[Effects of different culture conditions on isolation and expansion of stem cells from second-trimester amniotic fluids].
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 as 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 adipocyte.


RESULTS
(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 differentiate into adipocyte after induction. (3) In the group of bFGF, stem cells were isolated from 5 samples and expanded to nearly 10(7) 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.


CONCLUSIONS
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.
Liu
Hui
H
Department of Obstetrics and Gynecology, Chinese PLA General Hospital, Beijing 100853, China.
Liu
Da-qing
DQ
Guan
Li-dong
LD
Yan
Zhi-feng
ZF
Wang
Jing
J
He
Li-juan
LJ
Lü
Yang
Y
Nan
Xue
X
Li
Ya-li
YL
Pei
Xue-tao
XT
D000653:Amniotic Fluid / Q000166:cytology*; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D005434:Flow Cytometry; D006801:Humans; D011247:Pregnancy; D011262:Pregnancy Trimester, Second; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013194:Staining and Labeling; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects
D003470:Culture Media; D016222:Fibroblast Growth Factor 2