CN104450612A - Method for separating and identifying human amniotic mesenchymal stem cells - Google Patents
Method for separating and identifying human amniotic mesenchymal stem cells Download PDFInfo
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- CN104450612A CN104450612A CN201410741509.1A CN201410741509A CN104450612A CN 104450612 A CN104450612 A CN 104450612A CN 201410741509 A CN201410741509 A CN 201410741509A CN 104450612 A CN104450612 A CN 104450612A
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Abstract
The invention discloses a method for separating and identifying human amniotic mesenchymal stem cells. The method comprises the following steps: separating amniotic mesenchymal stem cells by adopting a tissue explants adherent method, and carrying out subculture; observing the cell form of cells from P1-P3 generations; detecting positive molecules and negative molecules on the surfaces of cell membranes by virtue of a flow cytometer and detecting the cell proliferation situation by adopting a six-pore plate method. According to the method, the cells separated from amniotic membrane tissues are hAMSCs, so that basis is provided to further studies on the hAMSCs in biological and immunological properties, differentiative capacity and karyotype stability and in serving as seed cells of clinical application and the like.
Description
Technical field
The present invention relates to a kind of Isolation and ldentification method of human amnion mesenchymal stem cell, belong to medical detection technology.
Background technology
Placenta combines by the embryophoric membrane of embryo and maternal uterine inner membrance the transitional organ exchanging material between the mothers and sons that grow up to, except containing except nurse cell, also containing a large amount of blood vessel and mesenchyme composition, placenta mesenchyma stem cell be in recent years newfound from placenta obtain a class stem cell, compared with the stem cell of extracting from Cord blood or marrow, not only stem cell population is many but also can be divided into the histocyte of multiple types, and placenta tissue derives from postpartum, it is collected process and is not caused any damage to parent and newborn infant, not by ethics and clinical medical restriction, in addition, researchist finds from Cell surface antigen analysis, placenta mesenchyma stem cell is the stem cell between embryonic stem cell and adult stem cell, due to its reduced immunogenicity, not easily repelled by the immunity system of acceptor, that desirable stem cell preserves and the structure of resource placenta that utilizes mainly haves three layers (amnion) chorion and decidua, blood vessel and red corpuscle are then rich in chorion and decidua, not easily separated, amnion is that one deck is easy to be separated, and structure simply organizes % if isolate mescenchymal stem cell from amnion, to be a good donor of mescenchymal stem cell, for investigator provides a simple pathway obtaining mescenchymal stem cell.
Summary of the invention
The object of the invention is: a kind of Isolation and ldentification method that human amnion mesenchymal stem cell is provided, human amnion mesenchymal stem cell can be gone out, to overcome the deficiencies in the prior art from from external successful separation and Culture.
Technical scheme of the present invention
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, adopt six well plate method to detect cell proliferative conditions with the positive molecule of flow cytomery surface of cell membrane and negative molecule.
Owing to adopting technique scheme; compared with prior art; the present invention by amnion tissue block is attached to culturing bottle carries out cultivation 7-10d after go out cell from tissue block surrounding growth; form of diverse; but based on spindle shape, after going down to posterity, growth rapidly, grows in flowing water shape; 3-6d reaches summit of growth, and this is consistent with the growth characteristics of bone marrow MSCs; Be passaged to for the 3rd generation, by flow cytometer detection cell membrane surface molecules, result display (cell high expression level CD44, CD105, CD73HE CD90, its phenotypic characteristic is similar to bone marrow MSCs; Illustrating that cell is isolated in this experiment from placenta amnion tissue is hAMSCs, for studying biology and immunological characteristic, differentiation capability, the karyotypic stability of hAMSCs further, and it can be used as the seed cell etc. of clinical application to provide the foundation.
Embodiment
The present invention is described in further detail below, but not as any limitation of the invention.
Embodiments of the invention:
1 materials and methods
1.1 Specimen origin
After placenta takes from health full term newborn infant Cesarean operation, contribute gained through family members' informed consent after signing " placenta processing protocol book ".
1.2 main raws and instrument
1.2.1 main raw
Permeability type Tissue Culture Flask (NEST), aseptic calibrated pipet, six orifice plates, centrifuge tube, bus obtain suction pipe, eye scissors, ophthalmic tweezers, operating scissors, penicillin bottle etc.
1.2.2 main agents
Cell culture fluid is that L-DMEM adds 10%FBS, 0.25% trypsinase, 5.5%NaHCO
3, green grass or young crops-chain enzyme mixed solution etc.
1.2.3 key instrument equipment
Bechtop (Suzhou purification) CO2 cell culture incubator, flow cytometer, inverted microscope, cryogenic freezing whizzer, just put microscope etc.
1.3 method
1.3.1 hAMSCs separation and Culture
Under aseptic condition, the placenta near umbilical cord place is cut, careful denuded amniotic membrane, and with stroke-physiological saline solution (NS) cleaning for several times until liquid is limpid, transfer them in penicillin bottle and be cut into about 1-2cm size with eye scissors, be laid in 25cm2 Tissue Culture Flask with ophthalmic tweezers gripping, carefully add nutrient solution along culturing bottle wall, make nutrient solution can cover amnion tissue block, be unlikely to again to make it floating, finally culturing bottle be placed in 37 degree of 5%CO2 incubators and cultivate.
1.3.2 hAMSCs Secondary Culture
After seeing and having Growth of Cells around tissue block, every 2d changes liquid 1 time, and when cell grows in the form of sheets, available aseptic PSB washes away amnion tissue block; After this, the next day change liquid 1 time, can go down to posterity when cell grows up to individual layer; First wash with aseptic PSB liquid when going down to posterity; After time, to remove residual nutrient solution, then use 0.25% trypsin solution (about 1.5ml) to digest about 3min in 37 degree, nutrient solution stops digestion, carefully blowing and beating cell with bus moral suction pipe makes it come off from culturing bottle, be transferred in 15min centrifuge tube, the centrifugal 3min of 800r/min, abandons supernatant, precipitation nutrient solution is resuspended, in evenly distribute to 2 culturing bottle, be placed in 37 degree of 5%CO2 incubators and cultivate, next day changes liquid.
1.3.3 hAMSCs phenotypic evaluation
The cell being passaged to P3 generation will be separated, according to flow cytometer detection test kit operation instruction and step operation, use flow cytomery cell membrane surface molecules, the positive molecule of detection comprises CD73-AP, CD90-FITC, CD105-Cy5.5 and negative molecule comprises CD11b-PE, CD19-PE, CD34-PE, CD45-PE, HLA-DR-PE.
The experiment 1.3.4 hAMSCs rises in value
The cell adopting six well plate method to detect cell proliferative conditions P3 generation is inoculated in six orifice plates, every porocyte quantity 1*10
3, be placed in 37 degree of 5%CO2 incubators and cultivate, the next day change liquid 1 time, grow up to until cell and get every day after individual layer, porocyte carries out tire and expects blue dyeing, with cell counting count board living cell counting number, and draws growth curve.
The present invention by amnion tissue block is attached to culturing bottle carries out cultivation 7-10d after go out cell from tissue block surrounding growth, form of diverse, but based on spindle shape; after going down to posterity, growth rapidly; grow in flowing water shape, 3-6d reaches summit of growth, and this is consistent with the growth characteristics of bone marrow MSCs; Be passaged to for the 3rd generation, by flow cytometer detection cell membrane surface molecules, result display (cell high expression level CD44, CD105, CD73HE CD90, its phenotypic characteristic is similar to bone marrow MSCs; Illustrating that cell is isolated in this experiment from placenta amnion tissue is hAMSCs, for studying biology and immunological characteristic, differentiation capability, the karyotypic stability of hAMSCs further, and it can be used as the seed cell etc. of clinical application to provide the foundation.
Claims (1)
1. the Isolation and ldentification method of a human amnion mesenchymal stem cell, it is characterized in that: adopt tissue explants adherent method to isolate amnion mesenchymal stem cell and Secondary Culture, P1-P3 is carried out to the observation of cellular form for cell, adopt six well plate method to detect cell proliferative conditions with the positive molecule of flow cytomery surface of cell membrane and negative molecule.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201410741509.1A CN104450612A (en) | 2014-12-08 | 2014-12-08 | Method for separating and identifying human amniotic mesenchymal stem cells |
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| CN201410741509.1A CN104450612A (en) | 2014-12-08 | 2014-12-08 | Method for separating and identifying human amniotic mesenchymal stem cells |
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| CN104450612A true CN104450612A (en) | 2015-03-25 |
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| CN201410741509.1A Pending CN104450612A (en) | 2014-12-08 | 2014-12-08 | Method for separating and identifying human amniotic mesenchymal stem cells |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109652362A (en) * | 2017-10-12 | 2019-04-19 | 北京弘润天源基因生物技术有限公司 | A kind of method that umbilical cord film saves and prepares stem cell |
-
2014
- 2014-12-08 CN CN201410741509.1A patent/CN104450612A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106754674B (en) * | 2016-12-09 | 2019-09-20 | 博雅干细胞科技有限公司 | The method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion |
| CN109652362A (en) * | 2017-10-12 | 2019-04-19 | 北京弘润天源基因生物技术有限公司 | A kind of method that umbilical cord film saves and prepares stem cell |
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Application publication date: 20150325 |
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