WO2021237762A1 - 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 - Google Patents

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 Download PDF

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WO2021237762A1
WO2021237762A1 PCT/CN2020/093716 CN2020093716W WO2021237762A1 WO 2021237762 A1 WO2021237762 A1 WO 2021237762A1 CN 2020093716 W CN2020093716 W CN 2020093716W WO 2021237762 A1 WO2021237762 A1 WO 2021237762A1
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stem cells
mesenchymal stem
medium
cells
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French (fr)
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张炳强
陈梦梦
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Qingdao Restore Biotechnology Co Ltd
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Qingdao Restore Biotechnology Co Ltd
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Priority to US17/634,553 priority Critical patent/US20230107321A1/en
Priority to EP20937954.4A priority patent/EP4159842A4/en
Priority to KR1020227004427A priority patent/KR20230016160A/ko
Priority to JP2022507329A priority patent/JP7390473B2/ja
Publication of WO2021237762A1 publication Critical patent/WO2021237762A1/zh
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Priority to US19/274,695 priority patent/US20250388865A1/en
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Definitions

  • the present invention relates to the field of biotechnology, in particular to a method for in vitro screening, activation, expansion, cryopreservation and cell bank establishment of mesenchymal stem cells.
  • Stem cells are the biggest hot spot in the biological world in recent years, and their development will provide revolutionary technical means in the medical field.
  • Stem cells are a kind of primitive cells with self-replication ability and multi-differentiation potential. Under certain conditions, they can differentiate into a variety of functional cells, which can be used to treat leukemia, congenital metabolic diseases, certain solid tumors, diabetes, and heart disease. And many diseases such as cerebral palsy have very broad medical uses.
  • Stem cells are the progenitor cells of these cells, and the medical profession also calls them "universal cells”.
  • MSC Mesenchymal stromal/stem cell
  • MSC is currently the safest, most effective and widely used adult stem cell in treatment, mainly derived from bone marrow, fat, umbilical cord, placenta, amniotic membrane and so on. Compared with other stem cells, MSC has the advantages of easy access, easy in vitro culture, long-term passage stability, low immunogenicity, and strong tissue repair ability. In addition, MSC is derived from adult cells, and can even be obtained from the patient's own body, rather than embryonic or fetal stem cells, so it does not involve moral and ethical issues. After continuous passage and cryopreservation, MSC still has the potential of multi-directional differentiation and self-replication. Studies have shown that human bone marrow MSC can be passaged in vitro for more than 40 generations and still maintain the characteristics of stem cells.
  • MSC has many functions and is widely used. Its main function is to carry out cell transplantation therapy. It can also be used as an ideal target cell for gene therapy. It also has certain applications in biological tissue engineering and immunotherapy. In recent years, MSC has been widely used in experimental and clinical research, and a large number of studies have revealed that it is used in cardiovascular, nervous system, motor system, digestive system, autoimmune diseases, blood system, urinary system, ophthalmology, orthopedics and other system diseases. Application value in diagnosis and treatment.
  • the present invention aims to solve the technical problems existing in the prior art. For this reason, the present invention proposes a method for in vitro screening, activation, expansion, cryopreservation and cell bank establishment of mesenchymal stem cell bodies (MSC).
  • the method has high efficiency, fast speed and safety in screening, activating, and expanding MSCs. High performance and low cost, and a large number of functionally activated MSCs obtained by screening and expansion can establish a cell bank for long-term storage, and still maintain good cell viability after resuscitation.
  • the invention provides a method for in vitro screening, activation, expansion, cryopreservation and cell bank establishment of mesenchymal stem cells. According to an embodiment of the present invention, the method includes:
  • the special primary screening medium for mesenchymal stem cells is 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- ⁇ , 2-8ng/ml rapamycin, 2-12ng/ml trametinib, 10-20ng/ml acetaminophen, 1-3ng/ml 5-HMF, 10-20ng/ml Serum-free complete medium for mesenchymal stem cells of chloroquine phosphate.
  • the special activation and expansion medium for mesenchymal stem cells is supplemented with 2-8ng/ml SCF, 1-4ng/ml bFGF, 10-20ng/ml paeoniflorin, 20-30ng/ml metformin hydrochloride, 1-4ng/ml Serum-free complete medium for mesenchymal stem cells of 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 mesenchymal stem cells contains 5-10% by volume of DMSO, 5-10% by volume of compound electrolyte injection, 5-10% by volume of hydroxyethyl starch 200/0.5 sodium chloride injection, 1 -2 vol% albumin, 1-2 vol% hydroxycamptothecin injection, 66-83 vol% mesenchymal stem cell serum-free complete medium.
  • the serum-free complete medium is TheraPEAK TM MSCGM-CD TM Medium , MesenPRO RS TM Medium, MSC Xeno-Free SFM or other commercially available serum-free media.
  • the mesenchymal stem cell culture is divided into the first stage of screening culture and the second stage of activation and large-scale expansion culture.
  • the two stages use different culture conditions.
  • the first stage of screening and culture is mainly to remove the original impurity cells. Regardless of the MSC derived from bone marrow, umbilical cord, or placenta, in the primary preparation process, more or less mixed cells such as blood cells and endothelial cells will be mixed.
  • the special primary screening medium for mesenchymal stem cells is specially added with cell growth-promoting factors and screening factors. SCF (Recombinant Human Stem Cell Factor) can stimulate cell proliferation and migration in vitro.
  • BMP-4 Recombinant Human Bone Morphogenetic Protein 4
  • TGF- ⁇ transforming growth factor
  • Acetaminophen is a common antipyretic and analgesic.
  • the present invention finds that after adding acetaminophen at an appropriate concentration, non-MSCs are obviously inhibited in primary culture, and the rate of apoptosis of hybrid cells is accelerated without affecting MSCs.
  • Rapamycin is a specific mTOR inhibitor with IC50 of 0.1nM. Rapamycin exerts an anti-tumor effect by inducing autophagy and inhibits tumor cell viability in a dose-dependent manner.
  • the low-dose rapamycin of the present invention can induce autophagy and apoptosis of non-MSCs in the primary generation.
  • the present invention found that very low doses (not higher than 20ng/ml) of chloroquine phosphate (conventional concentration of about 5ug/ml can be used as autophagy inhibitors and lysosome inhibitors), can significantly promote the proliferation of MSCs, and can be used as autophagy Inhibitors combat autophagy induced by rapamycin.
  • Trametinib is a reversible inhibitor of mitogen-activated extracellular signal-regulated kinase 1 (MEK1) and MEK2 activation and MEK1 and MEK2 kinase activity.
  • MEK protein is an upstream regulator of the extracellular signal-related kinase (ERK) pathway, which promotes cell proliferation.
  • 5-HMF (5-Hydroxymethylfurfural) has an antioxidant effect and can resist the oxidative damage caused by hydrogen peroxide.
  • the mechanism of action may be related to the decrease of nuclear factor kappa B protein expression and the increase of Bcl-2 protein expression by 5-HMF.
  • the second stage is activation and large-scale expansion culture.
  • SCF and bFGF basic fibroblast growth factor
  • Metformin has been around for 50 years since its inception. Because of its pharmacological effects in inhibiting hepatic glucose output and increasing the sensitivity of peripheral tissues to insulin, it has been widely used in type 2 diabetes, polycystic Treatment of metabolic diseases such as ovarian syndrome and obesity. In recent years, studies have found more and more effects of metformin.
  • the present invention finds for the first time that metformin hydrochloride can activate MSC, promote the proliferation and activation of stem cells, and significantly enhance the secretion ability of MSC factors.
  • Paeoniflorin (Paeoniflorin, PF) is the main effective component of the commonly used Chinese medicine Paeonia lactiflora, and it is a monoterpene glycoside compound.
  • researchers at home and abroad have carried out more in-depth studies on the pharmacological effects of paeoniflorin and found that paeoniflorin has anti-free radical damage, inhibits intracellular calcium overload and anti-neurotoxicity activities.
  • In vivo experiments have shown that it can reduce blood viscosity and resist Platelet aggregation, expansion of blood vessels, improvement of microcirculation, anti-oxidation, anti-convulsive and other biological effects, and have less toxic and side effects.
  • the present invention finds for the first time that paeoniflorin can activate MSCs, promote the proliferation and activation of stem cells, inhibit stem cell apoptosis, and significantly enhance the secretion ability of MSC factors.
  • CXCL10 CXC chemokine ligand-10, CXC chemokine ligand 10
  • IP-10 interferon-inducible protein-10
  • the present invention found for the first time that CXCL10 can activate MSC, promote stem cell proliferation and activation, inhibit stem cell apoptosis, and significantly enhance MSC factor secretion ability.
  • Forskolin forskolin
  • AC ubiquitous eukaryotic adenylate cyclase
  • cAMP cyclic adenosine monophosphate
  • Literature studies have found that Forskolin can promote the proliferation of olfactory ensheathing cells cultured in vitro and also induce stem cell differentiation.
  • the present invention finds that suitable concentration of Forskolin can promote MSC proliferation and activation, inhibit stem cell apoptosis, and promote the expression of MSC surface markers.
  • the special freezing solution for mesenchymal stem cells of the present invention is specially added with 5-10% by volume of compound electrolyte injection, which can better maintain the crystal osmotic pressure.
  • compound electrolyte injection Especially adding 5-10% by volume of hydroxyethyl starch 200/0.5 sodium chloride injection and 1-2% by volume of albumin can better maintain the colloidal osmotic pressure.
  • Adding 1-2% by volume of hydroxycamptothecin injection can greatly increase the cell viability after resuscitation.
  • the present invention By using the present invention to screen, activate, and expand MSCs, a large number of standardized, high-quality, and highly active MSCs can be obtained.
  • the invention has the advantages of high screening efficiency, fast amplification speed, high safety and low cost.
  • the present invention establishes a corresponding cell bank, categorizes large-scale stem cell storage, and has a long effective storage time. After resuscitation, the cells still maintain good cell viability and the cell recovery rate is high, thereby meeting the demand for a large number of stem cells in clinical treatment.
  • Figure 1 is a flow chart of the method of the present invention.
  • Fig. 2 shows adipose MSC (200 ⁇ ) obtained by culturing for 14 days with the method of the present invention.
  • Figure 3 is a comparison of the growth rate of stem cells between the method of the present invention and the conventional culture method.
  • the present invention provides a method for in vitro screening, activation, expansion, cryopreservation of mesenchymal stem cells and establishment of a cell bank thereof.
  • the method includes: using a primary screening medium dedicated for mesenchymal stem cells to carry out the first stage of screening and culture to obtain purification MSC; use the special activation expansion medium for mesenchymal stem cells to carry out the second stage activation and large-scale expansion culture of the purified mesenchymal stem cells to obtain a large number of activated MSCs; use the special freezing liquid for mesenchymal stem cells to freeze Store stem cells and store them according to ABO/RH typing and HLA typing, establish a searchable information file, and construct a mesenchymal stem cell bank.
  • the special primary screening medium for mesenchymal stem cells is 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- ⁇ , 2-8ng/ml rapamycin, 2-12ng/ml trametinib, 10-20ng/ml paracetamol, 1-3ng/ml 5-HMF, Serum-free complete medium for mesenchymal stem cells with 10-20ng/ml chloroquine phosphate.
  • the special activation and expansion medium for mesenchymal stem cells is supplemented with 2-8ng/ml SCF, 1-4ng/ml bFGF, 10-20ng/ml paeoniflorin, 20-30ng/ml metformin hydrochloride, Serum-free complete medium for mesenchymal stem cells of 1-4ng/ml hydrocortisone, 2-4ng/ml CXCL10, 1-2ng/ml Forskolin, 1-3ng/ml 5-HMF, 10-20ng/ml chloroquine phosphate .
  • the serum-free complete medium used is TheraPEAK TM MSCGM -CD TM Medium, MesenPRO RS TM Medium, MSC Xeno-Free SFM or other commercially available serum-free media.
  • MSC in the first stage of the primary screening culture of mesenchymal stem cells, passage once every 2-3 days, and passage twice; in the process of activation and large-scale expansion of the second mesenchymal stem cells, every 2- Passage 3d once and pass it many times. Therefore, MSC can achieve high-purity large-scale amplification in a short time, and obtain sufficient functionally activated MSC for possible clinical treatment.
  • the special cryopreservation solution for mesenchymal stem cells contains 5-10% by volume of DMSO, 5-10% by volume of compound electrolyte injection, and 5-10% by volume of hydroxyethyl starch 200/0.5 chlorinated Sodium injection, 1-2% by volume of albumin, 1-2% by volume of hydroxycamptothecin injection, 66-83% by volume of mesenchymal stem cell serum-free complete medium.
  • the cryopreservation concentration of mesenchymal stem cells is (1 ⁇ 10 7 -5 ⁇ 10 8) /ml.
  • the concentration of cryopreserved cells is high, suitable for large-scale cryopreservation of stem cells, the cost of cryopreservation is low, and the effect of cell freezing is good, and the cell viability and cell yield after resuscitation are high.
  • the activated and expanded mesenchymal stem cells are stored according to ABO/RH typing and HLA typing, to establish a mesenchymal stem cell information archive for retrieval, and construct a mesenchymal stem cell bank.
  • adipose MSCs are separated and obtained.
  • Donor screening The collection hospital must sign an informed consent form with the donor, in triplicate. One copy for each of the donor and collection medical institution, and the other copy to the laboratory along with the specimen. The hospital inquires about the donor’s personal information, past treatment history, family genetic history, and whether there is a history of infectious diseases, hematopoietic or immune system abnormalities and other information by means of inquiries and forms.
  • the donor’s physical examination information should include the following items: HIV antibody, hepatitis B surface antigen and antibody, hepatitis C antibody, cytomegalovirus antibody, Treponema pallidum antibody, transaminase, etc.
  • the informed consent form, personal information collection form, inspection information, etc. must be numbered and sealed.
  • anyone who contacts the data shall not disclose their privacy without the consent of the donor or its authorized personnel. Save according to ABO/RH classification and HLA classification, and establish a database of stem cell donor archives that can be retrieved.
  • SVF Short Vascular Fraction
  • the vascular matrix component is the active ingredient extracted from the adipose tissue of the patient. It contains a variety of repairing cells, including endothelial cells, non-characteristic stromal cells, blood cells, and tissues.
  • Cell populations formed by type macrophages, hematopoietic progenitor cells and MSCs are used for culture respectively, the traditional serum-containing medium (DMEM+10% FBS+8ng/ml bFGF) culture, the serum-free medium MSCGM-CD TM medium culture and the method of the present invention.
  • DMEM+10% FBS+8ng/ml bFGF serum-free medium
  • MSCGM-CD TM medium culture the method of the present invention.
  • DMEM+10%FBS+8ng/ml bFGF Traditional serum-containing medium (DMEM+10%FBS+8ng/ml bFGF) culture: According to the number of SVF cells, add 20ml (DMEM+10%FBS+8ng/ml bFGF) at a density of 1.0 ⁇ 10 6 /ml, and inoculate One T175 culture flask, placed in a carbon dioxide incubator, culture conditions: (37 ⁇ 0.5)°C, carbon dioxide volume fraction (5 ⁇ 0.2)%. Change the fluid every 2-3d. At about 6 days, when the primary cultured cells reach 70%-80% confluence, they are passaged for 4 consecutive passages.
  • DMEM+10%FBS+8ng/ml bFGF 20ml (DMEM+10%FBS+8ng/ml bFGF) at a density of 1.0 ⁇ 10 6 /ml, and inoculate One T175 culture flask, placed in a carbon dioxide incubator, culture conditions: (37 ⁇ 0.5)°C
  • TheraPEAK TM MSCGM-CD TM Medium (brand LONZA, article number 00190632) culture: According to the number of SVF cells, add 20ml of MSCGM-CD TM medium at a density of 1.0 ⁇ 10 6 /ml, and inoculate 1 T175 culture flask , Placed in a carbon dioxide incubator, culture conditions: 37 ⁇ 0.5 °C, the volume fraction of carbon dioxide is 5 ⁇ 0.2%. Change the fluid every 2-3d. At about 6 days, when the primary cultured cells reached 80% confluence, they were passaged for 4 consecutive passages.
  • Subculture replacement culture system is a special activation expansion medium for mesenchymal stem cells, that is, 5ng/ml SCF, 2ng/ml bFGF, 10ng/ml paeoniflorin, 20ng/ml metformin hydrochloride, 2ng/ml hydrogenated Serum-free complete medium for mesenchymal stem cells containing pine, 3ng/ml CXCL10, 1ng/ml Forskolin, 1ng/ml 5-HMF, 10ng/ml chloroquine phosphate, continue to culture. Observe the cells every day, according to the color of the medium, change the medium every 2-3d and pass them down.
  • Nude mice tumorigenicity test SPF-grade female BALB/c nude mice, 4-6 weeks old, weighing 18-20g, raised in a rat cage with a lid in a laminar air flow rack, drinking water, standard feed and other contact with animals All products are sterilized.
  • the MSCs cultured for 28 days were inoculated subcutaneously into the ribs of nude mice at 3 ⁇ 10 7 /0.2ml, and labeled with picric acid. Tumor formation was observed for a period of 2 months.
  • the yield and viability results of the cells cultured and expanded for 14 days by the three methods 2 ⁇ 10 7 SVFs in the traditional serum-containing medium were cultured for 14 days to obtain 2.17 ⁇ 10 9 MSCs, with a viability of 94.36%; serum-free medium MSCGM- After 14 days of CD TM culture, 2.69 ⁇ 10 9 MSCs were obtained, with a viability rate of 96.71%; while the culture method of the present invention obtained 4.35 ⁇ 10 9 MSCs after 14 days of culture, with a viability of 98.73%.
  • the method of the present invention has a far better yield of MSC cultured than the other two current conventional culture methods.
  • the MSC suspension obtained for 14 days was commissioned to detect the antibody hepatitis B surface antigen, hepatitis C antigen, human immunodeficiency virus antibody, Treponema pallidum specific antibody, macrophage virus, mycoplasma, bacteria and endotoxin.
  • the test results were all negative. It shows that this batch of MSC is safe and did not cause pollution during the cultivation process.
  • the expressions of the positive index rate and the negative index rate of the MSC cell surface markers obtained are significantly better than the other two conventional culture methods.
  • the MSC obtained by the method of the present invention in Example 1 was cryopreserved, and the effects of cryopreservation were compared.
  • the grouping is as follows:
  • Cryopreservation solution 1 10% by volume of DMSO, 90% by volume of MesenPRO RS TM Medium;
  • Cryopreservation solution 2 5 vol% DMSO, 90 vol% MesenPRO RS TM Medium;
  • Cryopreservation solution 3 5% by volume of DMSO, 10% by volume of compound electrolyte injection, 75% by volume of MesenPRO RS TM Medium;
  • Cryopreservation solution 4 5% by volume of DMSO, 10% by volume of compound electrolyte injection, 10% by volume of hydroxyethyl starch 200/0.5 sodium chloride injection, 75% by volume of MesenPRO RS TM Medium;
  • Cryopreservation solution 5 5% by volume of DMSO, 10% by volume of compound electrolyte injection, 10% by volume of hydroxyethyl starch 200/0.5 sodium chloride injection, 2% by volume of albumin, 73% by volume of MesenPRO RS TM Medium;
  • Cryopreservation solution 6 (the special cryopreservation solution for mesenchymal stem cells of the present invention): 5% by volume of DMSO, 10% by volume of compound electrolyte injection, 10% by volume of hydroxyethyl starch 200/0.5 sodium chloride injection, 2 volumes % Albumin, 2% by volume hydroxycamptothecin injection, 71% by volume MesenPRO RS TM Medium;
  • Freeze MSC cells according to the following steps: After mixing the cryopreservation solution with the cells, quickly transfer it into the cryopreservation tube and put it in the freezing box at -80°C overnight, and transfer it to liquid nitrogen the next day. Among them, 1ml cryopreservation solution is used for every 5 ⁇ 10 7 MSC cells. MSC cells were cryopreserved for 90 days and then resuscitated. Detect the survival rate of cells before and after cryopreservation, and the recovery rate of cells after resuscitation. Specifically, the cell survival rate calculation method before cryopreservation and after cryopreservation and resuscitation is: [number of living cells/(number of living cells+number of dead cells)] ⁇ 100%. The calculation method of the recovery rate of cells after resuscitation is: (number of viable cells after resuscitation/number of viable cells during cryopreservation) ⁇ 100%.
  • cryopreservation solution 2 is better than that of cryopreservation solution 1, indicating that 5% DMSO concentration is the optimum concentration. Increasing the DMSO concentration will increase the cytotoxicity of DMSO, but will not improve the MSC cell viability and cells. Yield.
  • the cell viability and cell yield of cryopreservation solution 3 are better than those of cryopreservation solution 2, indicating that compound electrolyte injection has the effect of stabilizing and regulating the osmotic pressure of crystals, and can improve the survival rate and yield of cryopreserved cells after resuscitation.
  • cryopreservation solution 4 is better than that of cryopreservation solution 3, indicating that hydroxyethyl starch 200/0.5 sodium chloride injection has the effect of stabilizing and regulating colloidal osmotic pressure, which can improve the recovery of cryopreserved cells Live rate and yield.
  • the cell viability and cell yield of cryopreservation solution 5 are better than those of cryopreservation solution 4, indicating that albumin has the effect of stabilizing and regulating colloidal osmotic pressure, and can improve the survival rate and yield of cryopreserved cells after resuscitation.
  • cryopreservation solution 6 The cell viability and cell yield of cryopreservation solution 6 are better than those of cryopreservation solution 5, indicating that hydroxycamptothecin injection can increase the survival rate and yield of cryopreserved cells after resuscitation.
  • the cell viability and cell yield of cryopreservation solution 6 were significantly better than the other five cryopreservation solutions, and the MSC viability rate after resuscitation reached more than 99%, and the yield rate was more than 97%, indicating that the mesenchymal stem cell special freezers of the present invention
  • the stock solution is suitable for MSC freezing.
  • umbilical cord MSCs are obtained, and stored according to ABO/RH typing and HLA typing, and information about umbilical cord mesenchymal stem cells can be established for retrieval Archives to construct a cell bank of umbilical cord mesenchymal stem cells.
  • the hospital inquires about the donor’s personal information, past treatment history, family genetic history, and whether there is a history of infectious diseases, hematopoietic or immune system abnormalities and other information by means of inquiries and forms.
  • the hospital must sign an informed consent form with the donor to obtain the consent of the donor or its authorized personnel, and consult its physical examination data to obtain the physical examination information.
  • the donor’s physical examination information should include the following items: HIV antibody, hepatitis B surface antigen and antibody, hepatitis C antibody, cytomegalovirus antibody, Treponema pallidum antibody, transaminase, etc.
  • Personal information collection forms, informed consent forms, inspection information, etc. need to be numbered and sealed, and an archive information database of umbilical cord donors can be established for retrieval.
  • the specific preparation method includes the following steps, taking a 5cm umbilical cord as an example, the operation is as follows (all manipulation steps are completed on a clean workbench):
  • the umbilical cord is collected 5cm, stored in a vaccine box at a constant temperature of 2-8°C, and sent to the laboratory within 48 hours.
  • the umbilical cord takes the umbilical cord and place it in a 5cm petri dish, wash it, cut it into several sections, and remove the three middle blood vessels.
  • the white connective tissue located between the amniotic membrane and the blood vessels is Walton's glue. Use long-handled toothed forceps to tear it off and place it in a sterile petri dish.
  • tissue homogenate after enzyme digestion was added with 6 times the volume of normal saline in a volume ratio of 1:6, filtered through a sterile 200-mesh filter to remove the residue, centrifuged at 400g for 8min, discarded the supernatant, and obtained the lower layer of cells.
  • MSC special primary screening culture According to the cell count, add 50ml of MSC special primary screening culture at a density of 1.0 ⁇ 10 6 cells/ml, that is, add 8ng/ml SCF, 4ng/ml BMP-4, 30IU/ml IL-10 And 4ng/ml LIF, 4ng/ml TGF- ⁇ , 8ng/ml rapamycin, 12ng/ml trametinib, 20ng/ml paracetamol, 3ng/ml 5-HMF, 20ng/ml chloroquine phosphate MSC Xeno-Free SFM, inoculate 3 T175 culture flasks, place in a carbon dioxide incubator, culture conditions: (37 ⁇ 0.5)°C, carbon dioxide volume fraction (5 ⁇ 0.2)%, change the medium every 2-3d.
  • MSCs For qualified MSCs, use 1ml of the above-configured MSC special cryopreservation solution for every 5 ⁇ 10 7 MSC cells to perform the freezing operation, and save them according to the ABO/RH classification and HLA classification, and establish the umbilical cord room for retrieval Mesenchymal stem cell information archives to construct a bank of umbilical cord mesenchymal stem cells.
  • placental MSCs are obtained, stored according to ABO/RH classification and HLA classification, and information about placental mesenchymal stem cells can be established for retrieval Archives to construct a placental mesenchymal stem cell bank.
  • the hospital inquires about the donor’s personal information, past treatment history, family genetic history, and whether there is a history of infectious diseases, hematopoietic or immune system abnormalities and other information by means of inquiries and forms.
  • the hospital must sign an informed consent form with the donor to obtain the consent of the donor or its authorized personnel, and consult its physical examination data to obtain the physical examination information.
  • the donor’s physical examination information should include the following items: HIV antibody, hepatitis B surface antigen and antibody, hepatitis C antibody, cytomegalovirus antibody, Treponema pallidum antibody, transaminase, etc.
  • Personal information collection forms, informed consent forms, inspection information, etc. need to be numbered and sealed, and a placental donor archive information database that can be retrieved is established.
  • placental preservation solution 100mg penicillin, 100mg streptomycin, 2.5mg amphotericin, 0.5g heparin sodium injection, 2.5g human albumin in 500ml It is obtained in DMEM medium), it is advisable to have the placenta submerged, and stored in a vaccine box at a constant temperature of 2-8°C, and sent to the laboratory within 24 hours.
  • the filtrate was centrifuged at 1800 rpm for 10 min at room temperature, the supernatant was discarded, and the cell pellet was resuspended in an appropriate amount of physiological saline.
  • the volume ratio of the resuspension:HES was 4: 1 Add HES, centrifuge at 300 rpm for 10 min, collect the upper liquid, and obtain cells after centrifugation.
  • 2.4 Primary screening culture According to the cell count, add 50ml of MSC special primary screening culture at a density of 1.0 ⁇ 10 6 cells/ml, that is, 2ng/ml SCF, 2ng/ml BMP-4, 10IU/ml IL-10 are added And 1ng/ml LIF, 1ng/ml TGF- ⁇ , 2ng/ml rapamycin, 2ng/ml trametinib, 10ng/ml acetaminophen, 1ng/ml 5-HMF, 10ng/ml chloroquine phosphate MesenPRO RS TM Medium, inoculate 3 T175 culture flasks, place them in a carbon dioxide incubator, culture conditions: (37 ⁇ 0.5)°C, carbon dioxide volume fraction (5 ⁇ 0.2)%, change the medium every 2-3d.
  • 2.5 Passage activation and expansion about 5-7 days, when the primary cultured cells reach 80% confluence, aspirate and discard the old cell culture solution, digest and pass them, and replace the culture system with MSC-specific activation expansion medium after passaging, that is, add 2ng/ ml SCF, 1-4ng/ml bFGF, 10ng/ml paeoniflorin, 20ng/ml metformin hydrochloride, 1ng/ml hydrocortisone, 2ng/ml CXCL10, 1ng/ml Forskolin, 1ng/ml 5-HMF, 10ng/ml MesenPRO RS TM Medium of chloroquine phosphate, continue to cultivate. Observe the cells every day, according to the color of the medium, change the medium every 2-3d and pass them down.
  • Detect qualified MSC use 1ml of the above-configured MSC special freezing solution for every 5 ⁇ 10 7 MSC cells, perform freezing operation, save according to ABO/RH classification and HLA classification, and establish a placental compartment for retrieval Mesenchymal stem cell information archives to construct a placental mesenchymal stem cell bank.

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Abstract

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。

Description

[根据细则37.2由ISA制定的发明名称]　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 技术领域
本发明涉及生物技术领域，具体地，涉及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。
背景技术
干细胞作为近年来生物界的最大热点，其发展将为医疗领域提供革命性的技术手段。干细胞是一类具有自我复制能力和多向分化潜能的原始细胞，在一定条件下，它可以分化成多种功能细胞，可用于治疗白血病、先天性代谢疾病、某些实体肿瘤、糖尿病、心脏病和脑瘫等多种疾病，具有非常广阔的医疗用途。人体有220余种细胞，它们通过有机整合形成复杂的组织和器官，各有其特定功能，如心肌细胞的收缩功能、神经细胞的信息传递功能等。干细胞是这些细胞的祖细胞，医学界也称其为“万用细胞”。
间充质干细胞(Mesenchymal stromal/stem cell，MSC)来源于发育早期的中胚层和外胚层，属于多能干细胞，具有多向分化潜能，可分化为脂肪、骨、软骨、肌肉、肌腱、韧带、神经、肝、心肌、内皮等多种组织细胞。2006年，国际细胞治疗协会(ISCT)规范了MSC的定义。只有同时符合以下三个标准的细胞，才能称之为MSC：①贴壁生长；②细胞表面表达一些特异性抗原(标记物)；③具有向脂肪细胞、成骨细胞、软骨细胞分化的能力。MSC是目前在治疗方面最为安全有效和应用广泛的成体干细胞，主要来源于骨髓、脂肪、脐带、胎盘、羊膜等。与其他干细胞相比，MSC具有易获取、易体外培养、长期传代稳定性、低免疫原性、组织修复能力强等优势。此外，MSC来自于成年的细胞，甚至可以从患者自身体内获得，而非胚胎或胎儿干细胞，因而不涉及道德和伦理学的问题。MSC在经过连续传代和冷冻保存之后，仍具有多向分化和自我复制的潜能。研究表明人骨髓MSC可在体外传代40代以上仍保持干细胞特性。
MSC功能较多，应用广泛，主要功能是进行细胞移植治疗，亦可作为一种理想的靶细胞用于基因治疗，同时在生物组织工程和免疫治疗中也有一定的应用。近些年MSC被大量应用于实验和临床研究中，已有大量研究揭示了它在心血管、神经系统、运动系统、消化系统、自身免疫病、血液系统、泌尿系统、眼科、骨科等系统疾病的诊断和治疗上的应用价值。
2016年，中国公布首批30家干细胞临床研究机构，2019年起干细胞临床研究机构与项目备案将实行动态管理，截止到2019年9月，国家批准干细胞临床治疗研究医院增至106家，军队系统的医院批准的共12家，一共118家机构；备案项目增至62个，文献研究专利申请不断增长。截止到2020年3月，在ClinicalTrial.gov上注册的干细胞相关临床研究已达5432项，其中中国有469项，较多开展的城市为广州、北京、上海。
然而，获得更高品质的细胞是MSC治疗的关键。干细胞治疗的挑战在于干细胞产品十分复杂，细胞来源和生产工艺的差异对干细胞的质量和治疗效果的影响很大，这也是之前众多干细胞治疗临床试验结果不理想的主要原因。因此，如何获取大量的、标准化的、高质量、高活性的MSC成为制约干细胞行业发展最重要的因素，MSC体外筛选、激活、扩增、冻存、建库的方法需要改进。
发明内容
本发明旨在解决现有技术中存在的技术问题。为此，本发明提出了间充质干细胞体(MSC)体外筛选、激活、扩增、冻存及其细胞库建立的方法，该方法筛选、激活、扩增MSC的效率高、速度快、安全性高且成本低，并且筛选扩增获得的大量功能激活的MSC可建立细胞库进行长期保存，复苏后依然保持良好的细胞活性。
本发明提供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。根据本发明的实施例，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MSC；利用间充质干细胞专用激活扩增培养基将纯化的MSC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MSC；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。
所述间充质干细胞专用原代筛选培养基是添加了2-8ng/ml SCF、2-4ng/ml BMP-4、10-30IU/ml IL-10和1-4ng/ml LIF、1-4ng/ml TGF-β、2-8ng/ml雷帕霉素、2-12ng/ml曲美替尼、10-20ng/ml对乙酰氨基酚、1-3ng/ml 5-HMF、10-20ng/ml磷酸氯喹的间充质干细胞无血清完全培养基。
所述间充质干细胞专用激活扩增培养基是添加了2-8ng/ml SCF、1-4ng/ml bFGF、10-20ng/ml芍药苷、20-30ng/ml盐酸二甲双胍、1-4ng/ml氢化可的松、2-4ng/ml CXCL10、1-2ng/ml Forskolin、 1-3ng/ml 5-HMF、10-20ng/ml磷酸氯喹的间充质干细胞无血清完全培养基。
所述间充质干细胞专用冻存液是包含5-10体积％的DMSO、5-10体积％的复方电解质注射液、5-10体积％羟乙基淀粉200/0.5氯化钠注射液、1-2体积％的白蛋白、1-2体积％羟喜树碱注射液、66-83体积％的间充质干细胞无血清完全培养基。
所述间充质干细胞专用原代筛选培养基、间充质干细胞专用激活扩增培养基、间充质干细胞专用冻存液中，所述的无血清完全培养基为TheraPEAK TMMSCGM-CD TMMedium、MesenPRO RS TMMedium、
Figure PCTCN2020093716-appb-000001
MSC Xeno-Free SFM或市售其他类型无血清培养基。
本发明的方法，将间充质干细胞培养分为第一阶段筛选培养和第二阶段激活和大规模扩增培养，两个阶段利用不同的培养条件。
第一阶段筛选培养，主要是为了去掉原代中杂细胞。无论骨髓、脐带、胎盘来源的MSC，在原代制备过程中或多或少会混杂进血液细胞、内皮细胞等杂细胞。间充质干细胞专用原代筛选培养基特别添加了细胞促生长因子和筛选因子。SCF(Recombinant Human Stem Cell Factor)在体外，能刺激细胞增殖、迁移。BMP-4(Recombinant Human Bone Morphogenetic Protein 4)是一种有效的骨形成蛋白，同时为转化生长因子(TGF-β)超家族的一部分，在间质细胞形成和多个器官的发育过程中起作用。对乙酰氨基酚为常见解热镇痛药，本发明发现，添加适当浓度对乙酰氨基酚后，原代培养中非MSC明显抑制，杂细胞凋亡速度加快，对MSC无影响。雷帕霉素是一种特异性的mTOR抑制剂，IC50为0.1nM，雷帕霉素通过诱导自噬发挥抗肿瘤作用，以剂量依赖性方式抑制肿瘤细胞活力。本发明低剂量雷帕霉素可诱导原代中非MSC自噬和凋亡。本发明发现，极低剂量(不高于20ng/ml)的磷酸氯喹(常规浓度约为5ug/ml可作为自噬抑制剂和溶酶体抑制剂),可以显著促进MSC增值，可以作为自噬抑制剂对抗雷帕霉素诱导的自噬。曲美替尼(Trametinib)是丝裂原活化的细胞外信号调节激酶1(MEK1)和MEK2激活和MEK1和MEK2激酶活性的可逆性抑制剂。MEK蛋白质是细胞外信号相关激酶(ERK)通路的上游调节器，它促进细胞增殖。5-HMF(5-羟甲基糠醛)具有抗氧化作用，可对抗过氧化氢引起的氧化损伤，作用机制可能与5-HMF降低核因子κB蛋白表达、增加Bcl-2蛋白表达有关。
第二阶段是激活和大规模扩增培养。SCF、bFGF(basic fibroblast growth factor)在体外，能刺激细胞增殖、迁移。二甲双胍从问世至今，已经走过了50年的历程，因其具有抑制肝葡糖糖输出，增加外周组织对胰岛素的敏感性等多方面的药理作用，已被广泛应用于2型糖尿病、多囊卵巢综合征、肥胖等代谢性疾病的治疗。近年来研究发现二甲双胍越来越多的作用。本发明首次发现，盐酸二甲双胍可激活MSC，促进干细胞增值、活化，显著提升MSC因子分泌能力。芍药苷(Paeoniflorin，PF)为常用中药芍药的主要有效成分，是一种单萜类糖苷化合物。近年来，国内外学者对芍药苷的药理作用展开了较为深入的研究，发现芍药苷具有抗自由基损伤，抑制细胞内钙超载和抗神经毒性等活性，体内实验证明其有降低血液黏度、抗血小板聚集、扩张血管、改善微循环、抗氧化、抗惊厥等多种生物学效应，并且毒副作用较小。本发明首次发现，芍药苷可激活MSC，促进干细胞增值、活化，抑制干细胞凋亡，显著提升MSC因子分泌能力。CXCL10(CXC chemokine ligand-10，CXC趋化因子配体10)，即IP-10(interferon-inducible protein-10)，能够抑制造血细胞集落形成，趋化单核细胞、活化T细胞和自然杀伤细胞，刺激T细胞黏附内皮细胞以及自然杀伤细胞介导的细胞融解，抑制血管生成等，而本发明首次发现CXCL10可激活MSC，促进干细胞增值、活化，抑制干细胞凋亡，显著提升MSC因子分泌能力。Forskolin(毛喉萜)，是一种普遍存在的真核细胞腺苷酸环化酶(AC)激活剂，在细胞生理学研究中，通常用来提高cAMP水平。可通过其催化亚基直接激活腺苷酸环化酶(AC)，以增加细胞内环磷酸腺苷(cAMP)的水平。文献研究发现Forskolin，可促进体外培养的嗅鞘细胞增值，也诱导干细胞分化。本发明发现合适浓度的Forskolin可促进MSC增值、活化，抑制干细胞凋亡，促进MSC表面标记物的表达。
本发明间充质干细胞专用冻存液特别添加5-10体积％的复方电解质注射液，可以更好维持晶体渗透压。特别添加5-10体积％羟乙基淀粉200/0.5氯化钠注射液和1-2体积％的白蛋白，可以更好维持胶体渗透压。添加1-2体积％羟喜树碱注射液，可以大幅提升复苏后细胞活率。
利用本发明对MSC进行筛选、激活、扩增培养可得到大量的、标准化的、高质量、高活性的MSC。本发明具有筛选效率高、扩增速度快、安全性高和成本低等优点。且本发明建立相应的细胞库，分类进行大规模的干细胞存储，有效保存时间长，复苏后细胞依然保持良好的细胞活力，细胞回收率高，从而满足临床治疗中大量干细胞的需求。
附图说明
图1是本发明方法的流程图。
图2是本发明方法培养14d获得的脂肪MSC(200×)。
图3是本发明方法与常规培养方法干细胞增值速度对比。
具体实施方式
本发明提供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，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MSC；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MSC；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。
根据本发明的实施例，间充质干细胞专用原代筛选培养基是添加了2-8ng/ml SCF、2-4ng/ml BMP-4、10-30IU/ml IL-10和1-4ng/ml LIF、1-4ng/ml TGF-β、2-8ng/ml雷帕霉素、2-12ng/ml曲美替尼、10-20ng/ml对乙酰氨基酚、1-3ng/ml 5-HMF、10-20ng/ml磷酸氯喹的间充质干细胞无血清完全培养基。
根据本发明的实施例，间充质干细胞专用激活扩增培养基是添加了2-8ng/ml SCF、1-4ng/ml bFGF、10-20ng/ml芍药苷、20-30ng/ml盐酸二甲双胍、1-4ng/ml氢化可的松、2-4ng/ml CXCL10、1-2ng/ml Forskolin、1-3ng/ml 5-HMF、10-20ng/ml磷酸氯喹的间充质干细胞无血清完全培养基。
根据本发明的实施例，间充质干细胞专用原代筛选培养基、间充质干细胞专用激活扩增培养基、间充质干细胞专用冻存液中，所用的无血清完全培养基为TheraPEAK TMMSCGM-CD TMMedium、MesenPRO RS TMMedium、
Figure PCTCN2020093716-appb-000002
MSC Xeno-Free SFM或市售其他类型无血清培养基。
根据本发明的实施例，第一阶段间充质干细胞原代筛选培养中，每2-3d传代一次，传代2次；第二间充质干细胞激活和大规模扩增培养过程中，每2-3d传代一次，多次传代。因此，MSC可在短时间内实现高纯度规模化扩增，获得充足的功能激活的MSC，用于可能的临床治疗。
根据本发明的实施例，间充质干细胞专用冻存液是包含5-10体积％的DMSO、5-10体积％的复方电解质注射液、5-10体积％羟乙基淀粉200/0.5氯化钠注射液、1-2体积％的白蛋白、1-2体积％ 羟喜树碱注射液、66-83体积％的间充质干细胞无血清完全培养基。其中，间充质干细胞的冻存浓度为(1×10 7-5×10 8)/ml。因此，冻存的细胞浓度高，适于大规模干细胞的冻存，冻存成本低，并且细胞冻存效果好，复苏后细胞活率和细胞得率高。
根据本发明的实施例，将激活扩增的间充质干细胞，按ABO/RH分型和HLA分型进行保存，建立可供检索的间充质干细胞信息档案，构建间充质干细胞库。
下面将结合实施例对本发明的方案进行解释。下述实施例中所用方法如无特别说明均为常规方法，所需试剂耗材及实验仪器等均可通过商业途径购得。
实施例1
利用本发明的间充质干细胞体外筛选、激活、扩增的方法，分离获取脂肪MSC。
1供体筛选：采集医院须与供体签署知情同意书，一式3份。供体、采集医疗机构各一份，另一份随标本送交实验室。医院以询问和填表方式征询供体个人信息、过往治疗史、家族遗传史，以及是否有传染病史及造血或免疫系统的异常情况等信息。供体体检信息应包括如下项目：艾滋病病毒抗体、乙肝表面抗原和抗体、丙肝抗体、巨细胞病毒抗体、梅毒螺旋体抗体、转氨酶等。知情同意书、个人信息采集表、检查信息等需编号密封保存，任何接触资料的人员未经供体本人或其授权人员的同意，不得泄露其隐私。按ABO/RH分型和HLA分型进行保存，建立可供检索的干细胞供体档案信息库。
2脂肪干细胞的制备：实验用脂肪取自腹部吸脂手术者。注意预留5ml外周血做快检和血型鉴定。无菌条件下，获得脂肪生理盐水混合物20mL，离心，PBS清洗两遍去除麻醉药品及血细胞，获得纯度较高的脂肪颗粒。0.1％胶原酶37℃恒温摇床消化60min，1500r/min，离心10min，去上层未消化的脂肪组织及油脂，沉淀重悬200um滤器过滤，再次离心，红细胞裂解液裂解红细胞5min、磷酸盐缓冲液洗涤两遍，获得SVF。SVF(Stromal Vascular Fraction)，即血管基质组分，是从患者自体抽取的脂肪组织中提取有效成分，含有多种具有修复功能的细胞，是内皮细胞、非特征性的基质细胞、血液细胞、组织型巨噬细胞、造血祖细胞和MSC等形成的细胞群。分别应用下述三种方式进行培养，传统含血清培养基(DMEM+10％FBS+8ng/ml bFGF)培养、无血清培养基MSCGM-CD TM培养基培养和本发明方法培养。
2.1传统含血清培养基(DMEM+10％FBS+8ng/ml bFGF)培养：根据SVF细胞数，按照1.0×10 6/ml的密度加入(DMEM+10％FBS+8ng/ml bFGF)20ml，接种1个T175培养瓶，置于二氧化碳培养箱，培养条件：(37±0.5)℃，二氧化碳体积分数为(5±0.2)％。每隔2-3d换液一次。6d左右，原代培养细胞达70％～80％融合时，进行传代，连续传代4次。
2.2无血清培养基TheraPEAK TMMSCGM-CD TMMedium(品牌LONZA，货号00190632)培养：根据SVF细胞数，按照1.0×10 6/ml的密度加入MSCGM-CD TM培养基20ml，接种1个T175培养瓶，置于二氧化碳培养箱，培养条件：37±0.5℃，二氧化碳体积分数为5±0.2％。每隔2-3d换液一次。6d左右，原代培养细胞达80％融合时，进行传代，连续传代4次。
2.3本发明方法培养：
2.3.1根据SVF细胞数，按照1.0×10 6/ml的密度加入MSC专用原代筛选培养20ml，即添加了5ng/ml SCF、5ng/ml BMP-4、20IU/ml IL-10和2ng/ml LIF、2ng/ml TGF-β、5ng/ml雷帕霉素、8ng/ml曲美替尼、10ng/ml对乙酰氨基酚、2ng/ml 5-HMF、15ng/ml磷酸氯喹的MSCGM-CD TMMedium，接种1个T175培养瓶，置于二氧化碳培养箱，培养条件：37±0.5℃，二氧化碳体积分数为5±0.2％。其中，每日观察细胞，根据培养基的颜色，2-3d进行换液。5d左右，原代培养细胞达70％～80％融合时，进行传代1次。
2.3.2传代更换培养体系为间充质干细胞专用激活扩增培养基，即添加了5ng/ml SCF、2ng/ml bFGF、10ng/ml芍药苷、20ng/ml盐酸二甲双胍、2ng/ml氢化可的松、3ng/ml CXCL10、1ng/ml Forskolin、1ng/ml 5-HMF、10ng/ml磷酸氯喹的间充质干细胞无血清完全培养基，继续培养。每日观察细胞，根据培养基的颜色，每2-3d换液传代一次。
2.4上述是三个方法培养扩增14d时，将获得的MSC全部回收，并进行细胞计数，计算细胞得率。均留取10ml培养基(培养72h)上清Elisa法进行分泌细胞因子TGF-β、GM-CSF、IL-2、IL-10、VEGF、HGF、PDGF的检测。将回收的细胞用200ml生理盐水重悬，并加入10ml人血清白蛋白，混匀；并从中取10ml细胞悬液待检，剩余的细胞可注入回输袋中准备回输。从回输袋中抽取10ml细胞悬液进行检测：支原体、内毒素、微生物和病毒五项。剩余部分细胞继续培养至28d。
2.5流式细胞仪检测MSC表面标记物。三个方法培养14d获得的建充质干细胞1×10 5(100ul)， 分别加入20ul CD29、CD73、CD90、CD105、CD34、CD45、CD14、HLA-DR抗体,混匀，避光孵育30min，PBS洗涤两次，美国Backman Clouter FC500流式细胞仪检测。
2.6细胞增值速度对比：分别取上述三种方法培养收集到的培养14d的脂肪MSC，消化制作成单细胞悬液，按5×10 4/cm 2接种于96孔培养板中，每孔分别加上述三种培养液(其中本发明方法为添加间充质干细胞专用激活扩增培养基)100μl。同时取培养板一列加入生长培养基不加脂肪MSC，每孔培养液为100μl，作为空白对照组。每12h取4孔，MTT比色法测570nm波长光吸收值(D570值)，连续测3d，绘制细胞生长曲线。方法如下：终止培养时，每孔加入MTT溶液20μl(浓缩液5mg/ml)，37℃孵育4h，吸掉上清液，每孔加入150μl DMSO，室温振荡10min，选择570nm波长，调空白对照组一列均值为零，在酶联免疫检测仪上测定各孔的D570值，求其平均值。以时间为横轴，D570值为纵轴绘制细胞的生长曲线。
2.7裸鼠致瘤试验：SPF级雌性BALB/c裸鼠，4-6周龄，体重18-20g，于空气层流架中带盖鼠笼内饲养，饮用水、标准饲料及其它与动物接触品均经灭菌处理。将上述培养28d的MSC按3×10 7个/0.2ml接种裸鼠肋部皮下，用苦味酸标记，为期2个月观察成瘤情况。
3实验结果
3.1三种方法培养扩增14d的细胞得率和活率结果：传统含血清培养基2×10 7个SVF培养14d后获得2.17×10 9个MSC，活率94.36％；无血清培养基MSCGM-CD TM培养14d后获得2.69×10 9个MSC，活率96.71％；而本发明培养方法培养14d后获得4.35×10 9个MSC，活率98.73％。本发明方法培养MSC得率远优于其他两种目前常规培养方法。
3.2三种方法培养扩增14d培养基上清Elisa分泌因子检测结果如下表1所示。本发明方法培养获得的MSC细胞因子分泌能力强于其他两种常规培养方法。
表1.三种方法培养14d后培养基上清细胞因子检测结果
Figure PCTCN2020093716-appb-000003
Figure PCTCN2020093716-appb-000004
3.3三种方法培养扩增14d获得的MSC悬液委托检测平台检测了抗体乙肝表面抗原、丙肝抗原、人类免疫缺陷病毒抗体、梅毒螺旋体特异性抗体、巨噬细胞病毒以及支原体、细菌和内毒素，检测结果均呈阴性。说明该批次MSC是安全的，培养过程中没有造成污染。
3.4三种方法培养扩增14d获得的细胞流式检测结果：三种方法培养扩增获得的细胞均符合MSC表面标记物的特征，阳性指标为CD29、CD73、CD90、CD105结果如表2所示，阴性指标CD34、CD45、CD14、HLA-DR结果如表3所示。
表2.三种方法培养14d后细胞流式检测阳性指标结果
Figure PCTCN2020093716-appb-000005
表3.三种方法培养14d后后细胞流式阴性指标检测结果
Figure PCTCN2020093716-appb-000006
可以看出，本发明培养方法，获得的MSC细胞表面标记物阳性指标率和阴性指标率的表达均明显优于其他两种常规培养方法。
3.5三种方法培养扩增14d获得MSC分别在对应培养体系中细胞增值速度对比结果：如图3所示，可以看出，本发明方法培养MSC时，细胞增值速度明显优于其他两种常规培养方法。
3.6裸鼠致瘤实验结果：在2个月观察期内皮下注射0.2ml生理盐水和3×10 7个/0.2ml培养28d的MSC的两组小鼠均未见肿瘤形成。该结果说明本发明的方法即使培养到28d，MSC依然是安全 有效的，不会导致肿瘤的形成。培养扩增后的MSC不会在体内形成肿瘤。
实施例2
对实施例1中本发明方法获得的MSC进行冻存，比较冻存效果。分组如下：
冻存液1：10体积％的DMSO、90体积％的MesenPRO RS TMMedium；
冻存液2：5体积％的DMSO、90体积％的MesenPRO RS TMMedium；
冻存液3：5体积％的DMSO、10体积％的复方电解质注射液、75体积％的MesenPRO RS TMMedium；
冻存液4：5体积％的DMSO、10体积％的复方电解质注射液、10体积％羟乙基淀粉200/0.5氯化钠注射液、75体积％的MesenPRO RS TMMedium；
冻存液5：5体积％的DMSO、10体积％的复方电解质注射液、10体积％羟乙基淀粉200/0.5氯化钠注射液、2体积％的白蛋白、73体积％的MesenPRO RS TMMedium；
冻存液6(本发明间充质干细胞专用冻存液)：5体积％的DMSO、10体积％的复方电解质注射液、10体积％羟乙基淀粉200/0.5氯化钠注射液、2体积％的白蛋白、2体积％羟喜树碱注射液、71体积％的MesenPRO RS TMMedium；
按照以下步骤冻存MSC细胞：将冷冻保存液与细胞混匀后，速移入冻存管，并放入冻存盒中，-80℃过夜，次日转入液氮内。其中，每5×10 7个MSC细胞采用1ml冻存液。冷冻保存MSC细胞90d，然后进行复苏。检测冻存前后细胞的存活率，以及复苏后细胞回收率。具体地，冻存前以及冻存并复苏后的细胞存活率计算方法为：【活细胞数/(活细胞数+死细胞数)】×100％。复苏后细胞回收率的计算方法为：(复苏后活细胞数/冻存时活细胞数)×100％。
复苏MSC细胞检查结果：
表4.不同冻存液冻存复苏效果比较
Figure PCTCN2020093716-appb-000007
Figure PCTCN2020093716-appb-000008
如表4所示，复苏后细胞存活率均低于冻存前。冻存液2保存的细胞活率和细胞得率优于冻存液1，表明5％DMSO浓度是最适浓度，增加DMSO浓度将增加DMSO的细胞毒性作用，反而不能提高MSC细胞活率和细胞得率。冻存液3保存的细胞活率和细胞得率优于冻存液2，表明复方电解质注射液有稳定和调节晶体渗透压的作用，可以提高冻存细胞复苏后活率和得率。冻存液4保存的细胞活率和细胞得率优于冻存液3，表明羟乙基淀粉200/0.5氯化钠注射液有稳定和调节胶体渗透压的作用，可以提高冻存细胞复苏后活率和得率。冻存液5保存的细胞活率和细胞得率优于冻存液4，表明白蛋白有稳定和调节胶体渗透压的作用，可以提高冻存细胞复苏后活率和得率。冻存液6保存的细胞活率和细胞得率优于冻存液5，表明羟喜树碱注射液可以提高冻存细胞复苏后活率和得率。冻存液6保存的细胞活率和细胞得率明显优于其余5种冻存液，且复苏后MSC活率达到99％以上，得率97％以上，表明本发明的间充质干细胞专用冻存液适用于MSC冻存。
实施例3
利用本发明实施例的一种间充质干细胞体外筛选、激活、扩增的方法，获得脐带MSC，按ABO/RH分型和HLA分型进行保存，建立可供检索的脐带间充质干细胞信息档案，构建脐带间充质干细胞细胞库。
1供体筛选
医院以询问和填表方式征询供体个人信息、过往治疗史、家族遗传史，以及是否有传染病史及造血或免疫系统的异常情况等信息。医院须与供体签署知情同意书取得供体本人或其授权人员的同意，查阅其体检资料，获得体检信息。供体体检信息应包括如下项目：艾滋病病毒抗体、乙肝表面抗原和抗体、丙肝抗体、巨细胞病毒抗体、梅毒螺旋体抗体、转氨酶等。个人信息采集表、知情同意书、检查信息等需编号密封保存，建立可供检索的脐带供体档案信息库。
2具体制备方法包括以下步骤，以5cm脐带为例操作如下(所有操纵步骤均在洁净工作台完成)：
2.1运输脐带：上述供体筛选合格的，采集脐带5cm，2-8℃恒温保存在疫苗箱中，48h内送至实验室。
2.2分解脐带：取脐带置于5cm培养皿中，洗涤，将其剪成数段，并剔除中间三根血管。位于羊膜与血管之间的白色结缔组织即为华尔通氏胶，用长柄有齿镊将其撕下，放入无菌平皿中。用无菌长柄手术剪，在离心管中将华尔通氏胶剪切成1mm 3左右的组织匀浆块，剪切时间15-20min；加入与组织匀浆块等体积的加入与组织块等体积的1mg/mlⅡ型胶原酶(预热到37℃)，置于恒温振荡培养箱，37℃，200rpm，消化60-90min(每20min取出轻轻摇匀后放回去)，至脐带组织匀浆块呈靡状即可。将酶消化后的组织匀浆按照体积比1：6，加入6倍体积的生理盐水，通过无菌200目滤网过滤除去残留物，400g离心8min，弃上清，获得下层细胞。
2.3原代筛选培养：根据细胞计数，按照1.0×10 6个/ml的密度加入MSC专用原代筛选培养50ml，即添加了8ng/ml SCF、4ng/ml BMP-4、30IU/ml IL-10和4ng/ml LIF、4ng/ml TGF-β、8ng/ml雷帕霉素、12ng/ml曲美替尼、20ng/ml对乙酰氨基酚、3ng/ml 5-HMF、20ng/ml磷酸氯喹的
Figure PCTCN2020093716-appb-000009
MSC Xeno-Free SFM，接种3个T175培养瓶，置于二氧化碳培养箱，培养条件：(37±0.5)℃，二氧化碳体积分数为(5±0.2)％，每隔2-3d换液一次。
2.4传代激活扩增：5-7d左右，原代培养细胞达80％融合时，吸弃旧细胞培养液，消化传代，传代后更换培养体系为MSC专用激活扩增培养基，即添加了8ng/ml SCF、4ng/ml bFGF、20ng/ml芍药苷、30ng/ml盐酸二甲双胍、4ng/ml氢化可的松、4ng/ml CXCL10、2ng/ml Forskolin、3ng/ml 5-HMF、20ng/ml磷酸氯喹的
Figure PCTCN2020093716-appb-000010
MSC Xeno-Free SFM，继续培养。每日观察细胞，根据培养基的颜色，每2-3d换液传代一次。
3建立细胞库：按5体积％的DMSO、5体积％的复方电解质注射液、5体积％羟乙基淀粉200/0.5氯化钠注射液、1体积％的白蛋白、1体积％羟喜树碱注射液、83体积％的
Figure PCTCN2020093716-appb-000011
MSC Xeno-Free SFM配置间充质干细胞专用冻存液。收集上述激活扩增的脐带MSC。将MSC细胞悬液进行检测：支原体、内毒素、微生物和病毒五项。检测合格的MSC，每5×10 7个MSC细胞采用1ml上述配置好的MSC专用冻存液，进行冻存操作，按ABO/RH分型和HLA分型进行保存，建立可供检索的脐带间充质干细胞信息档案，构建脐带间充质干细胞库。
实施例4
利用本发明实施例的一种间充质干细胞体外筛选、激活、扩增的方法，获得胎盘MSC，按ABO/RH分型和HLA分型进行保存，建立可供检索的胎盘间充质干细胞信息档案，构建胎盘间充质干细胞细胞库。
1供体筛选
医院以询问和填表方式征询供体个人信息、过往治疗史、家族遗传史，以及是否有传染病史及造血或免疫系统的异常情况等信息。医院须与供体签署知情同意书取得供体本人或其授权人员的同意，查阅其体检资料，获得体检信息。供体体检信息应包括如下项目：艾滋病病毒抗体、乙肝表面抗原和抗体、丙肝抗体、巨细胞病毒抗体、梅毒螺旋体抗体、转氨酶等。个人信息采集表、知情同意书、检查信息等需编号密封保存，建立可供检索的胎盘供体档案信息库。
2胎盘MSC制备
2.1采集一份供体筛选合格者的完整胎盘，加入适量的胎盘保存液(100mg青霉素、100mg链霉素、2.5mg两性霉素、0.5g肝素钠注射液、2.5g人血白蛋白溶于500ml DMEM培养基中即得)，以浸没过胎盘为宜，2-8℃恒温保存在疫苗箱中，24h内送至实验室。
2.2吸弃胎盘保存液，加入适量体积的含双抗的生理盐水，反复振荡几次，直至胎盘表面的血渍洗涤干净。去除胎盘表面的羊膜和蜕膜，加入适量体积的含双抗的生理盐水，将胎盘组织剪碎，并将组织块内的血液清洗干净。
2.3消化胎盘：收集剪碎的胎盘组织块，加入与胎盘组织块等体积的含0.2％Ⅳ型胶原酶的DMEM(胶原酶Ⅳ终浓度为0.1％)，于37℃水浴摇床中消化20-30min，之后再加入胎盘组织块1/4体积的含0.25％胰蛋白酶的DMEM，于37℃水浴摇床中消化10-20min。将两次消化后胎盘组织块经200目分析筛过滤收集滤液，混匀后于1800rpm室温离心10min，弃上清，用适量的生理盐水重悬细胞沉淀，按照重悬液:HES体积比4:1加入HES，300rpm离心10min，收集上层液体，离心后获得细胞。
2.4原代筛选培养：根据细胞计数，按照1.0×10 6个/ml的密度加入MSC专用原代筛选培养50ml，即添加了2ng/ml SCF、2ng/ml BMP-4、10IU/ml IL-10和1ng/ml LIF、1ng/ml TGF-β、2ng/ml 雷帕霉素、2ng/ml曲美替尼、10ng/ml对乙酰氨基酚、1ng/ml 5-HMF、10ng/ml磷酸氯喹的MesenPRO RS TMMedium，接种3个T175培养瓶，置于二氧化碳培养箱，培养条件：(37±0.5)℃，二氧化碳体积分数为(5±0.2)％，每隔2-3d换液一次。
2.5传代激活扩增：5-7d左右，原代培养细胞达80％融合时，吸弃旧细胞培养液，消化传代，传代后更换培养体系为MSC专用激活扩增培养基，即添加了2ng/ml SCF、1-4ng/ml bFGF、10ng/ml芍药苷、20ng/ml盐酸二甲双胍、1ng/ml氢化可的松、2ng/ml CXCL10、1ng/ml Forskolin、1ng/ml 5-HMF、10ng/ml磷酸氯喹的MesenPRO RS TMMedium，继续培养。每日观察细胞，根据培养基的颜色，每2-3d换液传代一次。
3建立细胞库：按5体积％的DMSO、10体积％的复方电解质注射液、10体积％羟乙基淀粉200/0.5氯化钠注射液、2体积％的白蛋白、2体积％羟喜树碱注射液、71体积％的MesenPRO RS TMMedium配置间充质干细胞专用冻存液。收集上述激活扩增的胎盘MSC。将MSC细胞悬液进行检测：支原体、内毒素、微生物和病毒五项。检测合格的MSC，每5×10 7个MSC细胞采用1ml上述配置好的MSC专用冻存液，进行冻存操作，按ABO/RH分型和HLA分型进行保存，建立可供检索的胎盘间充质干细胞信息档案，构建胎盘间充质干细胞库。

Claims (5)

  1. 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，其特征在于，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。
  2. 根据权利要求1所述的方法，其特征在于，所述间充质干细胞专用原代筛选培养基是添加了2-8ng/ml SCF、2-4ng/ml BMP-4、10-30IU/ml IL-10和1-4ng/ml LIF、1-4ng/ml TGF-β、2-8ng/ml雷帕霉素、2-12ng/ml曲美替尼、10-20ng/ml对乙酰氨基酚、1-3ng/ml 5-HMF、10-20ng/ml磷酸氯喹的间充质干细胞无血清完全培养基。
  3. 根据权利要求1所述的方法，其特征在于，所述间充质干细胞专用激活扩增培养基是添加了2-8ng/ml SCF、1-4ng/ml bFGF、10-20ng/ml芍药苷、20-30ng/ml盐酸二甲双胍、1-4ng/ml氢化可的松、2-4ng/ml CXCL10、1-2ng/ml Forskolin、1-3ng/ml 5-HMF、10-20ng/ml磷酸氯喹的间充质干细胞无血清完全培养基。
  4. 根据权利要求1所述的方法，其特征在于，所述间充质干细胞专用冻存液是包含5-10体积％的DMSO、5-10体积％的复方电解质注射液、5-10体积％羟乙基淀粉200/0.5氯化钠注射液、1-2体积％的白蛋白、1-2体积％羟喜树碱注射液、66-83体积％的间充质干细胞无血清完全培养基。
  5. 根据权利要求2、3、4所述的方法，其特征在于，所述间充质干细胞专用原代筛选培养基、间充质干细胞专用激活扩增培养基、间充质干细胞专用冻存液中，所述的无血清完全培养基为TheraPEAK TMMSCGM-CD TMMedium、MesenPRO RS TMMedium、
    Figure PCTCN2020093716-appb-100001
    MSC Xeno-Free SFM或市售其他类型无血清培养基。
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