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  • Using the DEF-CS system to culture human iPS cells
  • Comparison of the Cellartis DEF-CS system with other vendors' human iPS cell culture systems
  • Reprogramming PBMCs
  • Reprogramming fibroblasts
Home › Learning centers › Stem cell research › Technical notes › Pluripotent stem cells › Comparison of the Cellartis DEF-CS system with other vendors' human iPS cell culture systems

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Tech Note

Direct comparison of the Cellartis DEF-CS system with different vendors' systems for culturing human pluripotent stem cells

Introduction Results Conclusions Methods References

Introduction  

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.

Results  

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).


Cell Culture

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).

Comparison of growth rate between culture systems

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).

Scatter plot of SSC versus FSC

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.

SSEA-4

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.

Conclusions  

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.

Methods  

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.

References  

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 »

Related Products

Cat. # Product Size License Quantity Details
Y30010 Cellartis® DEF-CS™ 500 Culture System 1 Kit USD $520.00

License Statement

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C001 This product is manufactured and sold by Takara Bio Europe AB based on a commercial license to certain intellectual property rights held by Wisconsin Alumni Research Foundation (“WARF”). This product is covered by one or more claims of U.S. Patent No. 7,514,260 and its foreign counterparts. The purchase of this product conveys to the buyer the non-transferable right to use the product for its intended use, strictly limited to purchaser’s own internal research. No other express or implied license is granted to the purchaser. Purchaser cannot have any right to use this product or its components in humans for any purposes including but not limited to diagnostics and/or therapeutics, or otherwise clinical trials. Purchase does not include any right to resell or transfer this product to a third party regardless of whether or not compensation is received. Purchasers wishing to use this product for purposes other than internal research use should contact us.

Cellartis DEF-CS 500 Culture System is a defined culture system for efficient expansion of undifferentiated human pluripotent stem cells. This kit includes basal medium, coating substrate, and additives.

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components You May Also Like Image Data

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Expansion potential of a characterized working bank of human induced pluripotent stem (iPS) cells in the Cellartis DEF-CS Culture System

Expansion potential of a characterized working bank of human induced pluripotent stem (iPS) cells in the Cellartis DEF-CS Culture System
Expansion potential of a characterized working bank of human induced pluripotent stem (iPS) cells in the Cellartis DEF-CS Culture System. The Cellartis DEF-CS Culture System can produce 2 x 109 human iPS cells within 4 passages (18–20 days) from frozen cells (2.0–2.5 x 106 cells).

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Robust growth of human induced pluripotent stem (iPS) cells in the Cellartis DEF-CS Culture System

Robust growth of human induced pluripotent stem (iPS) cells in the Cellartis DEF-CS Culture System
Robust growth of human induced pluripotent stem (iPS) cells in the Cellartis DEF-CS Culture System. The number of iPS cells was quantified after being cultured for three weeks using either the Cellartis DEF-CS Culture System, a reference feeder system, or four other stem cell culture systems.

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Human induced pluripotent stem cells (iPS) cells grown in the Cellartis DEF-CS Culture System have the highest proportion and intensity of markers of pluripotency

Human induced pluripotent stem cells (iPS) cells grown in the Cellartis DEF-CS Culture System have the highest proportion and intensity of markers of pluripotency
Human induced pluripotent stem cells (iPS) cells grown in the Cellartis DEF-CS Culture System have the highest proportion and intensity of markers of pluripotency. Quantitative analysis of TRA1-60 (Panel A) and SSEA4 (Panel B) expression was performed on human iPS cells after five weeks culture in either the Cellartis DEF-CS Culture System, a reference feeder cell containing system, or four different stem cell culture systems.

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Human iPS cells grown in the Cellartis DEF-CS Culture System look different from those grown with traditional aggregate culture techniques

Human iPS cells grown in the Cellartis DEF-CS Culture System look different from those grown with traditional aggregate culture techniques
Human iPS cells grown in the Cellartis DEF-CS Culture System look different from those grown with traditional aggregate culture techniques. Freshly passaged human iPS cells were cultured for 5 days in either the Cellartis DEF-CS Culture System, on feeder cells, in mTeSR 1 medium (STEMCELL Technologies), or in Essential 8 Medium (E8; Life Technologies).

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Human induced pluripotent stem (iPS) cells cultured long-term in the Cellartis DEF-CS Culture System retain a normal karyotype

Human induced pluripotent stem (iPS) cells cultured long-term in the Cellartis DEF-CS Culture System retain a normal karyotype
Human induced pluripotent stem (iPS) cells cultured long-term in the Cellartis DEF-CS Culture System retain a normal karyotype. The human iPS cell line ChiPSC18 was cultured for 20 passages in the Cellartis DEF-CS Culture System. Chromosomal analysis indicates that the cells retain a normal karyotype.

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Human induced pluripotent stem (iPS) cells can be passaged as single cells in the Cellartis DEF-CS Culture System

Human induced pluripotent stem (iPS) cells can be passaged as single cells in the Cellartis DEF-CS Culture System

Human induced pluripotent stem (iPS) cells can be passaged as single cells in the Cellartis DEF-CS Culture System. A single GFP-actin iPS cell was isolated and placed in the well of a culture dish. Twenty-four hours after seeding, morphology was assessed by fluorescence microscopy at 20x (Panel A) and 40x (Panel B) magnification. Sixteen days later, the single GFP-actin iPS cell had proliferated into numerous cells as evidenced by microscopic observation at 4x (Panel C), 10x (Panel D), 20x (Panel E), and 40x (Panel F) magnification.

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Human pluripotent stem cells remain undifferentiated when cultured in the Cellartis DEF-CS Culture System

Human pluripotent stem cells remain undifferentiated when cultured in the Cellartis DEF-CS Culture System

Human pluripotent stem cells remain undifferentiated when cultured in the Cellartis DEF-CS Culture System. Human iPS cells cultured for 23 passages in the Cellartis DEF-CS Culture System were characterized by Oct-4 staining (Panel A) and nuclear staining (Panel B).

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Y30010: Cellartis DEF-CS 500 Culture System

Y30010: Cellartis DEF-CS 500 Culture System

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