Effects of different culture conditions on isolation and expansion of stem cells from second-trimester amniotic fluids
10.3760/cma.j.issn.0529-567x.2009.04.001
- VernacularTitle:不同培养条件对孕中期羊水干细胞分离及扩增能力的影响
- Author:
Hui LIU
;
Daqing LIU
;
Lidong GUAN
;
Zhifeng YAN
;
Jing WANG
;
Lijuan HE
;
Yang LU
;
Xue NAN
;
Yali LI
;
Xuetao PEI
- Publication Type:Journal Article
- Keywords:
Amniotic fluid;
Stem cells;
Cells,cuitured;
Cytokines
- From:
Chinese Journal of Obstetrics and Gynecology
2009;44(4):241-245
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effects of different culture conditions on the isolation and expansion of stem cells from second-trimester amniotic fluids.Methods Amniotic fluids were obtained from 15 pregnant women undergone amniocenteses for medical indications between 16-24 gestation weeks by transabdominal amniocenteses from September 2007 to June 2008.Amniotic fluids(10-20 ml)samples were collected and each WaS cultured under different conditions or groups.(1)Low-glucose DMEM(LD) medium supplemented with 10%of fetal bovine serum(group of 10% FBS);(2)LD medium with 20%of FBS(group of 20%FBS);(3)LD medium with 15%of FBS and 4 ng/ml of basic fibroblast growth factor (group of bFGF);(4)LD medium with 10%of FBS as well ag the culture plate coated with gelatin(group of gelatin).The effects of different conditions were evaluated by comparing the number of primary colonies,the cell morphology and the ability of expansion.The isolated stem cells were identified by flow cytometry,RT-PCR and differentiation ability to edipocyte.Resuits (1)The success rates of primary culture of the group of 10%FBS,20%FBS,bFGF and gelatin were 60%,73%,73%and 60% respectively(P>0.05).The numbers of colonies were 0.9±0.5,2.6±1.5,2.9±1.5,1.1±0.8(P<0.01 when group of 10%FBS and gelatin compared with group of 20%FBS and bFGF);among the primary colonies,fibroblast-like colonies accounted for 46%,49%,64%.44%respectively(P>0.05).(2)The second passage cells obtained from all of these four groups could difierentiate into adipocyte after induction.(3)In the group of bFGF,stem cells were isolated from 5 samples and expanded to nearly 107 cells after 5 passages(P<0.01 compared with other groups).(4)Karyotype were normal in all samples.(5)Stem cells from bFGF group showed positive expression of SSEA-4.Oct-4 and Nanog gene detected by flow cytometry and RT-PCR.Conclusion Stem cells can be isolated from second-trimester amniotic fluids;moderate serum concentration and supplementation of bFGF can improve the efficiency of isolation and expansion of amniotic fluid of stem cells.
