CN114717185A - Separation and culture method of giant panda placenta mesenchymal stem cells - Google Patents
Separation and culture method of giant panda placenta mesenchymal stem cells Download PDFInfo
- Publication number
- CN114717185A CN114717185A CN202210331080.3A CN202210331080A CN114717185A CN 114717185 A CN114717185 A CN 114717185A CN 202210331080 A CN202210331080 A CN 202210331080A CN 114717185 A CN114717185 A CN 114717185A
- Authority
- CN
- China
- Prior art keywords
- mesenchymal stem
- placenta
- stem cells
- culture
- giant panda
- 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
- 210000002826 placenta Anatomy 0.000 title claims abstract description 61
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 57
- 241000282452 Ailuropoda melanoleuca Species 0.000 title claims abstract description 41
- 238000000926 separation method Methods 0.000 title claims abstract description 11
- 238000012136 culture method Methods 0.000 title claims abstract description 7
- 210000004027 cell Anatomy 0.000 claims abstract description 40
- 238000000034 method Methods 0.000 claims abstract description 29
- 208000025174 PANDAS Diseases 0.000 claims abstract description 16
- 208000021155 Paediatric autoimmune neuropsychiatric disorders associated with streptococcal infection Diseases 0.000 claims abstract description 16
- 240000004718 Panda Species 0.000 claims abstract description 16
- 235000016496 Panda oleosa Nutrition 0.000 claims abstract description 16
- 230000029087 digestion Effects 0.000 claims abstract description 15
- 238000005406 washing Methods 0.000 claims abstract description 13
- 102000029816 Collagenase Human genes 0.000 claims abstract description 12
- 108060005980 Collagenase Proteins 0.000 claims abstract description 12
- 229960002424 collagenase Drugs 0.000 claims abstract description 12
- 230000004069 differentiation Effects 0.000 claims abstract description 11
- 238000012258 culturing Methods 0.000 claims abstract description 10
- 230000000877 morphologic effect Effects 0.000 claims abstract description 9
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- 238000007710 freezing Methods 0.000 claims abstract description 6
- 230000008014 freezing Effects 0.000 claims abstract description 6
- 238000011084 recovery Methods 0.000 claims abstract description 3
- 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
- 210000001519 tissue Anatomy 0.000 claims description 12
- 239000001963 growth medium Substances 0.000 claims description 11
- 210000005059 placental tissue Anatomy 0.000 claims description 11
- 230000003169 placental effect Effects 0.000 claims description 10
- 238000002791 soaking Methods 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 9
- 230000006698 induction Effects 0.000 claims description 9
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 8
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 7
- 239000002458 cell surface marker Substances 0.000 claims description 7
- DILRJUIACXKSQE-UHFFFAOYSA-N n',n'-dimethylethane-1,2-diamine Chemical compound CN(C)CCN DILRJUIACXKSQE-UHFFFAOYSA-N 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 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
- 238000007664 blowing Methods 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- 239000012091 fetal bovine serum Substances 0.000 claims description 6
- 229940049954 penicillin Drugs 0.000 claims description 6
- 229960005322 streptomycin Drugs 0.000 claims description 6
- 238000010009 beating Methods 0.000 claims description 5
- 239000002299 complementary DNA Substances 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 230000002293 adipogenic effect Effects 0.000 claims description 4
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 claims description 4
- 230000002648 chondrogenic effect Effects 0.000 claims description 4
- 230000002188 osteogenic effect Effects 0.000 claims description 4
- 239000002504 physiological saline solution Substances 0.000 claims description 4
- 238000010839 reverse transcription Methods 0.000 claims description 4
- 230000002194 synthesizing effect Effects 0.000 claims description 4
- 229930183010 Amphotericin Natural products 0.000 claims description 3
- QGGFZZLFKABGNL-UHFFFAOYSA-N Amphotericin A Natural products OC1C(N)C(O)C(C)OC1OC1C=CC=CC=CC=CCCC=CC=CC(C)C(O)C(C)C(C)OC(=O)CC(O)CC(O)CCC(O)C(O)CC(O)CC(O)(CC(O)C2C(O)=O)OC2C1 QGGFZZLFKABGNL-UHFFFAOYSA-N 0.000 claims description 3
- 108010019160 Pancreatin Proteins 0.000 claims description 3
- 238000011529 RT qPCR Methods 0.000 claims description 3
- 229940009444 amphotericin Drugs 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000012512 characterization method Methods 0.000 claims description 3
- 230000009089 cytolysis Effects 0.000 claims description 3
- 230000003203 everyday effect Effects 0.000 claims description 3
- 239000012894 fetal calf serum Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 3
- 210000003632 microfilament Anatomy 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 229940055695 pancreatin Drugs 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 238000003860 storage Methods 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 239000003550 marker Substances 0.000 claims description 2
- 210000000130 stem cell Anatomy 0.000 abstract description 5
- 238000003320 cell separation method Methods 0.000 abstract description 4
- 239000003242 anti bacterial agent Substances 0.000 abstract description 2
- 229940088710 antibiotic agent Drugs 0.000 abstract description 2
- 239000002771 cell marker Substances 0.000 abstract description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 229960001701 chloroform Drugs 0.000 description 3
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 3
- 229960003957 dexamethasone Drugs 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- DHCLVCXQIBBOPH-UHFFFAOYSA-N Glycerol 2-phosphate Chemical compound OCC(CO)OP(O)(O)=O DHCLVCXQIBBOPH-UHFFFAOYSA-N 0.000 description 2
- YASAKCUCGLMORW-UHFFFAOYSA-N Rosiglitazone Chemical compound C=1C=CC=NC=1N(C)CCOC(C=C1)=CC=C1CC1SC(=O)NC1=O YASAKCUCGLMORW-UHFFFAOYSA-N 0.000 description 2
- 230000009815 adipogenic differentiation Effects 0.000 description 2
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000009816 chondrogenic differentiation Effects 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000009818 osteogenic differentiation Effects 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- APIXJSLKIYYUKG-UHFFFAOYSA-N 3 Isobutyl 1 methylxanthine Chemical compound O=C1N(C)C(=O)N(CC(C)C)C2=C1N=CN2 APIXJSLKIYYUKG-UHFFFAOYSA-N 0.000 description 1
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 description 1
- 101100257359 Caenorhabditis elegans sox-2 gene Proteins 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 102100027754 Mast/stem cell growth factor receptor Kit Human genes 0.000 description 1
- 101710087603 Mast/stem cell growth factor receptor Kit Proteins 0.000 description 1
- 101100257363 Mus musculus Sox2 gene Proteins 0.000 description 1
- NPGIHFRTRXVWOY-UHFFFAOYSA-N Oil red O Chemical compound Cc1ccc(C)c(c1)N=Nc1cc(C)c(cc1C)N=Nc1c(O)ccc2ccccc12 NPGIHFRTRXVWOY-UHFFFAOYSA-N 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229960003942 amphotericin b Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 229960004586 rosiglitazone Drugs 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 210000003934 vacuole Anatomy 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Images
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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
-
- 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
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Hematology (AREA)
- Developmental Biology & Embryology (AREA)
- Urology & Nephrology (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Rheumatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention belongs to the field of stem cell separation and culture, and particularly relates to a panda placenta mesenchymal stem cell separation and culture method. The method comprises the following steps: step 1: aseptically collecting giant panda placenta; step 2: primarily separating the giant panda placenta mesenchymal stem cells by adopting a collagenase digestion method, and culturing the separated tissues in a culture dish; and step 3: performing cell morphological characteristic identification, surface mark identification and induced differentiation identification on the placenta mesenchymal stem cells cultured in the step 2; and 4, step 4: freezing and storing the cultured and subcultured placenta mesenchymal stem cells, and taking out the cells for cell recovery when in use. The invention obtains the highly purified giant panda placenta mesenchymal stem cells by adding high-concentration combined antibiotics and washing for many times when collecting the placenta, adopting a collagenase digestion method for separation culture and designing a giant panda placenta mesenchymal stem cell marker gene specific primer and the like.
Description
Technical Field
The invention belongs to the field of stem cell separation and culture, and particularly relates to a panda placenta mesenchymal stem cell separation and culture method.
Background
The giant pandas are species of the world for protecting the biodiversity, the Qinling mountain giant pandas are important subspecies populations of the giant pandas, have unique morphological characteristics and genetic characteristics, actively develop the work of preserving the germ plasm resources of the Qinling mountain giant pandas, establish the stem cell bank of the germ plasm resources of the Qinling mountain giant pandas, and have important functions and significance for protecting the genetic diversity of the giant pandas in China. The mesenchymal stem cells have the potential of multidirectional differentiation as the pluripotent stem cells, can be used as ideal stem cells for clinical treatment, and provide the best raw materials for clinical treatment of panda diseases. The placenta is used as a delivery waste product, and the damage to panda individuals can be used as the best mode for the source of the mesenchymal stem cells.
However, after the giant panda placenta is collected, it is often difficult to obtain high-purity giant panda placenta mesenchymal stem cells due to the problems of pollution, difficult cell emigration during tissue block culture and the like.
Disclosure of Invention
Aiming at the technical problems, the invention provides a separation and culture method of giant panda placenta mesenchymal stem cells, which comprises the steps of adding high-concentration combined antibiotics and washing for multiple times when collecting the placenta, adopting a collagenase digestion method for separation and culture, designing marker gene specific primers of the giant panda placenta mesenchymal stem cells and the like, and obtaining the high-purity giant panda placenta mesenchymal stem cells.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a giant panda placenta mesenchymal stem cell separation and culture method comprises the following steps:
step 1: aseptically collecting giant panda placenta;
step 2: primarily separating giant panda placenta mesenchymal stem cells by adopting a collagenase digestion method, and culturing the separated tissues in a culture dish;
and step 3: performing cell morphological characteristic identification, surface mark identification and induced differentiation identification on the placenta mesenchymal stem cells cultured in the step 2;
and 4, step 4: and (4) freezing and storing the cultured and passaged placenta mesenchymal stem cells, and taking out the placenta mesenchymal stem cells for cell recovery when in use.
Preferably, the specific operation of step 1 is:
step 1.1: storing the naturally delivered panda placenta in physiological saline containing 1% double antibody at 25 ℃, and transporting to a laboratory within 6h for primary cell separation, wherein the 1% double antibody is 100U/mL penicillin and 100U/mL streptomycin;
step 1.2: transferring the placenta tissue to a culture dish added with 75% alcohol in an ultra-clean workbench, soaking for 30s, taking out, washing with normal saline for 5-10 times, then soaking in 3-5% of triantion-containing normal saline for washing for 1min, transferring the placenta tissue to a new culture dish, and repeatedly soaking and washing with the 3-5% of triantion-containing normal saline for 10-15 times, wherein the triantion is penicillin, streptomycin and amphotericin;
step 1.3: step 1.2 after the wash was complete, the tissue was transferred to DMEN medium containing 1% double antibody, 20% fetal bovine serum.
Preferably, the specific operation of step 2 is:
step 2.1: cutting panda placenta tissue of step 1.3 into pieces of 1mm by using sterilized ophthalmic scissors3Adding 0.25% IV collagenase for digestion and blow beating for 40min, centrifuging at 1100rpm for 6min, removing supernatant, adding 1% double antibody and 20% fetal calf serum containing DMEN culture solution suspension cells, and transferring to a culture dish for culture;
step 2.2: observing the growth state of the cells every day, replacing the fresh culture medium in time, and carrying out digestion subculture by using 0.25% pancreatin diluted by 3-5 times when the cells grow to 80% confluence.
Preferably, the step 3 comprises:
3.1: cell morphological characterization
The placenta mesenchymal stem cells should show the adhesion capacity to a culture dish in the growth process, the cells are in a fusiform flat shape, the protrusion of a central nucleus is clear, the boundary is not obvious, and clear cell myofilament structures can be observed under a high power microscope;
3.2: surface marking identification
Step 3.2.1: extracting total RNA, collecting placenta mesenchymal stem cells, conducting Trizol lysis and room-temperature blowing for 5min, adding chloroform for centrifugation and layering, collecting a water sample layer, reducing the RNA through isopropanol precipitation, and extracting to obtain RNA;
step 3.2.2: the cDNA is synthesized by reverse transcription,
step 3.2.3: designing and synthesizing a giant panda placental mesenchymal stem cell surface marker gene primer, and identifying whether the giant panda placental mesenchymal stem cell surface marker gene primer is expressed or not through qRT-PCR;
3.3: and (3) induced differentiation identification, which comprises osteogenic induction culture result determination, adipogenic induction culture result determination and chondrogenic induction culture result determination.
Preferably, the cells are subjected to gradient cooling when being frozen, the cooling speed is-1 ℃/min to-2 ℃/min when the freezing is started, the speed can be increased when the temperature is lower than-25 ℃, and the cells are transferred to liquid nitrogen for storage after the temperature is reduced to-80 ℃.
Compared with the prior art, the invention has the beneficial effects that:
1. soaking the giant panda placenta in 75% alcohol for 30s and washing the giant panda placenta with 3% -5% physiological saline for multiple times, so that the pollution phenomenon generated during placenta collection can be effectively reduced, and the high-purity giant panda placenta mesenchymal stem cells can be obtained by adopting a 0.25% IV type collagenase digestion method;
2. adopting collagenase digestion method to successfully obtain the mesenchymal stem cells. In the existing process of separating cells by using a tissue block adherence method, no cells are emigrated after 10 days to 14 days after the tissue block adherence. Analysis comparing the differences of the two tests gives the following differences: 1) the placenta tissue should be transported to the laboratory quickly after delivery, and over 6 hours will affect the cell activity of the tissue; 2) the traditional tissue block separation method has low cell migration rate and long time consumption, the pandas are delivered naturally, the placenta is naturally exposed to the polluted environment, the placenta is inevitably affected in the process of carrying out sterile treatment on the placenta, the placenta tissue is mechanically sheared into small blocks and then digested by collagenase, the digestion time can be effectively reduced, and the incompletely digested tissue is inoculated on a culture dish again, so that the cells can be successfully migrated.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.
In the drawings:
FIG. 1 is a flow chart of a method of the present invention;
FIG. 2 is a morphological feature diagram of the placenta mesenchymal stem cells of Qinling mountain panda (a is multiplied by 100, b is multiplied by 40);
FIG. 3 is a picture of identification of osteogenic induced differentiation of mesenchymal stem cells of Qinling mountain panda placenta isolated and cultured according to the present invention;
FIG. 4 is a picture of identification of adipogenic induced differentiation of mesenchymal stem cells of Qinling mountain panda placenta isolated and cultured according to the present invention;
FIG. 5 is a drawing of identification of chondrogenic induced differentiation of mesenchymal stem cells of Qinling mountain panda placenta isolated and cultured in the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the present invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example (b):
referring to the attached figure 1, a method for separating and culturing giant panda placenta mesenchymal stem cells, which is a flow chart of the method, comprises the following steps:
step 1: aseptic collection of giant panda placenta comprises the following specific operations:
step 1.1: storing the naturally delivered panda placenta in physiological saline containing 1% double antibody at 25 ℃, and conveying the preserved panda placenta to a laboratory within 6h for primary cell separation;
step 1.2: transferring the placenta tissue to a culture dish added with 75% alcohol in an ultra-clean workbench, soaking for 30s, taking out, washing for 5-10 times by using normal saline, then soaking in 3-5% of three-resistant normal saline for washing for 1min, transferring the placenta tissue to a new culture dish, and repeatedly soaking and washing for 10-15 times by using the 3-5% of three-resistant normal saline, wherein the three-resistant is penicillin, streptomycin and amphotericin, wherein the penicillin content is 10kU/ml, the streptomycin content is 10mg/ml, and the amphotericin B content is 25 mu g/ml;
step 1.3: step 1.2 after the wash was complete, the tissue was transferred to DMEN medium containing 1% double antibody, 20% fetal bovine serum.
Step 2: primarily separating giant panda placenta mesenchymal stem cells by adopting a collagenase digestion method, and culturing the separated tissue in a culture dish, wherein the specific operation is as follows:
step 2.1: cutting panda placenta tissue of step 1.3 into pieces of 1mm by using sterilized ophthalmic scissors3Adding 0.25% IV collagenase for digestion and blow beating for 40min, centrifuging at 1100rpm for 6min, removing supernatant, adding 1% double antibody and 20% fetal calf serum containing DMEN culture solution suspension cells, and transferring to a culture dish for culture;
step 2.2: observing the growth state of the cells every day, replacing the fresh culture medium in time, and carrying out digestion subculture by using 0.25% pancreatin diluted by 3-5 times when the cells grow to 80% confluence.
And step 3: performing cell morphological characteristic identification, surface mark identification and induced differentiation identification on the placenta mesenchymal stem cells cultured in the step 2; the method comprises the following steps:
3.1: cell morphological characterization
The placenta mesenchymal stem cells should show the adhesion capacity to a culture dish in the growth process, the cells are in a fusiform flat shape, the protrusion of a central nucleus is clear, the boundary is not obvious, and the clear cell myofilament structure can be observed under a high power microscope, which is shown in figure 2;
3.2: surface marking identification
Step 3.2.1: extracting total RNA, collecting placenta mesenchymal stem cells, conducting Trizol lysis and room-temperature blowing for 5min, adding chloroform for centrifugation and layering, collecting a water sample layer, reducing the RNA through isopropanol precipitation, and extracting to obtain RNA; the method specifically comprises the following steps:
(1) washing mesenchymal stem cells by using precooled PBS, adding RNAtrizol for reacting for 5min, repeatedly blowing and beating until the cells are cracked into uniform solution, and collecting the uniform solution in a 1.5ml RNase-free EP tube;
(2) adding 200ul of trichloromethane, violently shaking for 15s to mix uniformly, standing at room temperature for 5min, centrifuging at 12000rpm/min in a 4 ℃ centrifuge for 15 min, and transferring the upper aqueous phase into an Eppendorf tube;
(3) adding isopropanol with the same volume, mixing uniformly, standing at room temperature for 10 minutes, centrifuging at 12000rpm/min in a centrifuge at 4 ℃ for 10 minutes, and observing that white precipitate is RNA;
(4) discarding the supernatant, placing on ice, adding 1ml of 75% alcohol, blowing, shaking, centrifuging at 7500rpm/min in a 4 ℃ centrifuge for 5min, discarding the supernatant, and repeating the 75% alcohol cleaning and centrifuging operation;
(5) drying the obtained RNA precipitate at room temperature, adding 30ul of RNase-free water when the RNA precipitate is transparent or semitransparent, repeatedly blowing and beating, subpackaging in a spectrophotometer for detection, and storing at-80 ℃.
Step 3.2.2: synthesizing cDNA by reverse transcription, adding water, a primer, a template RNA, dNTP, Buffer and reverse transcriptase with corresponding volumes according to the requirements of a commercial reverse transcription kit, setting a reverse transcription program according to the instruction, and amplifying to obtain the cDNA.
Step 3.2.3: designing and synthesizing a giant panda placental mesenchymal stem cell surface marker gene primer, and identifying whether the giant panda placental mesenchymal stem cell surface marker gene primer is expressed or not through qRT-PCR; the total RNA of the cultured cells in the step 3.2.1 and the cDNA reverse transcribed and synthesized in the step 3.2.2 are utilized to identify the expression stem cell marker genes CD34, CD90, SCFR and SOX-2 of the giant panda placenta mesenchymal stem cells by RT-PCR, and the sequences of primers are used to refer to the table 1.
TABLE 1 giant panda placenta mesenchymal stem cell surface marker gene primer sequence
3.3: inducing differentiation and identifying the cells,
3.1: determination of osteogenic Induction culture results
After the placenta mesenchymal stem cells are induced in an osteogenic differentiation culture medium for 21 days, the formation of a mineralized matrix is observed by alizarin red staining (figure 3), wherein the osteogenic differentiation culture medium is formed by adding 10% FBS, 0.1 mu M dexamethasone, 10mM beta-glycerophosphate and 2 mu M ascorbic acid into a basic culture medium, and the formation of the mineralized matrix can be observed by alizarin red staining.
3.2: determination of adipogenic Induction culture results
After the placenta mesenchymal stem cells are induced in a adipogenic differentiation culture medium for 21-28 days, lipid vacuoles can be observed after oil red O staining (figure 4), wherein the adipogenic differentiation culture medium is prepared by adding 10% FBS, 0.5mM 3-isobutyl-1-methylxanthine, 1 mu M dexamethasone, 0.5mM rosiglitazone and 10 mu g/mL insulin into a basic culture medium.
3.2: determination of chondrogenic induction culture results
After the placenta mesenchymal stem cells are induced in a chondrogenic differentiation culture medium for 21-28 days, the endogenic acid mucopolysaccharide in the cartilage tissue can be observed after the cells are stained by Alisin blue (figure 5), and the chondrogenic differentiation culture medium is formed by adding 10% FBS, 1% TGF-beta, 0.1 mu M dexamethasone, 3 mu M ascorbic acid, 1 mu M proline and 1 mu M sodium pyruvate into a DMEN culture solution.
And 4, step 4: separating and culturing the cultured placenta mesenchymal stem cells; the specific operation is as follows: when the cells are frozen, the gradient cooling is carried out, the cooling speed is-1 to-2 ℃/min when the freezing is started, the speed can be accelerated when the temperature is lower than-25 ℃, and the cells are transferred to liquid nitrogen for storage after the temperature is reduced to-80 ℃. 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 given by way of illustration of the principles of the present invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, and such changes and modifications are within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (5)
1. A separation and culture method of giant panda placenta mesenchymal stem cells is characterized in that: the method comprises the following steps:
step 1: aseptically collecting giant panda placenta;
step 2: primarily separating giant panda placenta mesenchymal stem cells by adopting a collagenase digestion method, and culturing the separated tissues in a culture dish;
and 3, step 3: performing cell morphological characteristic identification, surface marker identification and induced differentiation identification on the placenta mesenchymal stem cells cultured in the step 2;
and 4, step 4: and (4) freezing and storing the cultured and passaged placenta mesenchymal stem cells, and taking out the placenta mesenchymal stem cells for cell recovery when in use.
2. The method for separating and culturing giant panda placental mesenchymal stem cells according to claim 1, wherein the method comprises the following steps: the specific operation of the step 1 is as follows:
step 1.1: storing the naturally delivered panda placenta in physiological saline containing 1% double antibody at 25 ℃, and transporting to a laboratory within 6h for primary cell separation, wherein the 1% double antibody is 100U/mL penicillin and 100U/mL streptomycin;
step 1.2: transferring the placenta tissue to a culture dish added with 75% alcohol in an ultra-clean workbench, soaking for 30s, taking out, washing with normal saline for 5-10 times, then soaking in 3-5% of triantion-containing normal saline for washing for 1min, transferring the placenta tissue to a new culture dish, and repeatedly soaking and washing with the 3-5% of triantion-containing normal saline for 10-15 times, wherein the triantion is penicillin, streptomycin and amphotericin;
step 1.3: step 1.2 after the wash was complete, the tissue was transferred to DMEN medium containing 1% double antibody, 20% fetal bovine serum.
3. The method for separating and culturing giant panda placental mesenchymal stem cells according to claim 2, wherein the method comprises the following steps: the specific operation of the step 2 is as follows:
step 2.1: cutting panda placenta tissue of step 1.3 into pieces of 1mm by using sterilized ophthalmic scissors3Adding 0.25% IV collagenase for digestion and blow beating for 40min, centrifuging at 1100rpm for 6min, removing supernatant, adding 1% double antibody and 20% fetal calf serum containing DMEN culture solution suspension cells, and transferring to a culture dish for culture;
step 2.2: observing the growth state of the cells every day, replacing the fresh culture medium in time, and carrying out digestion subculture by using 0.25% pancreatin diluted by 3-5 times when the cells grow to 80% confluence.
4. The method for separating and culturing giant panda placental mesenchymal stem cells according to claim 3, wherein the method comprises the following steps: the step 3 comprises the following steps:
3.1: cell morphological characterization
The placenta mesenchymal stem cells should show the adhesion capacity to a culture dish in the growth process, the cells are in a fusiform flat shape, the protrusion of a central nucleus is clear, the boundary is not obvious, and clear cell myofilament structures can be observed under a high power microscope;
3.2: surface marking identification
Step 3.2.1: extracting total RNA, collecting placenta mesenchymal stem cells, conducting Trizol lysis and room-temperature blowing for 5min, adding chloroform for centrifugation and layering, collecting a water sample layer, reducing the RNA through isopropanol precipitation, and extracting to obtain RNA;
step 3.2.2: the cDNA is synthesized by reverse transcription,
step 3.2.3: designing and synthesizing a giant panda placental mesenchymal stem cell surface marker gene primer, and identifying whether the giant panda placental mesenchymal stem cell surface marker gene primer is expressed or not through qRT-PCR;
3.3: and (3) induced differentiation identification, which comprises osteogenic induction culture result determination, adipogenic induction culture result determination and chondrogenic induction culture result determination.
5. The method for separating and culturing giant panda placental mesenchymal stem cells according to claim 4, wherein the method comprises the following steps: the specific operation of the step 4 is as follows: when the cells are frozen, the gradient cooling is carried out, the cooling speed is-1 ℃/min to-2 ℃/min when the freezing is started, the speed can be increased when the temperature is lower than-25 ℃, and the cells are transferred to liquid nitrogen for storage after the temperature is reduced to-80 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210331080.3A CN114717185A (en) | 2022-03-31 | 2022-03-31 | Separation and culture method of giant panda placenta mesenchymal stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210331080.3A CN114717185A (en) | 2022-03-31 | 2022-03-31 | Separation and culture method of giant panda placenta mesenchymal stem cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114717185A true CN114717185A (en) | 2022-07-08 |
Family
ID=82238953
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210331080.3A Pending CN114717185A (en) | 2022-03-31 | 2022-03-31 | Separation and culture method of giant panda placenta mesenchymal stem cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114717185A (en) |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007069813A1 (en) * | 2005-12-16 | 2007-06-21 | Modern Cell & Tissue Technologies Inc. | Methods for culturing mesenchymal stem cell and compositions comprising mesenchymal stem cell for reparing skin defects |
| CN101270349A (en) * | 2008-03-20 | 2008-09-24 | 浙江大学 | Method for placenta mesenchyma stem cell separation and in vitro amplify cultivation |
| US20110217385A1 (en) * | 2008-10-10 | 2011-09-08 | Team Youn Biomedical Technology Co., Ltd. | Method for extracting mesenchymal stem cell from human or animal embryo and for extracting the secretion product thereof |
| US20120164114A1 (en) * | 2010-12-17 | 2012-06-28 | Abbot Stewart | Treatment of immune-related diseases and disorders using amnion derived adherent cells |
| CN104560870A (en) * | 2014-12-18 | 2015-04-29 | 江苏省北科生物科技有限公司 | Method for preparing decidua mesenchymal stem cell |
| CN106085952A (en) * | 2016-07-05 | 2016-11-09 | 四川华皓生物科技有限公司 | A kind of placental villi plate mescenchymal stem cell and extracting method thereof |
| CN106754674A (en) * | 2016-12-09 | 2017-05-31 | 博雅干细胞科技有限公司 | Method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion |
| WO2017096607A1 (en) * | 2015-12-11 | 2017-06-15 | 郭镭 | Method for separating and extracting huc-msc from outer layer of amniotic membrane tissue of umbilical cord |
| CN107338219A (en) * | 2017-02-06 | 2017-11-10 | 贵州泛特尔细胞生物技术有限公司 | A kind of separation of placenta mesenchyma stem cell and cultural method |
| RU2645255C1 (en) * | 2016-12-16 | 2018-02-19 | Федеральное государственное бюджетное учреждение "Национальный медицинский исследовательский центр акушерства, гинекологии и перинатологии имени академика В.И.Кулакова" Министерства здравоохранения Российской Федерации | Method for obtaining of biosafe culture of mesenchimal stem cells from human chorionic villae |
| CN109251890A (en) * | 2018-10-22 | 2019-01-22 | 中晶生物技术股份有限公司 | A kind of method and its application of high efficiency extraction endometrium mescenchymal stem cell |
| US20190269739A1 (en) * | 2016-11-03 | 2019-09-05 | Exostem Biotec Ltd. | Mesenchymal stem cells populations, their products, and use thereof |
| CN110317780A (en) * | 2019-07-17 | 2019-10-11 | 福建省海西细胞生物工程有限公司 | A kind of placenta decidua basalis mescenchymal stem cell preparation method |
| CN110964693A (en) * | 2019-12-27 | 2020-04-07 | 河北银丰鼎诚生物技术有限公司 | Separation method of umbilical cord mesenchymal stem cells |
-
2022
- 2022-03-31 CN CN202210331080.3A patent/CN114717185A/en active Pending
Patent Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007069813A1 (en) * | 2005-12-16 | 2007-06-21 | Modern Cell & Tissue Technologies Inc. | Methods for culturing mesenchymal stem cell and compositions comprising mesenchymal stem cell for reparing skin defects |
| CN101270349A (en) * | 2008-03-20 | 2008-09-24 | 浙江大学 | Method for placenta mesenchyma stem cell separation and in vitro amplify cultivation |
| US20110217385A1 (en) * | 2008-10-10 | 2011-09-08 | Team Youn Biomedical Technology Co., Ltd. | Method for extracting mesenchymal stem cell from human or animal embryo and for extracting the secretion product thereof |
| US20120164114A1 (en) * | 2010-12-17 | 2012-06-28 | Abbot Stewart | Treatment of immune-related diseases and disorders using amnion derived adherent cells |
| CN104560870A (en) * | 2014-12-18 | 2015-04-29 | 江苏省北科生物科技有限公司 | Method for preparing decidua mesenchymal stem cell |
| WO2017096607A1 (en) * | 2015-12-11 | 2017-06-15 | 郭镭 | Method for separating and extracting huc-msc from outer layer of amniotic membrane tissue of umbilical cord |
| CN106085952A (en) * | 2016-07-05 | 2016-11-09 | 四川华皓生物科技有限公司 | A kind of placental villi plate mescenchymal stem cell and extracting method thereof |
| US20190269739A1 (en) * | 2016-11-03 | 2019-09-05 | Exostem Biotec Ltd. | Mesenchymal stem cells populations, their products, and use thereof |
| CN106754674A (en) * | 2016-12-09 | 2017-05-31 | 博雅干细胞科技有限公司 | Method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion |
| RU2645255C1 (en) * | 2016-12-16 | 2018-02-19 | Федеральное государственное бюджетное учреждение "Национальный медицинский исследовательский центр акушерства, гинекологии и перинатологии имени академика В.И.Кулакова" Министерства здравоохранения Российской Федерации | Method for obtaining of biosafe culture of mesenchimal stem cells from human chorionic villae |
| CN107338219A (en) * | 2017-02-06 | 2017-11-10 | 贵州泛特尔细胞生物技术有限公司 | A kind of separation of placenta mesenchyma stem cell and cultural method |
| CN109251890A (en) * | 2018-10-22 | 2019-01-22 | 中晶生物技术股份有限公司 | A kind of method and its application of high efficiency extraction endometrium mescenchymal stem cell |
| CN110317780A (en) * | 2019-07-17 | 2019-10-11 | 福建省海西细胞生物工程有限公司 | A kind of placenta decidua basalis mescenchymal stem cell preparation method |
| CN110964693A (en) * | 2019-12-27 | 2020-04-07 | 河北银丰鼎诚生物技术有限公司 | Separation method of umbilical cord mesenchymal stem cells |
Non-Patent Citations (12)
| Title |
|---|
| ARAÚJO A 等: "Isolation of human mesenchymal stem cells from amnion, chorion, placental decidua and umbilical cord: comparison of four enzymatic protocols", 《BIOTECHNOL LETT》, vol. 40, no. 6, pages 989 - 998, XP036503985, DOI: 10.1007/s10529-018-2546-z * |
| LIU Y 等: "Isolation and characterization of mesenchymal stem cells from umbilical cord of giant panda", 《TISSUE CELL》, vol. 71, pages 1 - 7 * |
| SEMENOV O 等: "Multipotent mesenchymal stem cells from human placenta: critical parameters for isolation and maintenance of stemness after isolation", 《AM J OBSTET GYNECOL》, vol. 2019, no. 2, pages 215 - 13 * |
| 候宗柳 等主编: "《围产期成体干细胞基础与临床应用》", vol. 2016, 31 October 2016, 云南出版集团公司, pages: 5 * |
| 吴德沛 等主译: "《造血干细胞移植 原书第5版》", vol. 2020, 31 July 2020, 中国科学技术出版社, pages: 89 * |
| 张峰 等主编: "《细胞工程》", vol. 2014, 31 July 2014, 中国农业大学出版社, pages: 174 * |
| 张钧田 等主编: "《现代药理试验方法 上》", vol. 2012, 31 July 2012, 中国协和医科大学出版社, pages: 374 * |
| 沈柏均 等主编: "《实用细胞培养技术 第2版》", vol. 2016, 30 November 2016, 山东大学出版社, pages: 103 - 41 * |
| 陈凤 等: "人胎盘绒毛膜间充质干细胞分离培养方法研究", 《重庆医学》, vol. 51, no. 1, pages 122 - 126 * |
| 陈波: "人早孕蜕膜组织子宫内膜间质干细胞向子宫内膜上皮细胞的分化", 《中国组织工程研究》, vol. 20, no. 14, pages 2098 - 2103 * |
| 陈能文 著: "《大熊猫与生态文明》", vol. 2022, 31 January 2022, 华中科技大学出版社, pages: 151 * |
| 马永杰: "秦岭大熊猫间充质干细胞分离及种质资源保存", 《中国优秀硕士学位论文全文数据库 (电子数据）农业科技辑》, no. 1, pages 051 - 6 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107988153B (en) | Method for separating exosome from human umbilical cord blood mesenchymal stem cell source and used reagent | |
| Karamzadeh et al. | Isolation, characterization and comparative differentiation of human dental pulp stem cells derived from permanent teeth by using two different methods | |
| CN104560870B (en) | A kind of method for preparing decidua mescenchymal stem cell | |
| CN113249317A (en) | Isolated culture and amplification method and system for human umbilical cord mesenchymal stem cells | |
| WO2016049986A1 (en) | Method for separating umbilical cord mesenchymal stem cells | |
| JP2010527629A (en) | Method for isolating mesenchymal stem cells derived from placental chorionic plate membrane with high purity | |
| CN106635976B (en) | Method and kit for obtaining amniotic mesenchymal stem cells | |
| CN115074309A (en) | Method for simultaneously preparing single cell suspension and single cell nuclear suspension based on same tissue sample and application | |
| CN115058391A (en) | Culture method of hypoxic umbilical cord mesenchymal stem cells | |
| CN110872574B (en) | Efficient and reliable hESC-MSC preparation method | |
| CN112029717A (en) | Serum-free in vitro domestication culture of human mesenchymal stem cells | |
| CN115851587A (en) | Optimized culture medium, kit and culture method of human placenta-derived mesenchymal stem cells | |
| CN110846273A (en) | Adipose tissue-derived mesenchymal stem cell culture and trilineage differentiation induction method | |
| CN104805051B (en) | Method for inducing adipose-derived stem cells to differentiate into fibroblasts | |
| CN111925985B (en) | Domestication culture method of mesenchymal stem cells | |
| CN114717185A (en) | Separation and culture method of giant panda placenta mesenchymal stem cells | |
| CN111849878A (en) | Method for improving osteogenesis capacity of mesenchymal stem cells | |
| CN114107181B (en) | Sturgeon embryo cell line, culture medium and preparation method thereof | |
| CN110885784B (en) | Clinical application-grade adipose-derived stem cells and preparation method thereof | |
| CN110484491B (en) | Method for obtaining amniotic membrane and amniotic fluid derived endothelial progenitor cells and purification culture method thereof | |
| CN113430162A (en) | Model for intrauterine adhesion | |
| CN111979188A (en) | Placenta mesenchymal stem cell isolation culture amplification method | |
| CN106434546B (en) | Method for preparing mesenchymal stem cells by fully utilizing umbilical cord resources | |
| CN116814533B (en) | Cell culture medium for in vitro maintenance of phenotype and function of alveolar type II epithelial cells | |
| CN111040994A (en) | Method for efficiently separating adipose-derived mesenchymal stem cells |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20220708 |
|
| RJ01 | Rejection of invention patent application after publication |