RT Journal Article
SR Electronic
T1 Development of a modular automated system for maintenance and differentiation of adherent human pluripotent stem cells
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 079335
DO 10.1101/079335
A1 Duncan E. Crombie
A1 Maciej Daniszewski
A1 Helena H. Liang
A1 Tejal Kulkarni
A1 Fan Li
A1 Grace E. Lidgerwood
A1 Alison Conquest
A1 Damian Hernández
A1 Sandy S. Hung
A1 Katherine P. Gill
A1 Elisabeth De Smit
A1 Lisa S. Kearns
A1 Linda Clarke
A1 Valentin M. Sluch
A1 Xitiz Chamling
A1 Donald J. Zack
A1 Raymond C.B. Wong
A1 Alex W. Hewitt
A1 Alice Pébay
YR 2016
UL http://biorxiv.org/content/early/2016/10/05/079335.abstract
AB Patient-specific induced pluripotent stem cells (iPSCs) have tremendous potential for development of regenerative medicine, disease modelling and drug discovery. However, the processes of reprogramming, maintenance and differentiation are labour intensive and subject to inter-technician variability. To address these issues, we established and optimised protocols to allow for the automated maintenance of reprogrammed somatic cells into iPSCs to enable the large-scale culture and passaging of human pluripotent stem cells (PSCs) using a customized TECAN Freedom EVO. Generation of iPSCs was performed offline by nucleofection followed by selection of TRA-1-60 positive cells using a Miltenyi MultiMACS24 Separator. Pluripotency markers were assessed to confirm pluripotency of the generated iPSCs. Passaging was performed using an enzyme-free dissociation method. Proof of concept of differentiation was obtained by differentiating human PSCs into cells of the retinal lineage. Key advantages of this automated approach are the ability to increase sample size, reduce variability during reprogramming or differentiation, and enable medium to high-throughput analysis of human PSCs and derivatives. These techniques will become increasingly important with the emergence of clinical trials using stem cells.