patents.google.com

CN105420179A - Method for simultaneously extracting epithelial cells and mesenchymal stem cells from umbilical cord and placenta amnion tissues - Google Patents

️Wed Mar 23 2016

Method for simultaneously extracting epithelial cells and mesenchymal stem cells from umbilical cord and placenta amnion tissues Download PDF

Info

Publication number

CN105420179A

CN105420179A CN201510963676.5A CN201510963676A CN105420179A CN 105420179 A CN105420179 A CN 105420179A CN 201510963676 A CN201510963676 A CN 201510963676A CN 105420179 A CN105420179 A CN 105420179A Authority

China

Prior art keywords

cell

umbilical cord

placenta

amnion

cells

Prior art date

2015-12-17

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

Application number

CN201510963676.5A

Other languages

Chinese (zh)

Inventor

阎军

肖敏

任峰

Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

Stamm (tianjin) Biotechnology Research Co Ltd

Original Assignee

Stamm (tianjin) Biotechnology Research Co Ltd

Priority date (The priority date 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 date listed.)

2015-12-17

Filing date

2015-12-17

Publication date

2016-03-23

2015-12-17 Application filed by Stamm (tianjin) Biotechnology Research Co Ltd filed Critical Stamm (tianjin) Biotechnology Research Co Ltd

2015-12-17 Priority to CN201510963676.5A priority Critical patent/CN105420179A/en

2016-03-23 Publication of CN105420179A publication Critical patent/CN105420179A/en

Status Pending legal-status Critical Current

Links

210000001691 amnion Anatomy 0.000 title claims abstract description 55
210000002826 placenta Anatomy 0.000 title claims abstract description 51
210000003954 umbilical cord Anatomy 0.000 title claims abstract description 50
238000000034 method Methods 0.000 title claims abstract description 35
210000002919 epithelial cell Anatomy 0.000 title claims abstract description 26
210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 14
210000004027 cell Anatomy 0.000 claims abstract description 67
210000001519 tissue Anatomy 0.000 claims abstract description 27
239000000203 mixture Substances 0.000 claims abstract description 15
235000015097 nutrients Nutrition 0.000 claims abstract description 10
210000002966 serum Anatomy 0.000 claims abstract description 8
210000000130 stem cell Anatomy 0.000 claims description 38
239000002609 medium Substances 0.000 claims description 25
239000002953 phosphate buffered saline Substances 0.000 claims description 20
238000012258 culturing Methods 0.000 claims description 19
LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 17
208000030507 AIDS Diseases 0.000 claims description 12
230000001186 cumulative effect Effects 0.000 claims description 12
239000000243 solution Substances 0.000 claims description 12
210000003425 amniotic epithelial cell Anatomy 0.000 claims description 11
208000015181 infectious disease Diseases 0.000 claims description 10
239000007788 liquid Substances 0.000 claims description 10
IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 9
239000006144 Dulbecco’s modiﬁed Eagle's medium Substances 0.000 claims description 9
239000000654 additive Substances 0.000 claims description 9
230000000996 additive effect Effects 0.000 claims description 9
239000006285 cell suspension Substances 0.000 claims description 9
238000005138 cryopreservation Methods 0.000 claims description 9
238000002156 mixing Methods 0.000 claims description 9
230000029087 digestion Effects 0.000 claims description 7
IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
208000005176 Hepatitis C Diseases 0.000 claims description 6
102000004877 Insulin Human genes 0.000 claims description 6
108090001061 Insulin Proteins 0.000 claims description 6
108010019160 Pancreatin Proteins 0.000 claims description 6
102000004338 Transferrin Human genes 0.000 claims description 6
108090000901 Transferrin Proteins 0.000 claims description 6
210000004369 blood Anatomy 0.000 claims description 6
239000008280 blood Substances 0.000 claims description 6
244000309466 calf Species 0.000 claims description 6
230000004907 flux Effects 0.000 claims description 6
239000011521 glass Substances 0.000 claims description 6
239000003550 marker Substances 0.000 claims description 6
239000000463 material Substances 0.000 claims description 6
210000000496 pancreas Anatomy 0.000 claims description 6
229940055695 pancreatin Drugs 0.000 claims description 6
238000004659 sterilization and disinfection Methods 0.000 claims description 6
208000011580 syndromic disease Diseases 0.000 claims description 6
230000001464 adherent effect Effects 0.000 claims description 5
238000002474 experimental method Methods 0.000 claims description 5
230000012010 growth Effects 0.000 claims description 5
208000006379 syphilis Diseases 0.000 claims description 5
230000010261 cell growth Effects 0.000 claims description 4
238000006243 chemical reaction Methods 0.000 claims description 4
239000012634 fragment Substances 0.000 claims description 4
230000008569 process Effects 0.000 claims description 4
102100022464 5'-nucleotidase Human genes 0.000 claims description 3
241000894006 Bacteria Species 0.000 claims description 3
102100037241 Endoglin Human genes 0.000 claims description 3
102000003974 Fibroblast growth factor 2 Human genes 0.000 claims description 3
108090000379 Fibroblast growth factor 2 Proteins 0.000 claims description 3
102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 claims description 3
101000678236 Homo sapiens 5'-nucleotidase Proteins 0.000 claims description 3
101000881679 Homo sapiens Endoglin Proteins 0.000 claims description 3
101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 claims description 3
101000935043 Homo sapiens Integrin beta-1 Proteins 0.000 claims description 3
101000998011 Homo sapiens Keratin, type I cytoskeletal 19 Proteins 0.000 claims description 3
101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 claims description 3
102000008100 Human Serum Albumin Human genes 0.000 claims description 3
108091006905 Human Serum Albumin Proteins 0.000 claims description 3
102100025304 Integrin beta-1 Human genes 0.000 claims description 3
102100033420 Keratin, type I cytoskeletal 19 Human genes 0.000 claims description 3
102000011782 Keratins Human genes 0.000 claims description 3
108010076876 Keratins Proteins 0.000 claims description 3
241000204031 Mycoplasma Species 0.000 claims description 3
229930182555 Penicillin Natural products 0.000 claims description 3
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 3
BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 claims description 3
QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 3
102100033523 Thy-1 membrane glycoprotein Human genes 0.000 claims description 3
230000008859 change Effects 0.000 claims description 3
238000011109 contamination Methods 0.000 claims description 3
BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 claims description 3
238000000605 extraction Methods 0.000 claims description 3
238000007667 floating Methods 0.000 claims description 3
238000013467 fragmentation Methods 0.000 claims description 3
238000006062 fragmentation reaction Methods 0.000 claims description 3
230000007774 longterm Effects 0.000 claims description 3
229910052757 nitrogen Inorganic materials 0.000 claims description 3
229940049954 penicillin Drugs 0.000 claims description 3
238000004321 preservation Methods 0.000 claims description 3
230000001681 protective effect Effects 0.000 claims description 3
238000012797 qualification Methods 0.000 claims description 3
229920006395 saturated elastomer Polymers 0.000 claims description 3
229910052711 selenium Inorganic materials 0.000 claims description 3
239000011669 selenium Substances 0.000 claims description 3
229940091258 selenium supplement Drugs 0.000 claims description 3
238000010583 slow cooling Methods 0.000 claims description 3
229960001471 sodium selenite Drugs 0.000 claims description 3
239000011781 sodium selenite Substances 0.000 claims description 3
235000015921 sodium selenite Nutrition 0.000 claims description 3
238000011146 sterile filtration Methods 0.000 claims description 3
230000001954 sterilising effect Effects 0.000 claims description 3
QTENRWWVYAAPBI-YCRXJPFRSA-N streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.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](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O.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](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O QTENRWWVYAAPBI-YCRXJPFRSA-N 0.000 claims description 3
239000000126 substance Substances 0.000 claims description 3
230000004083 survival effect Effects 0.000 claims description 3
238000012360 testing method Methods 0.000 claims description 3
VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 claims description 3
238000000684 flow cytometry Methods 0.000 claims description 2
239000003153 chemical reaction reagent Substances 0.000 abstract description 5
238000002360 preparation method Methods 0.000 abstract description 3
108091005804 Peptidases Proteins 0.000 abstract description 2
238000004113 cell culture Methods 0.000 abstract description 2
102000035195 Peptidases Human genes 0.000 abstract 1
239000003102 growth factor Substances 0.000 abstract 1
235000019833 protease Nutrition 0.000 abstract 1
238000005070 sampling Methods 0.000 abstract 1
230000009182 swimming Effects 0.000 abstract 1
239000010410 layer Substances 0.000 description 6
241000192700 Cyanobacteria Species 0.000 description 5
102000029816 Collagenase Human genes 0.000 description 3
108060005980 Collagenase Proteins 0.000 description 3
229960002424 collagenase Drugs 0.000 description 3
230000004069 differentiation Effects 0.000 description 3
108090000790 Enzymes Proteins 0.000 description 2
102000004190 Enzymes Human genes 0.000 description 2
210000003995 blood forming stem cell Anatomy 0.000 description 2
230000001413 cellular effect Effects 0.000 description 2
230000000694 effects Effects 0.000 description 2
210000001671 embryonic stem cell Anatomy 0.000 description 2
238000002955 isolation Methods 0.000 description 2
210000001161 mammalian embryo Anatomy 0.000 description 2
238000000926 separation method Methods 0.000 description 2
206010008190 Cerebrovascular accident Diseases 0.000 description 1
KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
229920001917 Ficoll Polymers 0.000 description 1
108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
239000004365 Protease Substances 0.000 description 1
102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
102000013275 Somatomedins Human genes 0.000 description 1
208000006011 Stroke Diseases 0.000 description 1
230000009286 beneficial effect Effects 0.000 description 1
230000008901 benefit Effects 0.000 description 1
210000004271 bone marrow stromal cell Anatomy 0.000 description 1
210000004556 brain Anatomy 0.000 description 1
239000006143 cell culture medium Substances 0.000 description 1
210000000170 cell membrane Anatomy 0.000 description 1
230000004663 cell proliferation Effects 0.000 description 1
238000005119 centrifugation Methods 0.000 description 1
239000003795 chemical substances by application Substances 0.000 description 1
210000001136 chorion Anatomy 0.000 description 1
210000004252 chorionic villi Anatomy 0.000 description 1
238000010276 construction Methods 0.000 description 1
210000003785 decidua Anatomy 0.000 description 1
230000007812 deficiency Effects 0.000 description 1
230000003412 degenerative effect Effects 0.000 description 1
238000011161 development Methods 0.000 description 1
201000010099 disease Diseases 0.000 description 1
208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
238000005516 engineering process Methods 0.000 description 1
210000002304 esc Anatomy 0.000 description 1
238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
210000003958 hematopoietic stem cell Anatomy 0.000 description 1
230000001900 immune effect Effects 0.000 description 1
230000000302 ischemic effect Effects 0.000 description 1
210000004698 lymphocyte Anatomy 0.000 description 1
210000003716 mesoderm Anatomy 0.000 description 1
201000001119 neuropathy Diseases 0.000 description 1
230000007823 neuropathy Effects 0.000 description 1
239000002417 nutraceutical Substances 0.000 description 1
235000021436 nutraceutical agent Nutrition 0.000 description 1
238000011275 oncology therapy Methods 0.000 description 1
208000033808 peripheral neuropathy Diseases 0.000 description 1
210000005059 placental tissue Anatomy 0.000 description 1
230000002062 proliferating effect Effects 0.000 description 1
239000002356 single layer Substances 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
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
- 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
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
- 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
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
- C12N2500/24—Iron; Fe chelators; Transferrin
- C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
- 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
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
- 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
- C12N2509/10—Mechanical dissociation

Landscapes

Health & Medical Sciences (AREA)
Life Sciences & Earth Sciences (AREA)
Engineering & Computer Science (AREA)
Biomedical Technology (AREA)
Developmental Biology & Embryology (AREA)
Organic Chemistry (AREA)
Chemical & Material Sciences (AREA)
Zoology (AREA)
Biotechnology (AREA)
Wood Science & Technology (AREA)
Bioinformatics & Cheminformatics (AREA)
Genetics & Genomics (AREA)
Microbiology (AREA)
Cell Biology (AREA)
Reproductive Health (AREA)
Biochemistry (AREA)
General Engineering & Computer Science (AREA)
General Health & Medical Sciences (AREA)
Gynecology & Obstetrics (AREA)
Pregnancy & Childbirth (AREA)
Hematology (AREA)
Rheumatology (AREA)
Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention relates to a method for simultaneously extracting epithelial cells and mesenchymal stem cells from umbilical cord and placenta amnion tissues. The method comprises the steps of (1) sampling umbilical cord and placenta amnia and inoculating; (2) performing amplifying and passage on mesenchymal stem cells and amnion epithelial cells in a culture flask; (3) identifying the mesenchymal stem cells and the amnion epithelial cells: 1) when culture cells is transferred to the third generation, identifying the purity of culture cells after trypsinization; 2) verifying the cell purity to be 90 percent or more, namely the standard is met, and a mixture of the epithelial cells and the mesenchymal stem cells is obtained. The method is low in cost, simple and rapid, umbilical cord and placenta amnia are cut into tiny pieces by using a tissue cutting machine, which is conducive to swimming out of the tissues after the cells are directly inoculated in the culture flask; the cost is greatly lowered due to the fact that no proteinase reagent is used, and the preparation time is reduced, so that large-scale cell preparation in short time is realized; the method uses cell culture mediums being added with growth factors and other nutrients, and serum is saved.

Description

A kind of method organizing extraction epithelial cell and mescenchymal stem cell simultaneously from umbilical cord and placenta amnion

Technical field

The invention belongs to technical field of cell culture, especially a kind of method organizing extraction epithelial cell and mescenchymal stem cell simultaneously from umbilical cord and placenta amnion.

Background technology

Umbilical cord and placenta belong to embryo's surrounding tissue, and umbilical cord tissue is the source of mescenchymal stem cell (mesenchymalstemcells, MSCs).Placenta is made up of placenta amnion, chorion and basal decidua.Placenta contains a large amount of multiple stem cells, placenta amnion epithelial cell (amnioticepithelialcells, AECs) there is the characteristic of multiple stem cell, wherein there are some Subaerial blue green algae, its characteristic is embryonic stem cell (embryonicstemcell, ESCs) closely.Placenta mesenchyma stem cell is then present under amniotic epithelial cells layer.Amnion originates from the epithelial lining of embryo, its occur early than interior, in, outer three germinal layers, therefore, the differentiation capability of the sub-totipotent cell of placenta is better than the stem cell originating from other three germinal layers.Many experiments prove that they are more easily induced to differentiate the various cells becoming body, and the potential therefore for clinical treatment is just larger, also just has larger market.Compared to umbilical cord, it is fewer that the work obtaining stem cell from placenta is also carried out, and therefore the meaning of this development project is just more great.Chorionic villi of placenta is also containing a large amount of mescenchymal stem cells and hemopoietic stem cell (Hematopoieticstemcells, HSCs); But because its volume is very large, be not easy to draw materials, so be the last selection utilizing placenta tissue.

Human mesenchymal stem cell (MSC) and epithelial stem cell (ESC) are implanted in treatment various diseases and comprise treatment degenerative neuropathy, newborn baby ischemic and Adult Human Brain apoplexy, and have in cancer therapy and study widely, and obtain clinical application in multiple field.

By retrieval, find following several sections of patent publication us relevant to present patent application:

1, Human plactnta Subaerial blue green algae and stem cell bank construction process (CN103966159A) thereof, provide a kind of Human plactnta Subaerial blue green algae, it derives from human placenta's amnion of stripping, for the cellular features contained by the epithelial lining of amnion and mesenchyme layer, have employed the method be progressively separated, decrease the pollution of amnion mesenchymal confluent monolayer cells to the sub-totipotent cell of placenta to greatest extent, effectively improve the dryness of the sub-totipotent cell of Human plactnta.This invention additionally provides a kind ofly to be prepared Human plactnta Subaerial blue green algae from placenta amnion and sets up the method for stem cell bank, this invention is after peeling off human placenta's amnion and be cut into 5 × 5cm tissue block, with mesenchyme layer, the method be progressively separated be have employed to the epithelial lining of amnion, first protein enzyme solution digestion is carried out, its protein enzyme solution used contains trypsinase, Collagenase II, and EDTA.Then use Ficoll lymphocyte separation medium gradient centrifugation further, gather cell between two-layer liquid level to reach the object of a kind of Human plactnta Subaerial blue green algae of separation and Culture.

The step of the separate tissue of foregoing invention method is many, and a large amount of a variety of reagent be used to comprise expensive collagenase II.

2, a kind of Isolation and ldentification method (CN104450612A) of human amnion mesenchymal stem cell, disclose a kind of Isolation and ldentification method of human amnion mesenchymal stem cell, tissue explants adherent method is adopted to isolate amnion mesenchymal stem cell and Secondary Culture, P1-P3 is carried out to the observation of cellular form for cell, six well plate method are adopted to detect cell proliferative conditions with the positive molecule of flow cytomery surface of cell membrane and negative molecule, it is hAMSCs that cell is isolated in this experiment from placenta amnion tissue, for studying biology and the immunological characteristic of hAMSCs further, differentiation capability, karyotypic stability, and it can be used as the seed cell etc. of clinical application to provide the foundation.This invention, after stripping human placenta amnion, is cut into very tiny fritter (about 1-2cm) with eye scissors, then gripping be laid in Tissue Culture Flask, make cell vacillate out to realize adherent growth and propagation from tissue block in cultivation.When Growth of Cells merges in blocks, wash away amnion tissue block by sterile phosphate buffered saline (PBS).

Use a large amount of reagent to comprise expensive collagenase although aforesaid method avoids, the tissue block of plantation is still larger, and in-house cell can not be allowed the most effectively to vacillate out.And manual to shear and lay also very consuming time, be used in placenta amnion draw materials fashion can, but being used in umbilical cord draws materials just unactual, because the amount of umbilical cord tissue is much bigger, and very hard.

Through technical comparison, present patent application and above-mentioned two sections of patent publication us exist larger different.

Summary of the invention

The object of the invention overcomes the deficiencies in the prior art part, provides a kind of low cost, organizes the method simultaneously extracting epithelial cell and mescenchymal stem cell quickly and easily from umbilical cord and placenta amnion.

To achieve these goals, the technical solution adopted in the present invention is as follows:

Organize the method simultaneously extracting epithelial cell and mescenchymal stem cell from umbilical cord and placenta amnion, step is as follows:

(1) umbilical cord and placenta amnion are drawn materials and are inoculated:

1. high-temperature sterilization instruments and large glass dish, simultaneously disinfection by ultraviolet light Bechtop;

2. substratum is prepared: the composition of perfect medium is: DMEM/F12 adds the mixing additive of the foetal calf serum of perfect medium cumulative volume 5%, the Regular Insulin of perfect medium cumulative volume 1%, siderophilin and selenium, in perfect medium, final concentration is that in the Urogastron of 20 ngs/ml and perfect medium, final concentration is the basic fibroblast growth factor of 20 ngs/ml, sterile filtration, 4 DEG C of preservations;

In described DMEM/F12, the volume ratio of DMEM:F12 is 1:1; Described mixing additive and the final concentration in mixing additive are Regular Insulin 5 mcg/ml, siderophilin 10 mcg/ml and Sodium Selenite 5 ngs/ml;

3. strictly screen donor, blood testing gets rid of transmissible disease;

4. umbilical cord and placenta is transported in low temperature ice box: after mechanically peel placenta amnion, umbilical cord and placenta amnion are placed in large glass dish respectively, following steps are all aseptically carried out, umbilical cord is cut into the segment that 1cm is long, placenta amnion is cut into the square that about 5X5cm is long, with the Streptomycin sulphate cleansing tissue twice of the penicillin+cumulative volume 1% of phosphate-buffered saline+cumulative volume 1%, the blood of wash clean umbilical cord;

5. umbilical cord and placenta amnion tissue are placed in the phosphate-buffered saline of 4 DEG C, cell survival is had influence on to prevent from being organized in overheated when machinery shreds, using-system cutting machine is broken into the fragment of 1-3mm respectively, use low speed disrupting tissue, keep cytoactive simultaneously, obtain broken umbilical cord and amnion tissue;

6. the umbilical cord of fragmentation and amnion tissue are transferred in T175 Tissue Culture Flask respectively, and with organizing scraper to be laid at the bottom of culturing bottle, carefully add a small amount of perfect medium, make nutrient solution can cover broken umbilical cord and amnion tissue block, be unlikely to again to make it floating, be placed in 37 DEG C, saturated humidity 5%CO ₂cultivate in incubator;

(2) mescenchymal stem cell and amniotic epithelial cells increase and go down to posterity in culturing bottle:

1. observe visible cell in culturing process to vacillate out gradually from tissue block, and realize adherent growth and propagation further, cultivate 3-4 days, when Growth of Cells merge in blocks time, wash away remaining tissue by sterile phosphate buffered saline, be all changed to fresh complete medium continue cultivate;

2. within after this every 4 days, change a nutrient solution until cell covers with 90-100% culturing bottle floorage, just carry out trysinization and go down to posterity;

3. trysinization is gone down to posterity: abandon nutrient solution, cell is washed 2 times with PBS, every T175 is that the pancreas enzyme-EDTA of 0.25% was 37 DEG C of digestion 10 minutes with 10 milliliters of mass percents, after cell suspension each T175 add 1 milliliter of foetal calf serum with in and pancreatin reaction, 1000 revs/min centrifugal 10 minutes, settling flux, in perfect medium, goes down to posterity in new T175 culturing bottle in 1:4 cell count ratio;

(3) the qualification of mescenchymal stem cell and amniotic epithelial cells:

1. when the third generation is passed in trysinization, the purity with flow cytometry culturing cell:

The marker of amniotic epithelial cells comprises: Keratin sulfate CK19, CD29, and CD34; The marker of placenta and umbilical cord mesenchymal stem cells comprises: CD73, CD90, and CD105;

2. flow cytometer checking cell purity reaches more than 90%, and common transmissible disease is got rid of in chemical examination, and gets rid of bacterium, mycoplasma contamination, is namely conformance with standard, obtains the mixture of epithelial cell and mescenchymal stem cell.

And, described step (1) 3. transmissible disease for comprising acquired immune deficiency syndrome (AIDS), first, second, hepatitis C and syphilis; Or, described step (3) 2. transmissible disease for comprising acquired immune deficiency syndrome (AIDS), first, second, hepatitis C and syphilis.

And, described step (1) 5. in low speed be 400-800 rev/min.

And, the freezen protective of the mixture of described epithelial cell and mescenchymal stem cell and method for resuscitation, step is as follows:

(1) get the mixture of epithelial cell and mescenchymal stem cell, wash twice to wash FBS off with PBS, 0.25% pancreas enzyme-EDTA digestion, every T175 with 10 milliliters, 37 DEG C, 10 minutes, after cell suspension each T175 add 1 milliliter of FBS with in and pancreatin react.Centrifugal 10 minutes at 1000 revs/min;

(2) press cell density (1-3) X10 again ⁷/ ml is suspended in cells frozen storing liquid, is dispensed in 2ml cryopreservation tube, uses cell cryopreservation box frozen at-80 DEG C of refrigerator slow coolings, transfers to that liquid nitrogen container is medium-term and long-term to be saved backup after one day;

Described cells frozen storing liquid composition: the DMSO of total mass 20%, the human serum albumin of total mass 20%, the 60%M199 substratum of total mass;

(3), before using cell, thawed rapidly in 37 DEG C of water-baths by cell in cryopreservation tube, centrifugal 10 minutes at 1000 revs/min, settling flux is in PBS, and being prepared into cell density is 1x10 ⁸the cell suspension of/ml, to be implanted in interior experiment and clinical application as comprising, is kept at 4 DEG C before use always.

The advantage that the present invention obtains and positively effect are:

Present method cost is low, simple and quick, umbilical cord and placenta amnion are shredded into minimum fragment (being less than 2-3mm) by using-system cutting machine, much smaller than method any at present, be very beneficial to cell after being directly inoculated into culturing bottle and vacillate outside tissue, thus realize the growth of note wall; Do not use any protease reagent, thus reduce costs significantly, reduce preparation time, increase yield, prepared by mass cell become a reality; The method uses adds somatomedin and other nutraceutical complete cell culture medium, thus reduce foetal calf serum (FBS) concentration to 5%, but obtain the culture effect using 10%FBS at the same level and better with routine, save serum, but unlike most of serum-free culture, cause cell proliferation to slow down or differentiation ahead of time.

Embodiment

Below by specific embodiment, the invention will be further described, and following examples are descriptive, is not determinate, can not limit protection scope of the present invention with this.

The reagent used in the present invention, if no special requirements, is the common agents in this area; The method used in the present invention, if no special requirements, is the ordinary method in this area.

Organize the method simultaneously extracting epithelial cell and mescenchymal stem cell from umbilical cord and placenta amnion, step is as follows:

(1) umbilical cord and placenta amnion are drawn materials and are inoculated:

1. high-temperature sterilization instruments and large glass dish, simultaneously disinfection by ultraviolet light Bechtop;

3. strictly screen donor, blood testing is got rid of and is comprised acquired immune deficiency syndrome (AIDS), first, second, hepatitis C, the transmissible diseases such as syphilis;

4. umbilical cord and placenta is transported in low temperature ice box: after mechanically peel placenta amnion, umbilical cord and placenta amnion are placed in large glass dish respectively, following steps are all aseptically carried out, by the segment of umbilical cord scissors into about 1cm length, placenta amnion is cut into the square that about 5X5cm is long, with the Streptomycin sulphate cleansing tissue twice of the penicillin+cumulative volume 1% of phosphate-buffered saline+cumulative volume 1%, the blood of wash clean umbilical cord as far as possible;

5. umbilical cord and placenta amnion tissue are placed in the PBS of 4 DEG C, cell survival is had influence on to prevent from being organized in overheated when machinery shreds, using-system cutting machine is broken into minimum piece (fragment of about 1-3mm) respectively, use low speed (such as, 400-800 rev/min) disrupting tissue, keep cytoactive simultaneously, obtain broken umbilical cord and amnion tissue;

(2) mescenchymal stem cell and amniotic epithelial cells increase and go down to posterity in culturing bottle:

1. observe visible cell in culturing process to vacillate out gradually from tissue block, and realize adherent growth and propagation further, cultivate 3-4 days, when Growth of Cells merge in blocks time, wash away remaining tissue with aseptic PBS, be all changed to fresh complete medium continue cultivate;

2. within after this every 4 days, change a nutrient solution until cell covers with 90-100% culturing bottle floorage, just carry out trysinization and go down to posterity;

3. trysinization is gone down to posterity: abandon nutrient solution, washes cell 2 times with PBS, and 0.25% pancreas enzyme-EDTA (every T175 is with 10 milliliters) was 37 DEG C of digestion 10 minutes.After cell suspension each T175 add 1 milliliter of FBS with in and pancreatin reaction, 1000 revs/min are centrifugal 10 minutes, and settling flux, in perfect medium, goes down to posterity in new T175 culturing bottle in 1:4 cell count ratio;

(3) the qualification of mescenchymal stem cell and amniotic epithelial cells:

1., when the third generation is passed in trysinization, the purity of culturing cell is identified with flow cytometer (FACS):

The marker of amniotic epithelial cells comprises: Keratin sulfate CK19, CD29, and CD34; The marker of placenta and umbilical cord mesenchymal stem cells comprises: CD73, CD90, and CD105;

2. flow cytometer checking cell purity reaches more than 90%, and chemical examination eliminating comprises acquired immune deficiency syndrome (AIDS), first, second, the transmissible disease that hepatitis C etc. are common, and gets rid of bacterium, mycoplasma contamination, is namely conformance with standard, obtains the mixture of epithelial cell and mescenchymal stem cell.

Umbilical cord cells cultivates the mescenchymal stem cell of generation more than 90%, and amnion cell cultivates generation mescenchymal stem cell and epithelial cell (wherein containing the sub-totipotent cell of part), because these two kinds of cells are play synergy in most of clinical application, and be combined utilization, so there is no need to be separated, as long as it is conformance with standard that the summation of these two kinds of cells accounts for more than 90% of total cell count, what net result obtained is purer mescenchymal stem cell and mescenchymal stem cell and epithelial cell mixed culture, can according to clinical needs choice for use.

The freezen protective of the mixture of above-mentioned epithelial cell and mescenchymal stem cell and method for resuscitation, step is as follows:

(1) get the mixed culture of epithelial cell and mescenchymal stem cell, twice is washed to wash FBS off, 0.25% pancreas enzyme-EDTA digestion (every T175 is with 10 milliliters), 37 DEG C with PBS, 10 minutes, after cell suspension each T175 add 1 milliliter of FBS with in and pancreatin reaction.1000 revs/min centrifugal 10 minutes;

Described cells frozen storing liquid composition: the DMSO of total mass 20%, the human serum albumin of total mass 20%, the 60%M199 substratum of total mass;

Claims (4)

1. organize the method simultaneously extracting epithelial cell and mescenchymal stem cell from umbilical cord and placenta amnion, it is characterized in that: step is as follows: