Korean J Reprod Med.  2009 Dec;36(4):311-319.

Efficient Culture Method for Early Passage hESCs after Thawing

• Baek JA
• Kim HS
• Seol HW
• Seo J
• Jung J
• Yoon BA
• Park YB
• Oh SK
• Ku SY
• Kim SH
• Choi YM
• Moon SY

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.

Keyword

Human embryonic stem cells (hESCs); Cryopreservation-Thawing; Early passage; Mechanical separation method

MeSH Terms

Cryopreservation
Embryonic Stem Cells
Freezing
Handling (Psychology)
Humans
Vitrification