title: 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 modeling, and drug discovery. However, the processes of reprogramming, maintenance, and differentiation are labor intensive and subject to intertechnician variability. To address these issues, we established and optimized 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.
authors.affiliation: 1 Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of Melbourne, East Melbourne, Victoria, Australia.|Co-first authors.
authors.forename: Duncan E
authors.initials: DE
authors.lastname: Crombie
authors.affiliation: 1 Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of Melbourne, East Melbourne, Victoria, Australia.|Co-first authors.
authors.forename: Maciej
authors.initials: M
authors.lastname: Daniszewski
authors.affiliation: 1 Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of Melbourne, East Melbourne, Victoria, Australia.
authors.forename: Helena H
authors.initials: HH
authors.lastname: Liang
authors.affiliation: 1 Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of Melbourne, East Melbourne, Victoria, Australia.
authors.forename: Tejal
authors.initials: T
authors.lastname: Kulkarni
authors.affiliation: 2 School of Medicine, Menzies Institute for Medical Research, University of Tasmania, Hobart, Tasmania, Australia.|3 State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Centre, Sun Yat-sen University, Guangzhou, Guangdong, China.
authors.forename: Fan
authors.initials: F
authors.lastname: Li
authors.affiliation: 1 Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of Melbourne, East Melbourne, Victoria, Australia.
authors.forename: Grace E
authors.initials: GE
authors.lastname: Lidgerwood
authors.affiliation: 1 Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of Melbourne, East Melbourne, Victoria, Australia.
authors.forename: Alison
authors.initials: A
authors.lastname: Conquest
authors.affiliation: 1 Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of Melbourne, East Melbourne, Victoria, Australia.
authors.forename: Damian
authors.initials: D
authors.lastname: Hernández
authors.affiliation: 1 Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of Melbourne, East Melbourne, Victoria, Australia.
authors.forename: Sandy S
authors.initials: SS
authors.lastname: Hung
authors.affiliation: 1 Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of Melbourne, East Melbourne, Victoria, Australia.
authors.forename: Katherine P
authors.initials: KP
authors.lastname: Gill
authors.affiliation: 1 Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of Melbourne, East Melbourne, Victoria, Australia.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: De Smit
authors.affiliation: 1 Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of Melbourne, East Melbourne, Victoria, Australia.
authors.forename: Lisa S
authors.initials: LS
authors.lastname: Kearns
authors.affiliation: 1 Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of Melbourne, East Melbourne, Victoria, Australia.
authors.forename: Linda
authors.initials: L
authors.lastname: Clarke
authors.affiliation: 4 Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Valentin M
authors.initials: VM
authors.lastname: Sluch
authors.affiliation: 4 Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Xitiz
authors.initials: X
authors.lastname: Chamling
authors.affiliation: 4 Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA.|5 Departments of Neuroscience, Molecular Biology and Genetics, and Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Zack
authors.affiliation: 1 Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of Melbourne, East Melbourne, Victoria, Australia.
authors.forename: Raymond C B
authors.initials: RCB
authors.lastname: Wong
authors.affiliation: 1 Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of Melbourne, East Melbourne, Victoria, Australia.|2 School of Medicine, Menzies Institute for Medical Research, University of Tasmania, Hobart, Tasmania, Australia.|Co-senior authors.
authors.forename: Alex W
authors.initials: AW
authors.lastname: Hewitt
authors.affiliation: 1 Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of Melbourne, East Melbourne, Victoria, Australia.|Co-senior authors.
authors.forename: Alice
authors.initials: A
authors.lastname: Pébay
mesh_terms: D001331:Automation; D002448:Cell Adhesion; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line; D065150:Cellular Reprogramming; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D012160:Retina / Q000166:cytology
keywords: automated cell culture platform; automation; human pluripotent stem cells; iPSCs; liquid handling; passaging; retinal cell differentiation