA typing, a mesenchymal stem cell information file for retrieval is established, and a mesenchymal stem cell bank is constructed.

The scheme of the invention will be explained with reference to the examples. The methods used in the following examples are conventional methods unless otherwise specified, and the required reagent consumables and laboratory instruments and the like are commercially available.