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 (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.

Figure 1. EDTA dissociation is an effective method for pluripotent stem cell passaging

a)Summary of cell survival efficiency by different methods. After dissociation, enzyme/mechanic-generated large aggregates survive efficiently; individualized cells die at clonal density, but survive in the presence of inhibitors or high loading density; EDTA-generated small aggregates survive efficiently by high local density without the help of drug treatment. b)Human ESC colony morphology after plating. H1 cells were partially dissociated with EDTA, and they attached to the plate in a few minutes, spread in 2 h, and survived as colonies (24 h). Scale bars, 50 μm. c)Comparison of cell survival of different passaging methods. Cells were dissociated by TrypLE, EDTA or Dispase and added onto Matrigel-coated plates; next, live cells were counted after 24 h. Asterisk (*) signifies that the survival by EDTA differed significantly (P < 0.05, n=3) from TrypLE at 24 h.

Figure 2. iPSC derivation from human fibroblast cells in chemically defined medium

a)In the EDTA/E8-based iPSC derivation procedure, EDTA is the major handling method necessary for most cell culture stages. In comparison, the traditional procedure uses multiple dissociation methods (Supplementary Fig. 4a). b)A brief summary of the steps described in this protocol. c) Cell morphology changes in iPSC derivation process. Scale bars, 40 μm. d) FACS analysis of pluripotency markers on iPSCs derived from fibroblasts. Cells were derived in defined medium, and then expanded by the EDTA method. IgG control: unfilled peak; FITC-conjugated antibody: green peak. Antibodies: OCT4-Alexa-488 (Millipore, cat. no. FCMAB113A4); SSEA4-FITC (Biolegend, cat. no. 330410); Tra1-60-FITC (Millipore, cat. no. FCMAB115F); msIGg-FITC and nonimmune control (Millipore, cat. no. MABC006F). e)Immunostaining of pluripotency markers on iPSCs derived from fibroblasts. Left, phase-contrast images; right, fluorescence images. Scale bars, 20μm. Antibodies: OCT4-Alexa-488 (Millipore, cat. no. FCMAB113A4); SSEA4-FITC (Biolegend, cat. no. 330410); Tra1-60-PE (Biolegend, cat. no. 330610).