Improved cryopreservation of human embryonic stem cells with trehalose.
Human embryonic stem (ES) cells have been established either from fresh or frozen embryos. The recovery rates of undifferentiated human ES cells after cryopreservation with conventional slow-rate freezing and rapid-thawing methods are relatively low. The purpose of this study was to improve cryopreservation efficiency by modifying conventional methods with addition of trehalose. Immature oocytes donated from patients undergoing IVF treatment were utilized to generate blastocysts. One human ES cell line (named hES1) was established and characterized in detail. The hES1 cells expressed regular human ES cell markers, including stage-specific embryonic antigens SSEA-3, SSEA-4, tumour rejection antigens TRA-1-60, TRA-1-81 and octamer-binding transcription factor Oct-4 with high levels of alkaline phosphatase and telomerase activities. Cells could be differentiated to form teratomas in vivo. With slow-rate freezing and rapid-thawing methods modified by adding trehalose, the recovery rate of undifferentiated hES1 cells has been greatly improved from 15 to 48%. Cells retained pluripotency with normal karyotype after thawing. The results indicated that the use of trehalose is efficient and convenient for cryopreservation of human ES cells.
Wu
Chun Fang
CF
Shanghai Key Laboratory of Tissue Engineering, Shanghai 9th People's Hospital, Shanghai 2nd Medical University, Shanghai 200011, China.
Tsung
Hsiao Chien
HC
Zhang
Wen Jie
WJ
Wang
Yan
Y
Lu
Jun Hong
JH
Tang
Zheng Ya
ZY
Kuang
Yan Pin
YP
Jin
Wei
W
Cui
Lei
L
Liu
Wei
W
Cao
Yi Lin
YL
D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D002460:Cell Line; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D018076:DNA, Complementary / Q000235:genetics; D004622:Embryo, Mammalian / Q000166:cytology*; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D016513:Mice, SCID; D039904:Pluripotent Stem Cells; D020133:Reverse Transcriptase Polymerase Chain Reaction; D033581:Stem Cell Transplantation; D014183:Transplantation, Heterologous; D014199:Trehalose
D003451:Cryoprotective Agents; D018076:DNA, Complementary; D014199:Trehalose