CN102321575B - Separation and culture method of human umbilical mesenchymal cell - Google Patents
Separation and culture method of human umbilical mesenchymal cell Download PDFInfo
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- CN102321575B CN102321575B CN2011103009057A CN201110300905A CN102321575B CN 102321575 B CN102321575 B CN 102321575B CN 2011103009057 A CN2011103009057 A CN 2011103009057A CN 201110300905 A CN201110300905 A CN 201110300905A CN 102321575 B CN102321575 B CN 102321575B
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Abstract
The invention discloses a separation and culture method of human umbilical mesenchymal cells; the method adopts collagenase IV and pancreatin to digest and treat human umbilical cord living tissue, and the separated human umbilical mesenchymal cells are inoculated in a culture dish pre-coated by gelatin for amplification. The separation and culture method of the invention has the advantages of simple operations and easy popularization, and can conveniently obtain human umbilical mesenchymal cells with low immunogenicity.
Description
Technical field
The present invention relates to a kind of separation and cultural method of human umbilical cord mesenchymal cell.
Background technology
Limit because of embryonic stem cell receives ethics, Japanese scholar Shinya Yamanaka was the pluripotency differentiated stem cells with the mouse fibroblast cell reprogrammed in 2006, and this cell has the triploblastica of being divided into cell ability in theory.Japanese scholar Shinya Yamanaka in 2007 and American scholar T hompson are the pluripotency differentiated stem cells with HSF's reprogrammed; The whole world starts pluripotency differentiated stem cells reprogrammed upsurge subsequently; But reprogrammed cell source receives puzzlements such as difference between individuals, immunoreation, animal derived materials pollution at present; Therefore; Need seek a kind of easy acquisition, not have the reprogrammed cell source of problems such as ethics restriction, immunoreation and animal derived pollution, clinical to guarantee that the pluripotency differentiated stem cells can be applied to safely, at large.
Summary of the invention
The object of the present invention is to provide a kind of separation and cultural method of human umbilical cord mesenchymal cell; It has simple to operate, be easy to advantages such as popularization; Can obtain the human umbilical cord mesenchymal cell of low immunogenicity easily; It is as reprogrammed cell source, and reprogrammable is the pluripotency differentiated stem cells, supplies basis and clinical study to use.
The objective of the invention is to realize through following technical scheme: a kind of separation method of human umbilical cord mesenchymal cell may further comprise the steps:
1) gets people's umbilical cord living tissue, clean, chopping;
2) add collagenase IV, place CO
2Carry out digestion process in the incubator, add the DMEM/F12 substratum then and stop digestion, centrifugal, abandon supernatant;
3) add pancreatin, place CO
2Carry out digestion process in the incubator, add the DMEM/F12 substratum then and stop digestion;
4) filter, getting filtrating and carrying out centrifugally, abandon supernatant after centrifugal, add DMEM/F12 substratum re-suspended cell then.
Further, in described step 2) in, the concentration of collagenase IV is 1mg/ml～3mg/ml, the time of digestion process is 3.5～4.5 hours.
Collagenase IV can digest cell down from tissue; Have advantages such as action temperature and the damage of, pair cell be less, experiment shows, adopting concentration is the collagenase IV digestion 3.5～4.5 hours of 1mg/ml～3mg/ml; The individual cells amount that obtains is maximum, and cell viability is best; If reduce the concentration of collagenase IV, the time of prolongation digestion process, perhaps improve the concentration of collagenase IV, the time of shortening digestion process, all be unfavorable for the maximization of individual cells quantity and cell viability.
Further, in described step 3), the concentration of pancreatin is 0.05%～0.50%, and the time of digestion process is 10～20 minutes.
Further, also being added with concentration in the described pancreatin is 0.02% EDTA.
Separating unite the often growth of the cell obtain through collagenase IV, is not unicellular growth.And pancreatin is the complex body of plurality of enzymes, and it can be an individual cells with tissue differentiation, for the individual cells adherent growth.In pancreatin, adding EDTA is for chelating calcium ions and magnesium ions (because calcium ions and magnesium ions can suppress the active of pancreatin and destroy intercellular connecting structure); Further improve the activity of pancreatin; Make cell can be dispersed into individual cells, thereby obtain the umbilical cord mesenchyma cell of quantity and the maximized single adherent growth of vigor.
Further, also be added with 10%FBS, 1% non-essential amino acid, 1%L-glutaminate, 1% green grass or young crops-Streptomycin sulphate and 1mM 2 mercapto ethanol in the described DMEM/F12 substratum.
Further, said step 2) and the centrifugal condition in the step 4) be: 1500rpm～3000rpm, 5～10 minutes.Adopt this centrifugal condition, it is dead in centrifugal process to reduce primary cell to greatest extent.
Further, in described step 2) and step 3) in, CO
2The culture condition of incubator is: 37 ℃, 5%CO
2
Further, in described step 4), the employing diameter is that woollen goods filter screen of 40 μ m filters.
Woollen goods filter screen has slides advantages such as glutinous, soft, that drape is good, the pair cell damage is little; And the diameter of the filter opening diameter of 40 μ m and umbilical cord mesenchyma cell is suitable; Can only let single umbilical cord mesenchyma cell pass through; Other is not then can not passing through of individual cells by digestion, thereby has improved the purity of cell.
A kind of cultural method of human umbilical cord mesenchymal cell may further comprise the steps:
A. adopt gelatin that petridish is encapsulated processing, then with no Ca
2+And Mg
2+The PBS damping fluid this petridish is washed;
B. get the cell that obtains according to separation method according to the invention, be inoculated in the petridish after step a handles;
C. treat cell confluent growth to 85%～90% o'clock to carry out had digestive transfer culture.
Further, in described step a, adopting concentration is that 0.01%～0.02% gelatin encapsulated 20～30 minutes petridish.
Further, in described step b, cell is with 0.5 * 10
5～1.0 * 10
5Concentration be inoculated in the petridish after step a handles.
The present invention adopts enzyme digestion to obtain the human umbilical cord mesenchymal cell; That this method has is simple to operate, easy master, be convenient to advantages such as popularization; And the human umbilical cord mesenchymal cellular immunization source property that obtains is low; Non-animal derived sexual cell pollutes, and does not have the danger of propagating the xenogenesis pathogenic agent, for human umbilical cord mesenchymal cell reprogrammed in the future is that place mat is carried out in the application of pluripotency differentiated stem cells.
Embodiment
The preparation of DMEM/F12 substratum: in the DMEM/F12 basic medium, add 10%FBS, 1% non-essential amino acid, 1%L-glutaminate, 1% green grass or young crops-Streptomycin sulphate and 1mM 2 mercapto ethanol.
Get 1～3 centimetre of broken palace and produce nearly fetus end people umbilical cord living tissue, adopt no Ca
2+And Mg
2+The PBS damping fluid people's umbilical cord living tissue is cleaned, and with people's umbilical cord living tissue chopping size to 0.5～1 millimeter.
The human umbilical tissue piece that shreds is tiled to the petridish of 10cm, and adding 10ml concentration is the collagenase IV of 2mg/ml, places 37 ℃, 5%CO
2In the incubator 4 hours, add the DMEM/F12 substratum then and stop digestion, under 3000rpm centrifugal 8 minutes, inhale and abandon supernatant.
Adding 8ml concentration is 0.25% pancreatin (containing 0.02% EDTA), places 37 ℃, 5%CO
2In the incubator 15 minutes, add the DMEM/F12 substratum then and stop digestion, and to use the aperture be that woollen goods filter screen of 40 μ m filters, get filtrating under 1500rpm centrifugal 8 minutes, inhale and abandon supernatant, add the DMEM/F12 substratum, re-suspended cell.
Get the petridish of 10cm, using concentration in advance is that 0.01% gelatin encapsulated 30 minutes, then with no Ca
2+And Mg
2+PBS damping fluid washing 2 times.
With 1 * 10
5Concentration, with the cell suspension inoculation that obtains in this petridish.Change liquid weekly 2 times,, carry out had digestive transfer culture until cell confluent growth to 85%.
Claims (11)
1. the separation method of a human umbilical cord mesenchymal cell may further comprise the steps:
1) gets people's umbilical cord living tissue, clean, chopping;
2) add the collagenase IV, place CO
2Carry out digestion process in the incubator, add the DMEM/F12 substratum then and stop digestion, centrifugal, abandon supernatant;
3) add pancreatin, place CO
2Carry out digestion process in the incubator, add the DMEM/F12 substratum then and stop digestion;
4) filter, getting filtrating and carrying out centrifugally, abandon supernatant after centrifugal, add DMEM/F12 substratum re-suspended cell then.
2. separation method according to claim 1 is characterized in that: in described step 2) in, the concentration of collagenase IV is 1mg/ml～3mg/ml, the time of digestion process is 3.5～4.5 hours.
3. separation method according to claim 1 is characterized in that: in described step 3), the concentration of pancreatin is 0.05%～0.50%, and the time of digestion process is 10～20 minutes.
4. separation method according to claim 3 is characterized in that: also be added with concentration in the described pancreatin and be 0.02% EDTA.
5. separation method according to claim 1 is characterized in that: also be added with 10%FBS, 1% non-essential amino acid, 1%L-glutaminate, 1% green grass or young crops-Streptomycin sulphate and 1mM 2 mercapto ethanol in the described DMEM/F12 substratum.
6. separation method according to claim 1 is characterized in that: described step 2), the centrifugal condition in the step 4) is 1500rpm～3000rpm, and 5～10 minutes.
7. separation method according to claim 1 is characterized in that: in described step 2) and step 3) in, CO
2The culture condition of incubator is 37 ℃, 5%CO
2
8. separation method according to claim 1 is characterized in that: in described step 4), the employing diameter is that woollen goods filter screen of 40 μ m filters.
9. the cultural method of a human umbilical cord mesenchymal cell may further comprise the steps:
A. adopt gelatin that petridish is encapsulated processing, then with no Ca
2+And Mg
2+The PBS damping fluid this petridish is washed;
B. adopt the described separation method of claim 1, from people's umbilical cord living tissue, separate and obtain the human umbilical cord mesenchymal cell, and be inoculated in the petridish after step a handles;
C. treat cell confluent growth to 85%～90% o'clock to carry out had digestive transfer culture.
10. cultural method according to claim 9 is characterized in that: in described step a, adopting concentration is that 0.01%～0.02% gelatin encapsulated 20～30 minutes petridish.
11. cultural method according to claim 9 is characterized in that: in described step b, cell is with 0.5 * 10
5～1.0 * 10
5Concentration be inoculated in the petridish after step a handles.
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| CN2011103009057A CN102321575B (en) | 2011-10-08 | 2011-10-08 | Separation and culture method of human umbilical mesenchymal cell |
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| CN102321575B true CN102321575B (en) | 2012-11-21 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103045537A (en) * | 2012-12-11 | 2013-04-17 | 新疆医科大学第一附属医院 | Method for obtaining mesenchymal cell of umbilical cord |
| CN103602635B (en) * | 2013-11-19 | 2016-03-16 | 北京赛贝生物技术有限公司 | The cultural method of reprogrammed substratum, its preparation method and reprogrammed cell |
| CN105420181A (en) * | 2015-12-09 | 2016-03-23 | 南方医科大学南方医院 | Method for separating in-vitro mesenchymal cells |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1410531A (en) * | 2002-11-20 | 2003-04-16 | 上海市第一人民医院 | Culturing method of human peripheral blood vessel endothelial ancestry cell |
| CN101298606A (en) * | 2008-02-14 | 2008-11-05 | 天津环宇商桥商务信息咨询有限公司 | Preparation, storage and use of umbilical cord and placenta mesenchymal stem cell for clinical therapy |
| CN101492654A (en) * | 2008-03-17 | 2009-07-29 | 协和干细胞基因工程有限公司 | Method for using umbilical stalk placenta to prepare mesenchyma stem cell |
| CN102127522A (en) * | 2010-12-27 | 2011-07-20 | 协和干细胞基因工程有限公司 | Human umbilical mesenchymal stem cell and preparation method thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1410531A (en) * | 2002-11-20 | 2003-04-16 | 上海市第一人民医院 | Culturing method of human peripheral blood vessel endothelial ancestry cell |
| CN101298606A (en) * | 2008-02-14 | 2008-11-05 | 天津环宇商桥商务信息咨询有限公司 | Preparation, storage and use of umbilical cord and placenta mesenchymal stem cell for clinical therapy |
| CN101492654A (en) * | 2008-03-17 | 2009-07-29 | 协和干细胞基因工程有限公司 | Method for using umbilical stalk placenta to prepare mesenchyma stem cell |
| CN102127522A (en) * | 2010-12-27 | 2011-07-20 | 协和干细胞基因工程有限公司 | Human umbilical mesenchymal stem cell and preparation method thereof |
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