Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions

Abstract

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 (iPSCs). 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 pluripotent stem cells in E8 medium.

Ips Progression a Decade Devoted

“…Freshly isolated fibroblasts in E8-based fibroblast medium on vitronectin have consistently reprogrammed at a higher efficiency (60 to ~1,000 iPS cell colonies per 106 transfected fibroblasts) than the commercially available established stem cell lines [63]. E8 medium supporting higher reprogramming efficiencies for both viral and episomal approaches has been validated [64].…”

“…Dual advantage of E8 medium and the EDTA dissociation method has unveiled a faster expansion of large cell volume cultures. Furthermore, EDTA dissociation has enabled enrichment of potential iPSC's in an overcrowded reprogramming culture in which otherwise a routine secondary passaging is needed [64,65]. Sigma aldreich's PluriSTEM and human ES/iPS medium is a specially formulated defined media utilizing activin-A, tgfB1 and bFGF which aids in promoting stem cell self-renewal with increased cell viability and enhanced cell proliferation in single cell passaging.…”

Good Cell Culture Practice for stem cells and stem-cell-derived models

“…For routine culture of iPSCs, passaging can be achieved using chemicals, enzymes or mechanical means to facilitate cell detachment (Beers et al, 2012). The approach selected depends on the cell grade (e.g., research, manufacture, therapy), culture conditions (e.g., growth medium, surface matrix), and current state of the culture (high/low passage, extent of differentiation, etc.…”

“…The aforementioned reagents are best when splitting colonies as clumps, whereas trypsin is often selected to create single cell suspensions. In some conditions cell detachment may result in significant loss of cell vigor and viability, which may be reduced by the addition of Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitors (Beers et al, 2012). However, the impact of routine use of ROCK inhibitors in culture media is yet to be determined in longer term passaging.…”

Myocardial Reprogramming Medicine: The Development, Application, and Challenge of Induced Pluripotent Stem Cells

“…These limitations do not apply to ESCs or iPSCs which can be propagated indefinitely. Most laboratories can expand various iPSCs robustly using commercially available products, including feeder and feederfree protocols [164,165]. They are also able to generate large numbers of unambiguous cardiomyocytes with defined induction protocols.…”