The human iPS cell line 253G1 was thawed, seeded at a cell density of 1–3 x 104 cells/cm2, and maintained for five weeks in the DEF-CS system, or culture systems from four different vendors (vendor 1, 2, 3, and 4). The iPS cells were cultured on feeder-free coatings specific for each culture system and were handled according to each manufacturer's recommendations. As a control, 253G1 iPS cells were cultured on a mitomycin C-treated STO feeder-cell layer.
Growth rate and flow cytometry
After three weeks of adaptation to each culture system, growth rates of 253G1 iPS cells were calculated for the next 20 days by plotting the number of cells against days in each culture system. Briefly, 253G1 iPS cells from each culture system were detached using the reagents recommended by each vendor. To count cells, single-cell suspensions were generated by introducing a second digestion step for the aggregate cell systems using TrypLE Select enzyme (Life Technologies). Cell number in the single-cell suspensions was calculated manually. After five weeks of culture, the iPS cells were collected and incubated with TRA-1-60 and SSEA-4 antibodies conjugated to Alexa Fluor 488 and phycoerythrin, respectively. The labeled cells were analyzed by flow cytometry (FC); side scatter (SSC), and forward scatter (FSC), and percentages of TRA-1-60- and SSEA-4-positive cells were quantified for cells grown in the different culture systems.