Human embryonic stem cells (hESCs) have the potential for use in regenerative medicine and in the field of basic research. Therefore, effective cryopreservation and storage of hESCs are important for preservation of newly established cell line for various purposes. Despite poor survival and slow recovery after thawing, the conventional slow freezing method is most commonly used for cryopreservation of hESCs due to its simplicity and ease of use for freezing a large number of hESCs appropriate to clinical applications. Here we controlled the clump size (Group Ⅰ; 400～450 ㎛, Group Ⅱ; 800～900 ㎛, and Group Ⅲ; 1500～1700 ㎛) of hESCs at 5 days after plating using a glass pipette during cryopreservation in order to obtain a larger amount of hESCs after thawing. Attachment rates differed significantly (P<0.05) in each of the three groups and the average of attachment rate of GroupⅡ was highest in SNUhES4 and H1. In particular, the attachment rate of Group Ⅱ in SNUhES3 showed a significant improvement with ROCK inhibitor Y-27632. These results indicate that clump size and cell-cell adhesions of GroupⅡ are appropriate for cryopreservation compared to the Group Ⅰ and Group Ⅲ. This method increased cell viability and reduced the recovery time leading to various experiments, and therefore has an advantage for use with hESCs like newly established in particular. We demonstrated that use of this effective cryopreservation method with control of the clump size of hESCs can effectively improve the attachment rate and survival of post-thaw hESCs with and without Y-27632.

• Young Min Choi(Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Department of Obstetrics and Gynecology, Seoul National University College of Medicine)
• Shin Yong Moon(Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Department of Obstetrics and Gynecology, Seoul National University College of Medicine)
• Seok Hyun Kim(Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Department of Obstetrics and Gynecology, Seoul National University College of Medicine)
• Seung-Yup Ku(Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Department of Obstetrics and Gynecology, Seoul National University College of Medicine)
• Sun Kyung Oh(Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Department of Obstetrics and Gynecology, Seoul National University College of Medicine)
• Hee-Sun Kim(Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Department of Obstetrics and Gynecology, Seoul National University College of Medicine)
• Bo Ae Yoon(Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University)
• Jin Ah Baek(Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University)
• Hye Won Seol(Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University)
• Juwon Jung(Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University)