CN110540959A - Umbilical cord mesenchymal stem cell isolation culture amplification method - Google Patents
Umbilical cord mesenchymal stem cell isolation culture amplification method Download PDFInfo
- Publication number
- CN110540959A CN110540959A CN201910947859.6A CN201910947859A CN110540959A CN 110540959 A CN110540959 A CN 110540959A CN 201910947859 A CN201910947859 A CN 201910947859A CN 110540959 A CN110540959 A CN 110540959A
- Authority
- CN
- China
- Prior art keywords
- umbilical cord
- mesenchymal stem
- stem cells
- culture
- cord mesenchymal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000003954 umbilical cord Anatomy 0.000 title claims abstract description 157
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 96
- 238000000034 method Methods 0.000 title claims abstract description 46
- 230000003321 amplification Effects 0.000 title claims abstract description 25
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 25
- 238000002955 isolation Methods 0.000 title claims description 6
- 238000004140 cleaning Methods 0.000 claims abstract description 25
- 238000012258 culturing Methods 0.000 claims abstract description 15
- 238000000926 separation method Methods 0.000 claims abstract description 15
- 210000004027 cell Anatomy 0.000 claims description 63
- 210000001519 tissue Anatomy 0.000 claims description 51
- 239000000243 solution Substances 0.000 claims description 46
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 34
- 239000012894 fetal calf serum Substances 0.000 claims description 34
- 238000004113 cell culture Methods 0.000 claims description 17
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 13
- 238000001514 detection method Methods 0.000 claims description 13
- 229960001484 edetic acid Drugs 0.000 claims description 13
- 230000004927 fusion Effects 0.000 claims description 13
- 238000010008 shearing Methods 0.000 claims description 12
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 108010019160 Pancreatin Proteins 0.000 claims description 10
- 229940055695 pancreatin Drugs 0.000 claims description 10
- 238000013441 quality evaluation Methods 0.000 claims description 10
- 230000000694 effects Effects 0.000 claims description 9
- 210000001691 amnion Anatomy 0.000 claims description 8
- 230000004069 differentiation Effects 0.000 claims description 8
- 238000011156 evaluation Methods 0.000 claims description 8
- 210000003606 umbilical vein Anatomy 0.000 claims description 8
- 210000004369 blood Anatomy 0.000 claims description 7
- 239000008280 blood Substances 0.000 claims description 7
- 239000012535 impurity Substances 0.000 claims description 7
- 239000008055 phosphate buffer solution Substances 0.000 claims description 7
- 229930182555 Penicillin Natural products 0.000 claims description 6
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 6
- 210000004204 blood vessel Anatomy 0.000 claims description 6
- 239000002777 nucleoside Substances 0.000 claims description 6
- 150000003833 nucleoside derivatives Chemical class 0.000 claims description 6
- 229940049954 penicillin Drugs 0.000 claims description 6
- 229960005322 streptomycin Drugs 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 241000204031 Mycoplasma Species 0.000 claims description 5
- 230000036512 infertility Effects 0.000 claims description 5
- 238000003908 quality control method Methods 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 230000029087 digestion Effects 0.000 description 17
- 210000000130 stem cell Anatomy 0.000 description 12
- 238000011081 inoculation Methods 0.000 description 9
- 230000008569 process Effects 0.000 description 9
- 235000015110 jellies Nutrition 0.000 description 8
- 239000008274 jelly Substances 0.000 description 8
- 230000009286 beneficial effect Effects 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 230000035755 proliferation Effects 0.000 description 5
- 230000008827 biological function Effects 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 230000007774 longterm Effects 0.000 description 4
- 102100022464 5'-nucleotidase Human genes 0.000 description 3
- 102100037241 Endoglin Human genes 0.000 description 3
- 101000678236 Homo sapiens 5'-nucleotidase Proteins 0.000 description 3
- 101000881679 Homo sapiens Endoglin Proteins 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 2
- 102000006354 HLA-DR Antigens Human genes 0.000 description 2
- 108010058597 HLA-DR Antigens Proteins 0.000 description 2
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 2
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 2
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 2
- 101001046686 Homo sapiens Integrin alpha-M Proteins 0.000 description 2
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 2
- 102100022338 Integrin alpha-M Human genes 0.000 description 2
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 2
- 230000009815 adipogenic differentiation Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000009816 chondrogenic differentiation Effects 0.000 description 2
- 230000009194 climbing Effects 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000009818 osteogenic differentiation Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000001644 umbilical artery Anatomy 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000004115 adherent culture Methods 0.000 description 1
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical group C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 238000010322 bone marrow transplantation Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 102000038379 digestive enzymes Human genes 0.000 description 1
- 108091007734 digestive enzymes Proteins 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 210000001654 germ layer Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000032696 parturition Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 210000001428 peripheral nervous system Anatomy 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 210000001525 retina Anatomy 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 230000010415 tropism Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000002689 xenotransplantation Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/40—Nucleotides, nucleosides or bases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Developmental Biology & Embryology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Rheumatology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a separation, culture and amplification method of umbilical cord mesenchymal stem cells, which comprises the steps of 1) cleaning, 2) treating, 3) culturing and 4) inoculating and passaging.
Description
Technical Field
the invention relates to the technical field of stem cell isolated culture, in particular to an umbilical cord mesenchymal stem cell isolated culture amplification method.
background
Mesenchymal Stem Cells (MSCs) are a population of cells with the potential for self-renewal, high proliferation and multipotentiality, and are the cells of origin for animal organisms and various tissue organs. Recent studies have shown that mesenchymal stem cells are present in numerous adult tissues (e.g., bone marrow, muscle, gastrointestinal tract, skin, retina, peripheral and central nervous system, etc.). The MSCs have wide sources, are easy to separate and amplify in vitro, and have multidirectional differentiation potential to three germ layer cells; has low immunogenicity, no ability to stimulate allogeneic immune response, no killing by cytotoxic T cells and NK cells, and has repairing, tumor tropism and immunoregulation effects.
studies have shown that umbilical cord mesenchymal cells are mainly derived from umbilical cord Wharton' S jelly. The UC-MSCs has the advantages that: firstly, the umbilical cord is the waste after parturition, has convenient material taking, wide source and easy collection, preservation and refrigeration, and is not subject to the debate and limitation in ethics, morality and law; ② the medicine is relatively pure, and the infection rate of virus and pathogenic microorganism related to stem cell infusion is far lower than that of bone marrow transplantation; human UC-MSCs have rich, relatively original and strong differentiation capability, can be separated and cultured in vitro, has rapid amplification and stable biological performance, can still keep vigorous functions after multiple passage amplification, and can provide sufficient cell sources for experiments and clinics; the human UC-MSCs do not express or express histocompatibility markers in a low way, have low immunogenicity, do not have immunological rejection reaction or have weak reaction in xenotransplantation, can be used without strict matching, and is suitable for transplantation among different individuals; UC-MSCs can be directly obtained from umbilical cord of newborn, and stored for a long time without wound to organism. Therefore, UC-MSCs have been considered as a better source of MSCs in clinical and scientific work.
Human umbilical cord tissue is rich in Wharton's jelly, and how to efficiently separate umbilical cord mesenchymal stem cells from Wharton's jelly is a difficulty of main research at present and a key technology for restricting later clinical application and industrialization. The most common separation method for primary separation preparation of umbilical cord mesenchymal stem cells mainly comprises an enzyme digestion method and a tissue block adherent culture method.
The economic cost of enzyme digestion separation is high, the digestion time is long, generally 4-6 hours are needed, the longest digestion time is 16 hours, the size of a tissue block can influence the digestion effect of cells, if the digestion is insufficient, a sufficient number of cells cannot be obtained, and the digestion time is too long, the damage of digestive enzymes to the cells is too large, and a large number of cells die. Since the umbilical cord is mainly rich in Wharton's jelly, the digested liquid is very viscous and cells are not easily separated after centrifugation. The primary culture time of the conventional tissue block culture method is long, and the separation efficiency is influenced by the size and adherence of the tissue block which needs 14-20 days.
disclosure of Invention
The invention provides a separation, culture and amplification method of umbilical cord mesenchymal stem cells.
the scheme of the invention is as follows:
An umbilical cord mesenchymal stem cell isolation culture amplification method comprises the following steps:
1) Cleaning, namely acquiring a fresh umbilical cord under an aseptic condition, washing the surface of the umbilical cord by using a cleaning solution, and cleaning to remove residual blood and other impurities in blood vessels on the umbilical cord;
2) Treating, namely removing the umbilical vein and the amniotic membrane layer on the surface of the umbilical cord from the cleaned umbilical cord by using a scalpel, shearing the umbilical cord into thin strips, and shearing the thin strips into umbilical cord tissue blocks with the thickness of 1-3mm 3; the operation method is simple, the time is saved, only the umbilical cord is left as Wharton's jelly which is a main source of the umbilical cord mesenchymal stem cells;
3) Culturing, namely flatly paving the umbilical cord tissue block in a cell culture dish, volatilizing water, and adding the water into a culture solution containing 10-20% fetal calf serum for culturing; the operation is characterized in that the tissue block is small, the tissue is firmly attached to the wall, thereby being beneficial to the climbing of umbilical cord mesenchymal stem cells from the tissue block and also being capable of obtaining more primary cells;
4) Inoculating and subculturing, wherein after the umbilical cord tissue block primary isolated culture umbilical cord mesenchymal stem cells grow to 80-90% fusion, passage amplification culture can be carried out, umbilical cord mesenchymal stem cells are obtained, 0.0625% of pancreatin/EDTA (ethylene diamine tetraacetic acid)/digested cells are used for inoculating according to 3000-5000/cm 2, 95% fusion can be achieved 4-5 days after the umbilical cord mesenchymal stem cells are inoculated, and more than 95% of fused umbilical cord mesenchymal stem cells can be amplified to more than 25 generations. The digestion mode is low in pancreatin concentration, the damage to cells in the digestion process is small, the inoculation density can well control the growth cycle of the cells, and the umbilical cord mesenchymal stem cells can be amplified for more than 25 generations according to the digestion of the cells after passage of the inoculation density.
As a preferable technical scheme, the cleaning solution in the step 1) contains phosphate buffer solution of penicillin and streptomycin.
As a preferable technical scheme, the culture solution of 10-20% fetal calf serum in the step 3) contains the alpha-MEM basic culture solution containing nucleoside and the fetal calf serum, which is more beneficial to the separation and proliferation of stem cells, and can better maintain the dryness and biological functions of the stem cells in the long-term amplification culture process.
As a preferred technical scheme, the method further comprises a step 5) of performing quality evaluation on the mesenchymal stem cells obtained in the step 4), wherein the quality evaluation comprises cell number, cell activity, cell surface markers, differentiation function, sterility detection, mycoplasma detection and safety evaluation.
as a preferred technical scheme, in the step 3), the culture solution of 10-20% fetal calf serum is changed once every 3 days after the cells are cultured in the cell culture dish, then the culture solution of 10-20% fetal calf serum is changed every 2-3 days after the cells are attached to the wall, the umbilical cord mesenchymal stem cells can climb out of the umbilical cord tissue block after 4-5 days, and the umbilical cord mesenchymal stem cells can be fused by 80-90% after 7-10 days.
Preferably, the culture solution containing 10-20% fetal calf serum is added in the step 3) in a volume of 20-50 ml/dish.
the invention also provides a method for establishing the production specification, the quality control system and the evaluation system of the umbilical cord mesenchymal stem cells by adopting the umbilical cord mesenchymal stem cell separation culture amplification method as claimed in claim 1.
due to the adoption of the technical scheme, the umbilical cord mesenchymal stem cell separation culture amplification method comprises the steps of 1) cleaning, obtaining a fresh umbilical cord under an aseptic condition, washing the surface of the umbilical cord by using a cleaning solution, and cleaning to remove residual blood and other impurities on the umbilical cord; 2) treating, namely removing the umbilical vein and the amniotic membrane layer on the surface of the umbilical cord from the cleaned umbilical cord by using a scalpel, shearing the umbilical cord into thin strips, and shearing the thin strips into umbilical cord tissue blocks with the thickness of 1-3mm 3; 3) culturing, namely flatly paving the umbilical cord tissue block in a cell culture dish, volatilizing water, and adding the water into a culture solution containing 10-20% fetal calf serum for culturing; 4) inoculating and subculturing, wherein after the umbilical cord tissue block primary isolated culture umbilical cord mesenchymal stem cells grow to 80-90% fusion, passage amplification culture can be carried out, umbilical cord mesenchymal stem cells are obtained, 0.0625% of pancreatin/EDTA (ethylene diamine tetraacetic acid)/digested cells are used for inoculating according to 3000-5000/cm 2, 95% fusion can be achieved 4-5 days after the umbilical cord mesenchymal stem cells are inoculated, and more than 95% of fused umbilical cord mesenchymal stem cells can be amplified to more than 25 generations.
The invention has the advantages that:
The mesenchymal stem cells obtained by the tissue mass separation method have the advantages of simple method, low cost, good cell activity, high purity, high concentration of finally-harvested cells, simple method for obtaining the mesenchymal stem cells, capability of quickly and efficiently separating MSC (mesenchymal stem cell), short culture time, high concentration of finally-harvested cells, standardization, programming and normalization of the whole culture process of the umbilical cord mesenchymal stem cells, and guarantee of the cell quality completely meeting national promulgated stem cell preparation quality control and preclinical research guide principles (trial implementation), thereby providing clinical application with guarantee.
The method removes umbilical artery, vein and surface amniotic membrane, controls the size of tissue block to be 1-3mm3, adopts a cell culture dish to spread flat, and replenishes liquid after water is completely volatilized, thereby obviously improving the yield of primary cells.
drawings
FIG. 1 is a cut-to-thin strip view of the umbilical cord of example 3;
FIG. 2 is a diagram of umbilical cord tissue block adherence of example 3;
FIG. 3 is a climbing out of the tissue mass of umbilical cord mesenchymal stem cells of example 3;
FIG. 4 is a drawing of the flow-through detection of CD90 by umbilical cord mesenchymal stem cells P10 of example 3;
FIG. 5 is a drawing of the umbilical cord mesenchymal stem cell P10 generation flow detection CD105 of example 3;
FIG. 6 is a drawing of the flow assay CD73 of umbilical cord mesenchymal stem cells P10 of example 3;
FIG. 7 is a drawing of passage flow assay Negative of umbilical cord mesenchymal stem cells P10 of example 3;
FIG. 8 is a graph showing the identification of the multipotentiality of umbilical cord mesenchymal stem cells of example 4;
Figure 9 is a graph of umbilical cord mesenchymal stem cell growth of example 5.
Detailed Description
in order to make up for the above deficiencies, the present invention provides a method for separating, culturing and amplifying umbilical cord mesenchymal stem cells to solve the above problems in the background art.
an umbilical cord mesenchymal stem cell isolation culture amplification method comprises the following steps:
1) Cleaning, namely acquiring a fresh umbilical cord under an aseptic condition, washing the surface of the umbilical cord by using a cleaning solution, and cleaning to remove residual blood and other impurities in blood vessels on the umbilical cord;
2) Treating, namely removing the umbilical vein and the amniotic membrane layer on the surface of the umbilical cord from the cleaned umbilical cord by using a scalpel, shearing the umbilical cord into thin strips, and shearing the thin strips into umbilical cord tissue blocks with the thickness of 1-3mm 3; the operation method is simple, the time is saved, only the umbilical cord is left as Wharton's jelly which is a main source of the umbilical cord mesenchymal stem cells;
3) Culturing, namely flatly paving the umbilical cord tissue block in a cell culture dish, volatilizing water, and adding the water into a culture solution containing 10-20% fetal calf serum for culturing; the operation is characterized in that the tissue block is small, the tissue adheres to the wall firmly, cells can climb out of the tissue block, and more primary cells can be obtained;
4) Inoculating and subculturing, wherein he umbilical cord tissue block primary isolated culture umbilical cord mesenchymal stem cells grow to 80-90% fusion, passage amplification culture can be carried out, umbilical cord mesenchymal stem cells are obtained, 0.0625% of pancreatin/EDTA (ethylene diamine tetraacetic acid)/digested cells are used for inoculating according to 3000-5000/cm 2, 95% fusion can be achieved 4-5 days after the umbilical cord mesenchymal stem cells are inoculated, and more than 95% of fused umbilical cord mesenchymal stem cells can be amplified to more than 25 generations. The pancreatin concentration in the digestion mode has small damage to cells in the digestion process, the inoculation density can well control the growth cycle of the cells, and the umbilical cord mesenchymal stem cells can be amplified for more than 25 generations according to the digestion of the cells after passage of the inoculation density.
The cleaning solution in the step 1) contains phosphate buffer solution of penicillin and streptomycin.
The culture solution of 10-20% fetal calf serum in the step 3) contains alpha-MEM basic culture solution containing nucleoside and fetal calf serum, which is more beneficial to separation and proliferation of stem cells, and can better maintain the dryness and biological functions of the stem cells in the long-term amplification culture process.
further comprising the step 5) of performing quality evaluation on the mesenchymal stem cells obtained in the step 4), wherein the quality evaluation comprises cell number, cell activity, cell surface markers, differentiation function, sterility detection, mycoplasma detection and safety evaluation.
And 3) replacing the culture solution of 10-20% fetal calf serum once every 3 days of cell culture in the cell culture dish in the step 3), replacing the culture solution of 10-20% fetal calf serum every 2-3 days, allowing umbilical cord mesenchymal stem cells to climb out of the umbilical cord tissue block after attaching to the wall for 4-5 days, and allowing the umbilical cord mesenchymal stem cells to be fused by 80-90% after 7-10 days.
And 3) adding 20-50 ml/dish of culture solution containing 10-20% fetal calf serum.
The invention also provides a method for establishing the production specification, the quality control system and the evaluation system of the umbilical cord mesenchymal stem cells by adopting the umbilical cord mesenchymal stem cell separation culture amplification method as claimed in claim 1.
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
Example 1:
1) Cleaning, namely acquiring a fresh umbilical cord under an aseptic condition, washing the surface of the umbilical cord by using a cleaning solution, and cleaning to remove residual blood and other impurities in blood vessels on the umbilical cord;
2) Processing, namely removing the umbilical vein and the amniotic membrane layer on the surface of the umbilical cord from the cleaned umbilical cord by using a scalpel, shearing the umbilical cord into thin strips, and shearing the thin strips into umbilical cord tissue blocks with the diameter of 2mm 3; the operation method is simple, the time is saved, only the umbilical cord is left as Wharton's jelly which is a main source of the umbilical cord mesenchymal stem cells;
3) Culturing, namely flatly paving the umbilical cord tissue block in a cell culture dish, volatilizing water, and adding the umbilical cord tissue block into a culture solution containing 10% fetal calf serum for culturing; the operation is characterized in that the tissue block is small, the tissue adheres to the wall firmly, cells can climb out of the tissue block, and more primary cells can be obtained;
4) inoculating and subculturing, wherein after the umbilical cord tissue block primary isolated culture umbilical cord mesenchymal stem cells grow to 80% fusion, passage amplification culture can be carried out, umbilical cord mesenchymal stem cells are obtained, 0.0625% of pancreatin/EDTA (ethylene diamine tetraacetic acid) digestive cells are used for inoculating according to 3000-cm 2, 95% fusion can be achieved 4-5 days after the umbilical cord mesenchymal stem cells are inoculated, and more than 95% of fused umbilical cord mesenchymal stem cells can be amplified to more than 25 generations. The pancreatin concentration in the digestion mode has small damage to cells in the digestion process, the inoculation density can well control the growth cycle of the cells, and the umbilical cord mesenchymal stem cells can be amplified for more than 25 generations according to the digestion of the cells after passage of the inoculation density.
The cleaning solution in the step 1) contains phosphate buffer solution of penicillin and streptomycin.
The culture solution of 10-20% fetal calf serum in the step 3) contains alpha-MEM basic culture solution containing nucleoside and fetal calf serum, which is more beneficial to separation and proliferation of stem cells, and can better maintain the dryness and biological functions of the stem cells in the long-term amplification culture process.
Further comprising the step 5) of performing quality evaluation on the mesenchymal stem cells obtained in the step 4), wherein the quality evaluation comprises cell number, cell activity, cell surface markers, differentiation function, sterility detection, mycoplasma detection and safety evaluation.
And 3) replacing the culture solution of 10% fetal calf serum once every 3 days after cell culture in the cell culture dish in the step 3), replacing the culture solution of 10% fetal calf serum every 2-3 days after wall adhesion, showing that the umbilical cord mesenchymal stem cells climb out of the umbilical cord tissue block after 4-5 days of wall adhesion, and enabling the umbilical cord mesenchymal stem cells to be 80-90% fused after 7-10 days.
In the step 3), 20ml of culture solution containing 10% fetal calf serum is added in each dish.
Example 2:
An umbilical cord mesenchymal stem cell isolation culture amplification method comprises the following steps:
1) Cleaning, namely acquiring a fresh umbilical cord under an aseptic condition, washing the surface of the umbilical cord by using a cleaning solution, and cleaning to remove residual blood and other impurities in blood vessels on the umbilical cord;
2) processing, namely removing the umbilical vein and the amniotic membrane layer on the surface of the umbilical cord from the cleaned umbilical cord by using a scalpel, shearing the umbilical cord into thin strips, and shearing the thin strips into umbilical cord tissue blocks with the thickness of 3mm 3; the operation method is simple, the time is saved, only the umbilical cord is left as Wharton's jelly which is a main source of the umbilical cord mesenchymal stem cells;
3) culturing, namely flatly paving the umbilical cord tissue block in a cell culture dish, volatilizing water, and adding the umbilical cord tissue block into a culture solution containing 20% fetal calf serum for culturing; the operation is characterized in that the tissue block is small, the tissue adheres to the wall firmly, cells can climb out of the tissue block, and more primary cells can be obtained;
4) Inoculating and subculturing, wherein after the umbilical cord tissue block primary isolated culture umbilical cord mesenchymal stem cells grow to 90% fusion, passage amplification culture can be carried out, umbilical cord mesenchymal stem cells are obtained, 0.0625% of pancreatin/EDTA (ethylene diamine tetraacetic acid) are used for digesting the cells, and the cells are inoculated according to 5000/cm 2, the umbilical cord mesenchymal stem cells can reach 95% fusion after 4-5 days after inoculation, and the umbilical cord mesenchymal stem cells which are fused for more than 95% can be amplified to more than 25 generations. The pancreatin concentration in the digestion mode has small damage to cells in the digestion process, the inoculation density can well control the growth cycle of the cells, and the umbilical cord mesenchymal stem cells can be amplified for more than 25 generations according to the digestion of the cells after passage of the inoculation density.
the cleaning solution in the step 1) contains phosphate buffer solution of penicillin and streptomycin.
the culture solution of the 20% fetal calf serum in the step 3) contains the alpha-MEM basic culture solution containing nucleoside and the fetal calf serum, which is more beneficial to the separation and proliferation of stem cells, and can better maintain the dryness and biological functions of the stem cells in the long-term amplification culture process.
Further comprising the step 5) of performing quality evaluation on the mesenchymal stem cells obtained in the step 4), wherein the quality evaluation comprises cell number, cell activity, cell surface markers, differentiation function, sterility detection, mycoplasma detection and safety evaluation.
And 3) changing the culture solution of 20% fetal calf serum once every 3 days after cell culture in the cell culture dish in the step 3), then changing the culture solution of 20% fetal calf serum every 2-3 days after wall adhesion, showing that the umbilical cord mesenchymal stem cells climb out from the umbilical cord tissue block after 4-5 days of wall adhesion, and enabling the umbilical cord mesenchymal stem cells to reach 80-90% fusion after 7-10 days.
In the step 3), a culture solution containing 20% fetal calf serum is added in a volume of 50 ml/dish.
example 3:
After screening infectious diseases and the like, signing an informed consent and a donation agreement, aseptically collecting umbilical cords after delivery, flushing the surface with Phosphate Buffer Solution (PBS) containing penicillin and streptomycin under an aseptic condition, and cleaning to remove blood and other impurities remained in blood vessels.
Removing umbilical artery, removing umbilical vein and amniotic membrane layer on umbilical cord surface with surgical blade, cutting umbilical cord into thin strips (see figure 1), and cutting into tissue pieces with size of 1-3mm 3.
the tissue is spread in a cell culture dish and after the water is evaporated, complete culture solution containing 10-20% fetal calf serum is added.
Changing the liquid every 2-3 days after culturing the cells, wherein the cells can be seen to climb out of the tissue block after adhering the walls (shown in figure 2) for 4-5 days, and the umbilical cord mesenchymal stem cells can be fused by 80-90% after 7-10 days;
digesting the cells by 0.0625% of trypsin/EDTA, inoculating the cells by 3000-.
The culture solution of 20% fetal calf serum contains alpha-MEM basic culture solution containing nucleoside and fetal calf serum.
the umbilical cord simple mesenchymal stem cells obtained in the example 3 are subjected to umbilical cord mesenchymal stem cell dryness maintenance, and the stem cell surface antigens after the three culture solutions are cultured are subjected to surface marker logistics analysis, so that the results show that the obtained MSCs have good homogeneity. All express CD73, CD105, CD 90; does not express CD45, CD34, CD11b, CD19 and HLA-DR. The result is that the umbilical cord mesenchymal stem cells P10 flow-batch detection shows that the expression of CD73, CD90 and CD105 is positive, and the expression of Negative-CD 45, CD34, CD11b, CD19 and HLA-DR is Negative, and the result is shown in figure 4-figure 7.
Example 4:
performing multi-directional differentiation potential identification on the umbilical cord mesenchymal stem cells of example 3, wherein fig. 8A is an osteogenic differentiation control, and fig. 8B is alizarin red staining after osteogenic differentiation; FIG. 8C is a adipogenic differentiation control, and FIG. 8D is oil red "O" staining after adipogenic differentiation; fig. 8E is chondrogenic differentiation control and fig. 8F is axilliper blue staining after chondrogenic differentiation.
Example 5:
by adopting the technology, the umbilical cord mesenchymal stem cells in the embodiment 3 are expanded, wherein 140^106 cells can be separated from P1 generation, and 100 ten thousand times of expansion can be obtained from P5 generation.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (7)
1. An umbilical cord mesenchymal stem cell isolation culture amplification method is characterized by comprising the following steps:
1) cleaning, namely acquiring a fresh umbilical cord under an aseptic condition, washing the surface of the umbilical cord by using a cleaning solution, and cleaning to remove residual blood and other impurities in blood vessels on the umbilical cord;
2) treating, namely removing the umbilical vein and the amniotic membrane layer on the surface of the umbilical cord from the cleaned umbilical cord by using a scalpel, shearing the umbilical cord into thin strips, and shearing the thin strips into umbilical cord tissue blocks with the thickness of 1-3mm 3;
3) Culturing, namely flatly paving the umbilical cord tissue block in a cell culture dish, volatilizing water, and adding the water into a culture solution containing 10-20% fetal calf serum for culturing;
4) inoculating and subculturing, wherein after the umbilical cord tissue block primary isolated culture umbilical cord mesenchymal stem cells grow to 80-90% fusion, passage amplification culture can be carried out, umbilical cord mesenchymal stem cells are obtained, 0.0625% of pancreatin/EDTA (ethylene diamine tetraacetic acid)/digested cells are used for inoculating according to 3000-5000/cm 2, 95% fusion can be achieved 4-5 days after the umbilical cord mesenchymal stem cells are inoculated, and more than 95% of fused umbilical cord mesenchymal stem cells can be amplified to more than 25 generations.
2. the isolated culture and expansion method of umbilical cord mesenchymal stem cells according to claim 1, wherein the method comprises the following steps: the cleaning solution in the step 1) contains phosphate buffer solution of penicillin and streptomycin.
3. The isolated culture and expansion method of umbilical cord mesenchymal stem cells according to claim 1, wherein the method comprises the following steps: the culture solution of 10-20% fetal calf serum in the step 3) contains alpha-MEM basic culture solution containing nucleoside and fetal calf serum.
4. The isolated culture and expansion method of umbilical cord mesenchymal stem cells according to claim 1, wherein the method comprises the following steps: further comprising the step 5) of performing quality evaluation on the mesenchymal stem cells obtained in the step 4), wherein the quality evaluation comprises cell number, cell activity, cell surface markers, differentiation function, sterility detection, mycoplasma detection and safety evaluation.
5. the isolated culture and expansion method of umbilical cord mesenchymal stem cells according to claim 1, wherein the method comprises the following steps: and 3) replacing the culture solution of 10-20% fetal calf serum once every 3 days of cell culture in the cell culture dish in the step 3), replacing the culture solution of 10-20% fetal calf serum every 2-3 days, allowing umbilical cord mesenchymal stem cells to climb out of the umbilical cord tissue block after attaching to the wall for 4-5 days, and allowing the umbilical cord mesenchymal stem cells to be fused by 80-90% after 7-10 days.
6. The isolated culture and expansion method of umbilical cord mesenchymal stem cells according to claim 1, wherein the method comprises the following steps: and 3) adding 20-50 ml/dish of culture solution containing 10-20% fetal calf serum.
7. a method for establishing the production specification, quality control system and evaluation system of umbilical cord mesenchymal stem cells is characterized in that: the umbilical cord mesenchymal stem cell separation, culture and amplification method of claim 1 is adopted.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910947859.6A CN110540959A (en) | 2019-10-08 | 2019-10-08 | Umbilical cord mesenchymal stem cell isolation culture amplification method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910947859.6A CN110540959A (en) | 2019-10-08 | 2019-10-08 | Umbilical cord mesenchymal stem cell isolation culture amplification method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110540959A true CN110540959A (en) | 2019-12-06 |
Family
ID=68715238
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910947859.6A Pending CN110540959A (en) | 2019-10-08 | 2019-10-08 | Umbilical cord mesenchymal stem cell isolation culture amplification method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110540959A (en) |
Cited By (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111467376A (en) * | 2020-05-23 | 2020-07-31 | 湖南源品细胞生物科技有限公司 | Application of MSC (Mobile switching center) in relieving influence of cytokine storm |
| CN111529551A (en) * | 2020-05-23 | 2020-08-14 | 湖南源品细胞生物科技有限公司 | Application of MSC (mesenchymal stem cell) in adjusting number of plasma cells |
| CN111544452A (en) * | 2020-05-23 | 2020-08-18 | 湖南源品细胞生物科技有限公司 | Application of MSC (mesenchymal stem cell) in regulating number of CD4+ T cells |
| CN111557951A (en) * | 2020-05-23 | 2020-08-21 | 湖南源品细胞生物科技有限公司 | Novel method for treating virus infection patient by mesenchymal stem cells |
| CN111557950A (en) * | 2020-05-23 | 2020-08-21 | 湖南源品细胞生物科技有限公司 | Application of MSC (mesenchymal stem cell) in regulating and controlling NK (natural killer) cell gene expression |
| CN111568929A (en) * | 2020-05-23 | 2020-08-25 | 湖南源品细胞生物科技有限公司 | Application of MSC (mesenchymal stem cell) in regulating number of effector CD8+ T cells |
| CN111568930A (en) * | 2020-05-23 | 2020-08-25 | 湖南源品细胞生物科技有限公司 | Application of MSC (mesenchymal stem cell) in adjusting number of NK (natural killer) cells |
| CN111568927A (en) * | 2020-05-23 | 2020-08-25 | 湖南源品细胞生物科技有限公司 | Application of MSC (mesenchymal stem cell) in regulating number of memory B cells |
| CN111568931A (en) * | 2020-05-23 | 2020-08-25 | 湖南源品细胞生物科技有限公司 | Application of MSC (mesenchymal stem cell) in regulating and controlling human body effect CD4+ T memory cell gene expression |
| CN111568928A (en) * | 2020-05-23 | 2020-08-25 | 湖南源品细胞生物科技有限公司 | Application of MSC (mesenchymal stem cell) in regulating and controlling gene expression of effector CD8+ T cells |
| CN111979188A (en) * | 2020-08-25 | 2020-11-24 | 江苏赛亿细胞技术研究院有限公司 | Placenta mesenchymal stem cell isolation culture amplification method |
| CN111979189A (en) * | 2020-08-25 | 2020-11-24 | 江苏赛亿细胞技术研究院有限公司 | Isolated culture and amplification method of amniotic mesenchymal stem cells |
| CN112011505A (en) * | 2020-09-09 | 2020-12-01 | 广州同康生物科技有限公司 | Umbilical cord mesenchymal stem cell separation method |
| CN112029713A (en) * | 2020-08-25 | 2020-12-04 | 江苏赛亿细胞技术研究院有限公司 | Chorionic mesenchymal stem cell isolation culture amplification method |
| CN112043725A (en) * | 2020-09-17 | 2020-12-08 | 湖南源品细胞生物科技有限公司 | Application of mesenchymal stem cells in improvement of human body oxidative stress state |
| CN112126622A (en) * | 2020-08-27 | 2020-12-25 | 陕西佰傲干细胞再生医学有限公司 | Umbilical cord mesenchymal stem cell primary isolation culture method capable of improving yield |
| CN112159795A (en) * | 2020-10-13 | 2021-01-01 | 肖利霞 | Passage amplification method for umbilical cord mesenchymal stem cell working cells in public bank |
| CN112852726A (en) * | 2021-02-24 | 2021-05-28 | 河南省银丰生物工程技术有限公司 | Method for separating and amplifying mesenchymal stem cells |
| CN113061638A (en) * | 2021-03-06 | 2021-07-02 | 河北北科生物科技有限公司 | Stem cell quality evaluation system and application method |
| CN113201490A (en) * | 2021-05-26 | 2021-08-03 | 新乡医学院 | Method for sequentially culturing human umbilical cord mesenchymal stem cells by triple culture medium |
| CN113604428A (en) * | 2021-08-19 | 2021-11-05 | 上海纳米技术及应用国家工程研究中心有限公司 | Primary isolation method of human umbilical cord-derived mesenchymal stem cells |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101591644A (en) * | 2009-04-13 | 2009-12-02 | 中国人民解放军第三○二医院 | The preparation of umbilical cord-mesenchymal stem cells (UC-MSCs) used in clinical therapy and storage |
| CN101914496A (en) * | 2010-08-06 | 2010-12-15 | 青岛奥克生物开发有限公司 | Simplified method for preparing umbilical mesenchymal stem cells in large scale |
| CN101974484A (en) * | 2010-11-03 | 2011-02-16 | 江苏省北科生物科技有限公司 | Method for preparing human umbilical cord mesenchymal stem cells |
| CN102154200A (en) * | 2010-12-13 | 2011-08-17 | 中国人民解放军第三○二医院 | Preparation and storage of mesenchymal stem cells for clinical treatment |
| CN102191229A (en) * | 2010-03-16 | 2011-09-21 | 侯亚义 | Method for rapidly and effectively acquiring umbilical cord mesenchymal stem cell (MSC) |
| CN104630142A (en) * | 2015-02-06 | 2015-05-20 | 广州赛莱拉干细胞科技股份有限公司 | Separation and culture method of bovine umbilical cord mesenchymal stem cells |
| CN104762257A (en) * | 2015-04-13 | 2015-07-08 | 云南和泽西南生物科技有限公司 | Method for preparing mesenchymal stem cell from umbilical cord |
| CN106434547A (en) * | 2016-12-17 | 2017-02-22 | 重庆市干细胞技术有限公司 | Method for preparing umbilical cord mesenchymal stem cells |
| CN106497873A (en) * | 2016-12-08 | 2017-03-15 | 北京银丰鼎诚生物工程技术有限公司 | One kind prepares human umbilical cord mesenchymal stem cells factor secretion inducing culture medium on a large scale |
| CN110257327A (en) * | 2019-06-27 | 2019-09-20 | 重庆市细胞生物工程技术有限公司 | A kind of isolated culture method of umbilical cord mesenchymal stem cells |
| CN110305839A (en) * | 2019-08-02 | 2019-10-08 | 陕西佰傲干细胞再生医学有限公司 | Mesenchymal stem cell serum-free culture medium |
-
2019
- 2019-10-08 CN CN201910947859.6A patent/CN110540959A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101591644A (en) * | 2009-04-13 | 2009-12-02 | 中国人民解放军第三○二医院 | The preparation of umbilical cord-mesenchymal stem cells (UC-MSCs) used in clinical therapy and storage |
| CN102191229A (en) * | 2010-03-16 | 2011-09-21 | 侯亚义 | Method for rapidly and effectively acquiring umbilical cord mesenchymal stem cell (MSC) |
| CN101914496A (en) * | 2010-08-06 | 2010-12-15 | 青岛奥克生物开发有限公司 | Simplified method for preparing umbilical mesenchymal stem cells in large scale |
| CN101974484A (en) * | 2010-11-03 | 2011-02-16 | 江苏省北科生物科技有限公司 | Method for preparing human umbilical cord mesenchymal stem cells |
| CN102154200A (en) * | 2010-12-13 | 2011-08-17 | 中国人民解放军第三○二医院 | Preparation and storage of mesenchymal stem cells for clinical treatment |
| CN104630142A (en) * | 2015-02-06 | 2015-05-20 | 广州赛莱拉干细胞科技股份有限公司 | Separation and culture method of bovine umbilical cord mesenchymal stem cells |
| CN104762257A (en) * | 2015-04-13 | 2015-07-08 | 云南和泽西南生物科技有限公司 | Method for preparing mesenchymal stem cell from umbilical cord |
| CN106497873A (en) * | 2016-12-08 | 2017-03-15 | 北京银丰鼎诚生物工程技术有限公司 | One kind prepares human umbilical cord mesenchymal stem cells factor secretion inducing culture medium on a large scale |
| CN106434547A (en) * | 2016-12-17 | 2017-02-22 | 重庆市干细胞技术有限公司 | Method for preparing umbilical cord mesenchymal stem cells |
| CN110257327A (en) * | 2019-06-27 | 2019-09-20 | 重庆市细胞生物工程技术有限公司 | A kind of isolated culture method of umbilical cord mesenchymal stem cells |
| CN110305839A (en) * | 2019-08-02 | 2019-10-08 | 陕西佰傲干细胞再生医学有限公司 | Mesenchymal stem cell serum-free culture medium |
Non-Patent Citations (1)
| Title |
|---|
| 佚名: "Alpha MEM with Nucleosides", 《佚名》 * |
Cited By (22)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111467376A (en) * | 2020-05-23 | 2020-07-31 | 湖南源品细胞生物科技有限公司 | Application of MSC (Mobile switching center) in relieving influence of cytokine storm |
| CN111529551A (en) * | 2020-05-23 | 2020-08-14 | 湖南源品细胞生物科技有限公司 | Application of MSC (mesenchymal stem cell) in adjusting number of plasma cells |
| CN111544452A (en) * | 2020-05-23 | 2020-08-18 | 湖南源品细胞生物科技有限公司 | Application of MSC (mesenchymal stem cell) in regulating number of CD4+ T cells |
| CN111557951A (en) * | 2020-05-23 | 2020-08-21 | 湖南源品细胞生物科技有限公司 | Novel method for treating virus infection patient by mesenchymal stem cells |
| CN111557950A (en) * | 2020-05-23 | 2020-08-21 | 湖南源品细胞生物科技有限公司 | Application of MSC (mesenchymal stem cell) in regulating and controlling NK (natural killer) cell gene expression |
| CN111568929A (en) * | 2020-05-23 | 2020-08-25 | 湖南源品细胞生物科技有限公司 | Application of MSC (mesenchymal stem cell) in regulating number of effector CD8+ T cells |
| CN111568930A (en) * | 2020-05-23 | 2020-08-25 | 湖南源品细胞生物科技有限公司 | Application of MSC (mesenchymal stem cell) in adjusting number of NK (natural killer) cells |
| CN111568927A (en) * | 2020-05-23 | 2020-08-25 | 湖南源品细胞生物科技有限公司 | Application of MSC (mesenchymal stem cell) in regulating number of memory B cells |
| CN111568931A (en) * | 2020-05-23 | 2020-08-25 | 湖南源品细胞生物科技有限公司 | Application of MSC (mesenchymal stem cell) in regulating and controlling human body effect CD4+ T memory cell gene expression |
| CN111568928A (en) * | 2020-05-23 | 2020-08-25 | 湖南源品细胞生物科技有限公司 | Application of MSC (mesenchymal stem cell) in regulating and controlling gene expression of effector CD8+ T cells |
| CN111979188A (en) * | 2020-08-25 | 2020-11-24 | 江苏赛亿细胞技术研究院有限公司 | Placenta mesenchymal stem cell isolation culture amplification method |
| CN111979189A (en) * | 2020-08-25 | 2020-11-24 | 江苏赛亿细胞技术研究院有限公司 | Isolated culture and amplification method of amniotic mesenchymal stem cells |
| CN112029713A (en) * | 2020-08-25 | 2020-12-04 | 江苏赛亿细胞技术研究院有限公司 | Chorionic mesenchymal stem cell isolation culture amplification method |
| CN112126622A (en) * | 2020-08-27 | 2020-12-25 | 陕西佰傲干细胞再生医学有限公司 | Umbilical cord mesenchymal stem cell primary isolation culture method capable of improving yield |
| CN112126622B (en) * | 2020-08-27 | 2024-06-07 | 山东佰鸿干细胞生物技术有限公司 | Primary isolated culture method of umbilical cord mesenchymal stem cells capable of improving yield |
| CN112011505A (en) * | 2020-09-09 | 2020-12-01 | 广州同康生物科技有限公司 | Umbilical cord mesenchymal stem cell separation method |
| CN112043725A (en) * | 2020-09-17 | 2020-12-08 | 湖南源品细胞生物科技有限公司 | Application of mesenchymal stem cells in improvement of human body oxidative stress state |
| CN112159795A (en) * | 2020-10-13 | 2021-01-01 | 肖利霞 | Passage amplification method for umbilical cord mesenchymal stem cell working cells in public bank |
| CN112852726A (en) * | 2021-02-24 | 2021-05-28 | 河南省银丰生物工程技术有限公司 | Method for separating and amplifying mesenchymal stem cells |
| CN113061638A (en) * | 2021-03-06 | 2021-07-02 | 河北北科生物科技有限公司 | Stem cell quality evaluation system and application method |
| CN113201490A (en) * | 2021-05-26 | 2021-08-03 | 新乡医学院 | Method for sequentially culturing human umbilical cord mesenchymal stem cells by triple culture medium |
| CN113604428A (en) * | 2021-08-19 | 2021-11-05 | 上海纳米技术及应用国家工程研究中心有限公司 | Primary isolation method of human umbilical cord-derived mesenchymal stem cells |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110540959A (en) | Umbilical cord mesenchymal stem cell isolation culture amplification method | |
| CN106754674B (en) | The method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion | |
| CN105238751B (en) | Isolated culture method of umbilical cord tissue mesenchymal stem cells | |
| CN107299082B (en) | Method for separating placenta mesenchymal cells from tissues and culturing into mesenchymal stem cells | |
| CN109234229B (en) | Method for separating mesenchymal stem cells from placental blood vessels and digestive enzyme composition used in same | |
| CN101629165B (en) | Preparation method of original mesenchymal stem cell | |
| CN113249317A (en) | Isolated culture and amplification method and system for human umbilical cord mesenchymal stem cells | |
| CN106801032B (en) | Construction method of human amniotic epithelial stem cell bank | |
| CN104762257B (en) | A kind of method preparing mescenchymal stem cell from umbilical cord | |
| CN101591644A (en) | The preparation of umbilical cord-mesenchymal stem cells (UC-MSCs) used in clinical therapy and storage | |
| CN108220229A (en) | A kind of preparation method for improving umbilical cord derived mesenchymal stem cell primary cell yield | |
| CN106318906A (en) | Method for large-scale culture of human umbilical cord mesenchymal stem cells | |
| CN105238749A (en) | Method for resuscitating mesenchymal stem cells | |
| CN108865988A (en) | A kind of separation of human amnion mesenchymal stem cell, culture and purification process | |
| CN102154200A (en) | Preparation and storage of mesenchymal stem cells for clinical treatment | |
| CN107083359B (en) | Stem cell culture medium and stem cell separation method | |
| CN107354130B (en) | Human placenta chorion mesenchymal stem cell separation method | |
| CN102191229A (en) | Method for rapidly and effectively acquiring umbilical cord mesenchymal stem cell (MSC) | |
| CN109628388B (en) | Isolation of mesenchymal stem cells from placental blood vessels with digestive enzyme composition | |
| CN105505865A (en) | Separation method for umbilical cord mesenchymal stem cells | |
| CN112029713A (en) | Chorionic mesenchymal stem cell isolation culture amplification method | |
| CN104726403A (en) | Method for separating and culturing human placental-derived mesenchymal stem cell | |
| CN112522192A (en) | Separation culture method of dental pulp mesenchymal stem cells | |
| CN104818243A (en) | Separation method of placenta-derived fetal stem cells | |
| CN111979188A (en) | Placenta mesenchymal stem cell isolation culture amplification method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |