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  • ‹ Back to Pluripotent stem cells
  • 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 › Using the DEF-CS system to culture human iPS cells

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Using the DEF-CS system to culture human iPS cells

Introduction Results

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. The system includes all components needed for culture (i.e., media, additives, and coating compound). In this system, iPS cells grow in an undifferentiated, homogenous monolayer with a high consistent growth rate.

Results  

Cell morphology in the DEF-CS system

One advantage of the DEF-CS system is the unique potential to culture human iPS cells as a single, homogenous monolayer of undifferentiated cells. Because of this, human iPS cells grown in DEF-CS will look very different from those grown with traditional aggregate culture methods. Notably, there will be a larger cytoplasm-to-nucleus ratio, and cells at lower densities will exhibit a morphology that would otherwise indicate differentiation. However, despite this appearance, human iPS cells grown in DEF-CS media retain their pluripotency and are not differentiated. For reference, the image below depicts the expected morphologies of freshly passaged human iPS cells cultured for five days in either the DEF-CS system, on feeder cells, in mTeSR 1 medium (STEMCELL Technologies), or in Essential 8 Medium (E8; Life Technologies).

TN01

Observing cells grown in the DEF-CS system

When initially transferring iPS cells to this system, some cell characteristics might be different from previously used culture systems. The DEF-CS system utilizes single-cell passaging, and therefore the morphology of cells cultured in the DEF-CS system differs from that of cells cultured in systems using aggregate passaging methods. Second, newly passaged cells tend to spread out. However, when proliferating, the cells get denser, and the typical undifferentiated stem cell morphology (i.e., high nucleus-to-cytoplasm ratio, defined borders, and prominent nucleoli) appears. The images below show a human iPS cell line cultured in the DEF-CS system. A cell density of 5 x 104 cells/cm2 corresponds to newly passaged cells, while the higher cell densities are after several days in culture.

TN02

Transferring iPS cells to the DEF-CS system

Cells grown in other culture systems

Undifferentiated human iPS cells maintained in other culture systems can be readily transferred to the DEF-CS system. It takes 2–5 passages to adapt a cell line to the DEF-CS system. The standard DEF-CS culture protocol should be followed, although some modifications may increase the success of transfer:

  • The cells may benefit from a higher concentration of DEF-CS COAT-1 (coating substance). Use a dilution of 1:10 or 1:5 during the first passage to provide extra support during the transfer process.
  • A higher seeding density for the first passage (e.g., 8.0 x 104 cells/cm2) can also be beneficial to the cells.
  • Newly transferred cells might initially grow at a slightly slower rate. A suitable passage interval might, therefore, be between 3 and 7 days for the first passages. The cells are ready for passage when they have acquired the morphology in the images above (see 1.5 x 105 cells/cm2 and 2.0 x 105 cells/cm2). Even if the cells are sparse after 7 days in culture, passaging is still recommended.

Fresh iPS cell cultures

Fresh cultures can be transferred to the DEF-CS system at passage. The cells should be dissociated according to the protocol of the previous system and seeded as single cells or aggregates according to the DEF-CS protocol using a 1:1 split ratio.

Frozen iPS cells

Cryopreserved cells can be thawed directly into the DEF-CS system.

Learn more about the DEF-CS system »

Related Products

Cat. # Product Size Price License Quantity Details
Y30010 Cellartis® DEF-CS™ 500 Culture System 1 Kit $438.00

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 and its use are covered by one or more claims of patents owned by WARF, including U.S. Patent Nos. 7,514,260, 7,439,064, 7,005,252, 7,217,569 and their 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.

Notice to purchaser

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).


Cellartis DEF-CS 500 Culture System video protocol

Watch the video to learn how to culture human iPS cells

Learn more


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

Learn how the DEF-CS culture system outperforms other culture systems.

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