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Toxicol In Vitro. 2017 Dec;45(Pt 3):445-454. doi: 10.1016/j.tiv.2017.07.023. Epub 2017 Aug 15.

Towards optimisation of induced pluripotent cell culture: Extracellular acidification results in growth arrest of iPSC prior to nutrient exhaustion.

Author information

1
Medical University of Innsbruck, Department of Physiology and Medical Physics, Schöpfstraße 41, 6020 Innsbruck, Austria. Electronic address: Anja.Wilmes@i-med.ac.at.
2
Medical University of Innsbruck, Department of Physiology and Medical Physics, Schöpfstraße 41, 6020 Innsbruck, Austria.
3
Boehringer Ingelheim Pharma GmbH & Co.KG, Nonclinical Drug Safety Germany, 88397 Biberach an der Riss, Germany.
4
Medical University of Innsbruck, Division of Hygiene and Medical Microbiology, Schöpfstr, 41, 6020 Innsbruck, Austria.
5
Newcastle University, Institute of Genetic Medicine, Central Parkway, Newcastle upon Tyne, NE1 3BZ, UK.
6
Division of Molecular and Computational toxicology, Amsterdam Institute for Molecules, Medicines and Systems, Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands. Electronic address: p.jennings@vu.nl.

Abstract

Human induced pluripotent stem cells (iPSC) have the potential to radically reduce the number of animals used in both toxicological science and disease elucidation. One initial obstacle culturing iPSC is that they require daily medium exchange. This study attempts to clarify why and propose some practical solutions. Two iPSC lineages were fed at different intervals in a full growth area (FGA) or a restricted growth area (RGA). The FGA consisted of a well coated with Matrigel™ and the RGA consisted of a coated coverslip placed in a well. Glucose, lactate, extracellular pH and cell cycle phases were quantified. Without daily feeding, FGA cultured iPSC had significantly reduced growth rates by day 2 and began to die by day 3. In contrast, RGA cultured cells grew to confluence over 3days. Surprisingly, glucose was not exhausted under any condition. However, extracellular pH reached 6.8 after 72h in FGA cultures. Artificially reducing medium pH to 6.8 also inhibited glycolysis and initiated an increase in G0/G1 phase of the cell cycle, while adding an additional 10mM bicarbonate to the medium increased glycolysis rates. This study demonstrates that iPSC are highly sensitive to extracellular acidification, a likely limiting factor in maintenance of proliferative and pluripotent status. Culturing iPSC in RGA prevents rapid extracellular acidification, while still maintaining pluripotency and allowing longer feeding cycles.

KEYWORDS:

Cell cycle; Glucose; Growth arrest; Lactate; iPS; pH

PMID:
28821352
DOI:
10.1016/j.tiv.2017.07.023
[Indexed for MEDLINE]

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