Development of a modular automated system for maintenance and differentiation of adherent human pluripotent stem cells
Development of a modular automated system for maintenance and differentiation of adherent human pluripotent stem cells
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.
Sluch Valentin M.、Kearns Lisa S.、Clarke Linda、Li Fan、Hern¨￠ndez Damian、Zack Donald J.、P¨|bay Alice、Smit Elisabeth De、Daniszewski Maciej、Kulkarni Tejal、Crombie Duncan E.、Wong Raymond C.B.、Lidgerwood Grace E.、Hewitt Alex W.、Conquest Alison、Liang Helena H.、Chamling Xitiz、Gill Katherine P.、Hung Sandy S.
Wilmer Eye Institute, Johns Hopkins University School of MedicineCentre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of MelbourneCentre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of MelbourneSchool of Medicine, Menzies Institute for Medical Research, University of Tasmania||State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Centre, Sun Yat-sen UniversityCentre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of MelbourneWilmer Eye Institute, Johns Hopkins University School of MedicineCentre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of MelbourneCentre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of MelbourneCentre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of MelbourneCentre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of MelbourneCentre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of MelbourneCentre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of MelbourneCentre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of MelbourneCentre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of Melbourne||School of Medicine, Menzies Institute for Medical Research, University of TasmaniaCentre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of MelbourneCentre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of MelbourneWilmer Eye Institute, Johns Hopkins University School of MedicineCentre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of MelbourneCentre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of Melbourne
医学研究方法生物科学研究方法、生物科学研究技术生物工程学
human pluripotent stem cellsiPSCsautomationliquid handlingautomated cell culture platformpassagingretinal cell differentiation
Sluch Valentin M.,Kearns Lisa S.,Clarke Linda,Li Fan,Hern¨￠ndez Damian,Zack Donald J.,P¨|bay Alice,Smit Elisabeth De,Daniszewski Maciej,Kulkarni Tejal,Crombie Duncan E.,Wong Raymond C.B.,Lidgerwood Grace E.,Hewitt Alex W.,Conquest Alison,Liang Helena H.,Chamling Xitiz,Gill Katherine P.,Hung Sandy S..Development of a modular automated system for maintenance and differentiation of adherent human pluripotent stem cells[EB/OL].(2025-03-28)[2025-04-27].https://www.biorxiv.org/content/10.1101/079335.点此复制
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