Cryopreservation of human embryonic stem cells by vitrification.
BACKGROUND
The efficiency of traditional cryopreservation of human embryonic stem (ES) cells is low, and there have been few attempts to prove new cryopreservation methods effective. This study was designed to evaluate the efficiency of cryopreservation of human ES cells using vitrification method.


METHODS
Human ES cells clumped from an identical cell line were randomly allocated to be cryopreserved by vitrification or by slow freezing. The recovery rates, the growth and differentiation potential of thawed human ES cells were compared between these two groups. The pluripotency of human ES cells after thawing was identified.


RESULTS
Eighty-one point nine percent (59/72) of human ES cell clumps were recovered after vitrification, while only 22.8% (16/70) were recovered after slow freezing (P < 0.01). The colonies after vitrification manifested have not only faster growth but also a lower level of differentiation when compared to colonies subjected to the slow freezing protocol. However, the rates of growth and differentiation in undifferentiated colonies from both groups were identical to the rates in those of non-cryopreserved stem cells after a prolonged culture period. Passage 6 of vitrified human ES cells retained the properties of pluripotent cells, a normal karyotype and expressed the transcription factor OCT-4, stage specific expressed antigen-4 (SSEA-4) and SSEA-3. Teratoma growth of these cells demonstrated the ability to develop into all three germ layers.


CONCLUSIONS
Vitrification is effective in cryopreserving human ES cells. During a prolonged culture, human ES cells retain their pluripotency after cryopreservation.
Zhou
Can-quan
CQ
Reproductive Medical Center, First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China.
Mai
Qing-yun
QY
Li
Tao
T
Zhuang
Guang-lun
GL
D002454:Cell Differentiation; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D004622:Embryo, Mammalian / Q000166:cytology*; D006801:Humans; D009997:Osmotic Pressure; D013234:Stem Cells / Q000166:cytology*