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      Investigating the feasibility of scale up and automation of human induced pluripotent stem cells cultured in aggregates in feeder free conditions ☆

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          Highlights

          • •

            First published protocol for scalable automation of hiPSC in feeder-free conditions.

          • •

            Successful transfer of hiPSC between sites representative of research and manufacture.

          • •

            Comparability between manual and automated expansion protocols for hiPSC.

          Abstract

          The transfer of a laboratory process into a manufacturing facility is one of the most critical steps required for the large scale production of cell-based therapy products. This study describes the first published protocol for scalable automated expansion of human induced pluripotent stem cell lines growing in aggregates in feeder-free and chemically defined medium. Cells were successfully transferred between different sites representative of research and manufacturing settings; and passaged manually and using the CompacT SelecT automation platform. Modified protocols were developed for the automated system and the management of cells aggregates (clumps) was identified as the critical step. Cellular morphology, pluripotency gene expression and differentiation into the three germ layers have been used compare the outcomes of manual and automated processes.

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          Induced pluripotent stem cells from a spinal muscular atrophy patient.

          Allison Ebert, Junying Yu, Ferrill F. Rose … (2009)
          Spinal muscular atrophy is one of the most common inherited forms of neurological disease leading to infant mortality. Patients have selective loss of lower motor neurons resulting in muscle weakness, paralysis and often death. Although patient fibroblasts have been used extensively to study spinal muscular atrophy, motor neurons have a unique anatomy and physiology which may underlie their vulnerability to the disease process. Here we report the generation of induced pluripotent stem cells from skin fibroblast samples taken from a child with spinal muscular atrophy. These cells expanded robustly in culture, maintained the disease genotype and generated motor neurons that showed selective deficits compared to those derived from the child's unaffected mother. This is the first study to show that human induced pluripotent stem cells can be used to model the specific pathology seen in a genetically inherited disease. As such, it represents a promising resource to study disease mechanisms, screen new drug compounds and develop new therapies.
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            Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells.

            S Rashid, Sébastien Corbineau, Nick Hannan … (2010)
            Human induced pluripotent stem (iPS) cells hold great promise for advancements in developmental biology, cell-based therapy, and modeling of human disease. Here, we examined the use of human iPS cells for modeling inherited metabolic disorders of the liver. Dermal fibroblasts from patients with various inherited metabolic diseases of the liver were used to generate a library of patient-specific human iPS cell lines. Each line was differentiated into hepatocytes using what we believe to be a novel 3-step differentiation protocol in chemically defined conditions. The resulting cells exhibited properties of mature hepatocytes, such as albumin secretion and cytochrome P450 metabolism. Moreover, cells generated from patients with 3 of the inherited metabolic conditions studied in further detail (alpha1-antitrypsin deficiency, familial hypercholesterolemia, and glycogen storage disease type 1a) were found to recapitulate key pathological features of the diseases affecting the patients from which they were derived, such as aggregation of misfolded alpha1-antitrypsin in the endoplasmic reticulum, deficient LDL receptor-mediated cholesterol uptake, and elevated lipid and glycogen accumulation. Therefore, we report a simple and effective platform for hepatocyte generation from patient-specific human iPS cells. These patient-derived hepatocytes demonstrate that it is possible to model diseases whose phenotypes are caused by pathological dysregulation of key processes within adult cells.
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              Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions.

              Daniel Gulbranson, Guokai Chen, Andrew J. Thomson … (2012)
              This protocol describes an EDTA-based passaging procedure to be used with chemically defined E8 medium that serves as a tool for basic and translational research into human pluripotent stem cells (PSCs). In this protocol, passaging one six-well or 10-cm plate of cells takes about 6-7 min. This enzyme-free protocol achieves maximum cell survival without enzyme neutralization, centrifugation or drug treatment. It also allows for higher throughput, requires minimal material and limits contamination. Here we describe how to produce a consistent E8 medium for routine maintenance and reprogramming and how to incorporate the EDTA-based passaging procedure into human induced PSC (iPSC) derivation, colony expansion, cryopreservation and teratoma formation. This protocol has been successful in routine cell expansion, and efficient for expanding large-volume cultures or a large number of cells with preferential dissociation of PSCs. Effective for all culture stages, this procedure provides a consistent and universal approach to passaging human PSCs in E8 medium.
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                Author and article information

                Contributors
                Filipa A.C. Soares
                Journal
                Journal ID (nlm-ta): J Biotechnol
                Journal ID (iso-abbrev): J. Biotechnol
                Title: Journal of Biotechnology
                Publisher: Elsevier Science Publishers
                ISSN (Print): 0168-1656
                ISSN (Electronic): 1873-4863
                Publication date PMC-release: 10 March 2014
                Publication date (Print): 10 March 2014
                Volume: 173
                Issue: 100
                Pages: 53-58
                Affiliations
                [a ]Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute, Anne McLaren Laboratory for Regenerative Medicine and Department of Surgery, University of Cambridge, UK
                [b ]Centre for Biological Engineering, Loughborough University, UK
                [c ]Wellcome Trust Sanger Institute, Hinxton, UK
                Author notes
                [* ]Corresponding author at: Laboratory for Regenerative Medicine, MRC Centre for Stem Cell Biology and Regenerative Medicine, University of Cambridge, West Forvie Building, Robinson Way, CB2 0SZ Cambridge, UK. Tel.: +44 01223 747490. facs2@ 123456cam.ac.uk
                [1]

                Joint senior authors.

                Article
                Publisher ID: S0168-1656(13)00552-X
                DOI: 10.1016/j.jbiotec.2013.12.009
                PMC ID: 3969287
                PubMed ID: 24440272
                SO-VID: 350cd02d-ccbf-4489-bf1c-297b1279d213
                Copyright © © 2013 The Authors
                License:

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                Date received : 16 October 2013
                Date revision received : 23 November 2013
                Date accepted : 16 December 2013
                Categories
                Subject: Short Communication

                ScienceOpen disciplines: Biotechnology
                Keywords: induced pluripotent stem cells,feeder free,automation,scale up,manufacturing
                Data availability:
                ScienceOpen disciplines: Biotechnology
                Keywords: induced pluripotent stem cells, feeder free, automation, scale up, manufacturing

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