CN103361306A - Isolation and culture of multipotent stem cells in human and mammalian tissues - Google Patents
Isolation and culture of multipotent stem cells in human and mammalian tissues Download PDFInfo
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- CN103361306A CN103361306A CN2013101691144A CN201310169114A CN103361306A CN 103361306 A CN103361306 A CN 103361306A CN 2013101691144 A CN2013101691144 A CN 2013101691144A CN 201310169114 A CN201310169114 A CN 201310169114A CN 103361306 A CN103361306 A CN 103361306A
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Abstract
The invention relates to a method for isolation and culture of multipotent stem cells from human and mammalian tissues. A trypsin stem cell culture solution with low concentration is adopted for culturing and screening development totipotent potent stem cells in the tissues, wherein the stem cell culture solution removing serum can ensure that the multipotent stem cells are not differentiated during isolation and culture; and the low-concentration trypsin has an effect on screening the multipotent stem cells in the tissues. By using the method, marker genes Oct4 and SSEA expressing embryonic stem cells can be isolated and cultured from the human and the mammalian tissues, and the in-vitro suspension culture of the multipotent stem cells which can form embryoid bodies is further realized.
Description
Affiliated technical field
The present invention relates to a kind of from people and mammalian tissues organ separation and Culture pluripotent stem cell method.
Background technology
At present, known have immunomagnetic beads method, two phase culture methods from the people with the method that Mammals is subjected to separate pluripotent stem cell the second trimester amniotic fluid.And these two kinds of methods all can not be from people and mammalian tissues organ the separation and Culture pluripotent stem cell.The invention provides a kind of pluripotent stem cell isolation cultivation method, the method adopts lower concentration pancreatin nutrient solution cultivator and mammalian tissues primary cell, thus pluripotent stem cell in the separation and Culture tissue.
Summary of the invention
For separation and Culture pluripotent stem cell from people and mammalian tissues, the invention provides a kind of method, the method can be from people and mammalian tissues separation and Culture have the stem cell of totipotency potential.
The technical scheme of the present invention's separation and Culture pluripotent stem cell from people and mammalian tissues is: add lower concentration trypsinase in the stem cell nutrient solution of serum-free.Can obtain pluripotent stem cell with this nutrient solution cultivator and mammalian tissues primary cell.Wherein, the stem cell nutrient solution of serum-free can guarantee to histogen for cell cultivation process in pluripotent stem cell do not break up, lower concentration trypsinase to the tissue in pluripotent stem cell play a kind of screening effect.
The invention has the beneficial effects as follows, can be from people and mammalian tissues the separation and Culture pluripotent stem cell.The pluripotent stem cell that exists in people and the mammalian tissues is the desirable cell of research people and mammiferous numerous disease such as hereditary defect, tumour and exogenous virus infection mechanism.
Description of drawings
The present invention is further described below in conjunction with accompanying drawing and example
Fig. 1 is sheep fetal kidney tissue pluripotent stem cell;
Fig. 2 is the real-time quantitative amplification curve that sheep fetal kidney tissue pluripotent stem cell is expressed Oct4, SSEA-1, and wherein, 2.1 is reference gene glyceraldehyde 3-phosphate dehydro-genase gene (GAPDH), and 2.2 is SSEA-1, and 2.3 is Oct4;
Fig. 3 is that sheep fetal kidney tissue pluripotent stem cell In vitro Suspension is cultivated the embryoid that formed afterwards in 7 days.
Embodiment
The concrete steps that sheep fetal kidney tissue pluripotent stem cell obtains among Fig. 1 are:
1. dosing
Nutrient solution I:DMEM+1%L-glutamine+1% non-essential amino acid+1% Sodium.alpha.-ketopropionate+micro-beta-mercaptoethanol+5ng or 500u/ml LIF+5ng/ml bFGF are with the filter filtration of 0.22 μ l, 4 ℃ of preservations; Nutrient solution II: nutrient solution I+15%FBS; Nutrient solution III: nutrient solution I+0.01% trypsinase, with the filter filtration of 0.22 μ l, 4 ℃ of preservations.
2. sheep fetal kidney tissue pluripotent stem cell separation and Culture
Under the aseptic condition, adopt and organize mechanical separation method to separate, cultivate sheep fetal kidney tissue primary cell, nutrient solution II37 ℃ 5%CO
2Cultivated 48 hours under the saturated humidity condition; After cell clone occurs, wash 3 times with D-PBS, then add nutrient solution III, 37 ℃ of 5%CO
2Cultivated 18 hours under the saturated humidity condition; 1500r/min * 5min centrifugal collecting cell with nutrient solution II suspension cell, blows all, with 10
5Cell/ware is inoculated in the 6cm culture dish, occurs cell clone (Fig. 1) after 72 hours; Biography is frozen for subsequent use after 3～4 generations.
The concrete steps of Fig. 2 are
Extract sheep fetal kidney tissue totalRNA, carry out genomic dna before the reverse transcription and remove reaction, then its cDNA is synthesized in reverse transcription, confidential reference items are glyceraldehyde 3-phosphate dehydro-genase (GAPDH), primer information sees Table 1, Real Time PCR carries out 3 repetitions of each sample (Fig. 2) according to TaKaRa SYBR PremixEX TaqTM IIkit explanation.
Genomic dna is removed reaction:
Two-step approach real time PCR standard program:
Table 1Real Time PCR primer sequence
The concrete steps of Fig. 3 are
It is good to get growth conditions, form good the 5th generation sheep fetal kidney tissue pluripotent stem cell, when treating that Growth of Cells reaches 80% degree of converging, under the aseptic condition, D-PBS washes 3 times, trysinization suspension cell, 1500r/min * 5min centrifugal collecting cell, embryoid nutrient solution (DMEM+15%FBS) re-suspended cell, adjusting cell concn is 1 * 10
6/ ml; Add 15ml D-PBS in the 6cm culture dish, also drip at the ware lid inboard take 20ul as a droplet; The reversing of ware lid is covered on ware, in 37 ℃ of 5%CO
2Saturated humidity condition low suspension is cultivated; Behind the 7d, aseptic technique is collected droplet in the 6cm culture dish, to microscopically observe, (Fig. 3) takes pictures.
Claims (4)
1. the method for a separation and Culture pluripotent stem cell from people and mammalian tissues, it is characterized in that: people and mammalian tissues pluripotent stem cell with lower concentration pancreatin nutrient solution separation and Culture have totipotency potential.
According to claim 1 from people and mammalian tissues the method for separation and Culture pluripotent stem cell, it is characterized in that: lower concentration trypsinase stem cell nutrient solution can be from people and mammalian tissues the separation and Culture pluripotent stem cell.
According to claim 1 from people and mammalian tissues the method for separation and Culture pluripotent stem cell, it is characterized in that: the pluripotent stem cell with the method separation and Culture can be expressed Oct4, SSEA.
According to claim 1 from people and mammalian tissues the method for separation and Culture pluripotent stem cell, it is characterized in that: cultivate with the pluripotent stem cell In vitro Suspension of the method separation and Culture and can form embryoid.
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| CN103361306A true CN103361306A (en) | 2013-10-23 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101402943A (en) * | 2008-11-17 | 2009-04-08 | 中山大学中山眼科中心 | Method for in vitro abduction and cultivation of multi-potentiality stem cell |
| CN101864451A (en) * | 2009-04-15 | 2010-10-20 | 中国科学院上海生命科学研究院 | Hoofed mammal inducible multipotential stem cell and preparation method thereof |
| CN101984050A (en) * | 2009-09-01 | 2011-03-09 | 中国科学院广州生物医药与健康研究院 | Cell type used for producing induced pluripotent stem (iPS) cells and preparation method and application thereof |
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2013
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101402943A (en) * | 2008-11-17 | 2009-04-08 | 中山大学中山眼科中心 | Method for in vitro abduction and cultivation of multi-potentiality stem cell |
| CN101864451A (en) * | 2009-04-15 | 2010-10-20 | 中国科学院上海生命科学研究院 | Hoofed mammal inducible multipotential stem cell and preparation method thereof |
| CN101984050A (en) * | 2009-09-01 | 2011-03-09 | 中国科学院广州生物医药与健康研究院 | Cell type used for producing induced pluripotent stem (iPS) cells and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 王书军，等: "诱导的多潜能干细胞类胚体分化过程中残留未分化细胞的研究", 《中国美容医学》 * |
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