The DEF-CS system is a robust culture system for efficient expansion of human induced pluripotent stem (iPS) cells in a feeder-free and defined environment. With this system, the cells are passaged enzymatically and maintain pluripotency and a stable karyotype for more than 30 passages.

In this set of experiments, the DEF-CS system was directly compared to four vendors' feeder-free culture systems for expansion and maintenance of human iPS cells. A feeder cell-based culture system was used as a control. Pluripotency and growth rate analyses indicate that the Cellartis culture system provides robust growth while efficiently maintaining cells in an undifferentiated state.

The human iPS cell line 253G1 was grown in the DEF-CS system, various feeder-free culture systems from different vendors (vendor 1, 2, 3, and 4), or in a culture containing feeder cells.The cells were acclimated to each culture system for three weeks. Then, growth rate and pluripotency were characterized for cells growing in each system. The Cellartis DEF-CS culture system promoted a high and robust growth rate for the 253G1 iPS cells (Figure 1).

Figure 1. Comparison of growth rate between culture systems. Culture in Cellartis DEF-CS resulted in a high growth rate over the course of the experiment.

It has previously been shown that side scatter (SSC) is highly heterogeneous in undifferentiated pluripotent stem cells and predicts clonogenic self-renewal (Ramirez et al., 2013). Cells grown in the various culture systems were collected and analyzed by flow cytometry. Cells grown in the DEF-CS system displayed more heterogeneity in SSC than the cell populations grown in the other vendors' culture systems (Figure 2).

Figure 2. Scatter plot of side scatter (SSC) versus forward scatter (FSC) for 253G1-hiPS cells as analyzed by flow cytometry.

Cells were also analyzed for expression of the stem cell markers TRA-1-60 and SSEA-4. Flow cytometry analysis indicated that cells grown in the Cellartis DEF-CS culture system maintain the highest proportion of TRA-1-60+ and SSEA-4+ cells, markers indicative of pluripotency (Figures 3 and 4).

Figure 3. Expression of the pluripotency marker TRA-1-60. After five weeks in culture, TRA-1-60 expression in 253G1 iPS cells was analyzed by flow cytometry. Growth in the DEF-CS system resulted in cell populations with the highest proportion of TRA-1-60+ cells.

Figure 4. Expression of the pluripotency marker SSEA-4. After five weeks in culture, SSEA-4 expression in 253G1 iPS cells was analyzed by flow cytometry. Culture in the DEF-CS system resulted in a higher proportion of cells that express SSEA-4 than the majority of other systems tested.

The Cellartis DEF-CS culture system allowed for efficient and robust expansion of 253G1 iPS cells similar to other vendors' culture systems. Culture in the DEF-CS system resulted in a higher proportion of pluripotent stem cells that express the TRA-1-60 and SSEA-4 stem cell markers than the majority of the other systems tested. The highly heterogeneous SSC versus FSC pattern was similar for cells grown in the Cellartis DEF-CS system and the control feeder culture system, but was different for other vendors' culturing systems. Taken together, these data indicate that the DEF-CS system resulted in the most robust and stable growth and was best able to maintain human iPS cells in an undifferentiated state.

Cell culture

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.

Ramirez, J.-M. et al. Side scatter intensity is highly heterogeneous in undifferentiated pluripotent stem cells and predicts clonogenic self-renewal. Stem Cells Dev. 22, 1851–60 (2013).

Learn more about the DEF-CS culture system »