BIOTECHNOLOGY 63
Cell passaging solution P
Ying Nie, Patrick Walsh, Diana L Clarke, Jon Rowley and Thomas Fellner look at a new development in stem cells cultivation.
revailing human pluripotent stem cells (hPSC) cultivation
practices continuously enrich the undifferentiated cell population through visual selection and
mechanical transfer of small undifferentiated multi-cellular aggregates from hPSC colonies. Tis labour intensive method results in substantial post- detachment cell injury and
death, ultimately affecting the rate of cell expansion. Several alternative methods have been proposed and include the use of one or more enzymes to dissociate adherent hESCs and
Fig. 1. WA09 hPSCs cultured for more than 25 passages using a hypertonic citrate dissociation solution retain their pluripotentency and karyotypic normalcy. A) Immunohistochemical and B) flow cytometric analysis for stemness antigens such as OCT4, SOX2, NANOG, SSEA4, TRA160, TRA181. C) In vitro embryoid body pluripotential differentiation assay for the three embryonic germ layers indicated by smooth muscle actin (SMA, mesoderm), alpha feto protein (AFP, endoderm) and beta III tubulin (TUJ1, ectoderm). D) In vivo teratoma pluripotency assay depicting retinal pigmented epithelium (ectoderm), cartilage (mesoderm), and gut-like epithelium (endoderm). E) G-Banding analysis of WA09 cells after 25 passages in StemPro or 27 passages in mTeSR1 medium. Scale bars: 200μM. mTeSR1 is a registered trademark of WiCell Research Institute; StemPro is a registered trademark of Life Technologies Corporation.
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