Development of a modular automated system for maintenance and differentiation of adherent human pluripotent stem cells.

Duncan E Crombie; Maciej Daniszewski; Helena H Liang; Tejal Kulkarni; Fan Li; Grace E Lidgerwood; Alison Conquest; Damian Hernandez; Sandy S Hung; Katherine P Gill; Elisabeth De Smit; Lisa S Kearns; Linda Clarke; Valentin M Sluch; Xitiz Chamling; Donald J Zack; Raymond CB Wong; Alex Hewitt; Alice Pebay

doi:10.1101/079335

bioRxiv · 10.1101/079335

Development of a modular automated system for maintenance and differentiation of adherent human pluripotent stem cells.

Duncan E Crombie·Maciej Daniszewski·Helena H Liang·Tejal Kulkarni·Fan Li·Grace E Lidgerwood·Alison Conquest·Damian Hernandez·Sandy S Hung·Katherine P Gill·Elisabeth De Smit·Lisa S Kearns·Linda Clarke·Valentin M Sluch·Xitiz Chamling·Donald J Zack·Raymond CB Wong·Alex Hewitt·Alice Pebay

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Abstract

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.

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Duncan E Crombie, Maciej Daniszewski, Helena H Liang, Tejal Kulkarni, Fan Li, Grace E Lidgerwood, Alison Conquest, Damian Hernandez, Sandy S Hung, Katherine P Gill, Elisabeth De Smit, Lisa S Kearns, Linda Clarke, Valentin M Sluch, Xitiz Chamling, Donald J Zack, Raymond CB Wong, Alex Hewitt, Alice Pebay. 2016-10-05. Development of a modular automated system for maintenance and differentiation of adherent human pluripotent stem cells.. https://doi.org/10.1101/079335

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