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Korean Journal of Reproductive Medicine 2009;36(4):311-319.
Published online December 1, 2009.
Efficient Culture Method for Early Passage hESCs after Thawing.
Jin Ah Baek, Hee Sun Kim, Hye Won Seol, Jin Seo, Juwon Jung, Bo Ae Yoon, Yong Bin Park, Sun Kyung Oh, Seung Yup Ku, Seok Hyun Kim, Young Min Choi, Shin Yong Moon
1Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, 199-1, Dongsung Dong, Jongno Gu, Seoul, 110-810, Korea.
2Central Research Institute, Sam Jin Pharm. Co. Ltd., Hwasung 445-746, Korea.
3Department of Obstetrics and Gynecology, Seoul National University College of Medicine, 101 Daehang Ro, Jongno Gu, Seoul, 110-744, Korea.
Abstract
OBJECTIVE
Human embryonic stem cells (hESCs) have the capacity to differentiate into all of the cell types and therefore hold promise for cell therapeutic applications. In order to utilize this important potential of hESCs, enhancement of currently used technologies for handling and manipulating the cells is required. The cryopreservation of hESC colonies was successfully performed using the vitrification and slow freezing-rapid thawing method. However, most of the hESC colonies were showed extremely spontaneous differentiation after freezing and thawing. In this study, we were performed to rapidly collect of early passage hESCs, which was thawed and had high rate of spontaneously differentiation of SNUhES11 cell line. METHODS: Four days after plating, partially spontaneously differentiated parts of hESC colony were cut off using finely drawn-out dissecting pipette, which is mechanical separation method. RESULTS: After separating of spontaneously differentiated cells, we observed that removed parts were recovered by undifferentiated cells. Furthermore, mechanical separation method was more efficient for hESCs expansion after thawing when we repeated this method. The recovery rate after removing differentiated parts of hESC colonies were 55.0%, 74.5%, and 71.1% when we have applied this method to three passages. CONCLUSION: Mechanical separation method is highly effective for rapidly collecting and large volumes of undifferentiated cells after thawing of cryopreserved early passage hESCs.
Key Words: Human embryonic stem cells (hESCs); Cryopreservation-Thawing; Early passage; Mechanical separation method
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