Successful vitrification of human amnion-derived mesenchymal stem cells.
BACKGROUND
A cryopreservation protocol for human amnion-derived mesenchymal stem cells (HAMs) is required because these cells cannot survive for long periods in culture. The aim of this study was to determine whether vitrification is a useful freezing method for storage of HAMs.


METHODS
HAMs were cryopreserved using vitrification method. The morphology and viability of thawed HAMs was evaluated by Trypan Blue staining. The expression of several embryonic stem cell (ESC) markers was evaluated using flow cytometry, RT-PCR and immunocytochemistry. Von Kossa, Oil Red O and Alcian Blue staining were used to asses the differentiation potential of thawed HAMs.


RESULTS
The post-thawing viability of HAMs was 84.3 +/- 3.2% (Mean +/- SD, n = 10). The thawed HAMs showed morphological characteristics indistinguishable from the non-vitrified fresh HAMs. The expression of surface antigens (strong positive for CD44, CD49d, CD59, CD90, CD105 and HLA-ABC; weak positive for HLA-G; negative for CD31, CD34, CD45, CD106, CD117 and HLA-DR) and the expression of ESC markers [CK18, fibroblast growth factor-5, GATA-4, neural cell adhesion molecule, Nestin, Oct-4, stem cell factor, HLA-ABC, Vimentin, bone morphogenetic protein (BMP) 4, hepatocyte nuclear factor 4 alpha (HNF-4 alpha), Pax-6, alpha-fetoprotein, Brachyury, BMP-2, TRA-1-60, stage-specific embryonic antigen (SSEA-3, SSEA-4)] were maintained in the vitrified-thawed HAMs. The thawed HAMs retained ability to differentiate into osteoblasts, adipocytes and chondrocytes under appropriate culture conditions.


CONCLUSIONS
Our results suggest that vitrification is a reliable and effective method for cryopreservation of HAMs.
Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, 300 Gumi, Bundang, Seongnam, Gyeonggi 463-707, Korea.
Jeong Hee
JH
Moon
Jung Ryeol
JR
Lee
Byung Chul
BC
Jee
Chang Suk
CS
Suh
Seok Hyun
SH
Kim
Hyun Jung
HJ
Lim
Hae Kwon
HK
Kim
D000650:Amnion / Q000166:cytology*; D015703:Antigens, CD / Q000096:biosynthesis; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D005260:Female; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D011247:Pregnancy
D015703:Antigens, CD