Bulk vitrification of human embryonic stem cells.
BACKGROUND
The traditional vitrification method cannot keep up with the increased culture and propagation efficiency required to cryopreserve large quantities of vigorously proliferating human embryonic stem (HES) cells. In this study, we describe a newly invented vitrification carrier for cryopreserving large amount of HES cells and evaluate whether this bulk vitrification (BV) method is as effective as the popular open-pulled straw (OPS) vitrification method.


METHODS
HES cell clumps were harvested after passage and transferred to a cell strainer; only those clumps with a diameter more than 70 microm were included in the study and randomly selected to be cryopreserved by the BV method, OPS vitrification or slow freezing method. HES cell survival, growth and pluripotency were analyzed after thawing.


RESULTS
Bulk vitrification method with cell strainer could cryopreserve 136 +/- 23.4 cell clumps at one time (round), which was 30 times as high as those for OPS method (4 +/- 1.5). After thawing, bulk-vitrified HES cells exhibited high survival rate up to 94.3%, comparable with the OPS method. All surviving cell clumps generated HES cell colonies. Teratomas comprising all three primordial germ layers were formed in severe combined immunodeficient mice after subcutaneous injection of post-thawed, bulk-vitrified HES cell clumps, confirming pluripotency.


CONCLUSIONS
This new BV method could cryopreserve a large quantity of HES cell clumps at one time, which not only would satisfy routine cryopreservation of HES cell during daily culture process but also guarantee researchers have large quantity of efficiently cryopreserved HES cells ready for a scheduled study at any time.
Li
Tao
T
Reproductive Medicine Center, First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong 510080, People's Republic of China. zhoucanquan@gmail.com
Zhou
Canquan
C
Liu
Caixia
C
Mai
Qingyun
Q
Zhuang
Guanglun
G
D000818:Animals; D002454:Cell Differentiation; D002455:Cell Division; D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods* / Q000592:standards; D004622:Embryo, Mammalian / Q000166:cytology*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology; D006801:Humans; D007279:Injections, Subcutaneous; D051379:Mice; D016513:Mice, SCID; D039904:Pluripotent Stem Cells / Q000473:pathology; D033581:Stem Cell Transplantation / Q000379:methods; D013724:Teratoma / Q000209:etiology / Q000473:pathology