[Isolation, culture and cryopreservation of cells derived from fetal appendages].
OBJECTIVE
To investigate the optimal method for isolation, culture and cryopreservation of cells from fetal appendages, for the purpose of providing viable cells for tissue engineering, cell therapy and gene therapy.


METHODS
Trypsin dispersion method was used to isolate cells from human umbilical cord and placenta. The tissues from umbilical cord and placenta were cryopreserved with dimethylsulfoxide (DMSO) in different concentrations. Then the percentage of living cells in thawed tissues, and their micro-structure were observed and compared with fresh tissues under transmission electron microscope. The expression of cell immune phenotype before and after cryopreservation were detected with immuno-histochemistry method.


RESULTS
The percentage of living cells in human fresh umbilical cord was 67.0%, while that in cryopreserved umbilical cord was 23.4%, 55.5%, 48.8%, 31.8%, respectively in 5%, 10%, 15%, 20% of DMSO. The percentage of living cells in cryopreserved tissues was similar to that of fresh tissues when the volume percentage of DMSO was 10% (P > 0.05), and it was significantly different with that when volume percentage of DMSO was 5% and 20% (P < 0.01). The result by transmission electron microscope was coincident with the results shown above. The results were similar between placenta and umbilical cord. There was no obvious changes in immune phenotype of the tissue and cells after cryopreservation.


CONCLUSIONS
Cryopreservation with this method can isolate a large amount of cells from fetal appendages, with no changes in immune phenotype after cryopreservation, and the effect was best when the volume percentage of DMSO was 10%.
Yu
Xu-Ming
XM
Department of Burns, 2nd People's Hospital of Shenzhen, Shenzhen 518000, PR China.
Xue
Zhi-Gang
ZG
Dai
Guo-Sheng
GS
Xu
Xu
X
Chen
Wan-An
WA
Fan
Kun-Wu
KW
Liang
De-Sheng
DS
Xia
Jia-Hui
JH
D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D015925:Cryopreservation; D004121:Dimethyl Sulfoxide; D005260:Female; D005333:Fetus; D006801:Humans; D010920:Placenta / Q000166:cytology*; D014470:Umbilical Cord / Q000166:cytology
D004121:Dimethyl Sulfoxide