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Stem Cell Application Protocol

Transferring iPSCs from other feeder-free culture systems to the DEF-CS culture system

Introduction Materials required Protocol

Introduction  

Undifferentiated human iPS cells maintained in other feeder-free culture systems can be readily transferred to the DEF-CS culture system. Cryopreserved human iPS cells can be thawed directly using the DEF-CS culture system. Fresh cultures should be transferred on days when they would normally be passaged.

Materials required  

  • Cellartis DEF-CS 500 Culture System (Takara Bio, Cat. # Y30010)
    (includes COAT-1, Basal Medium, GF-1, GF-2, and GF-3)
  • TrypLE Select Enzyme (1X), no phenol red
    (Thermo Fisher Scientific, Cat. #12563011)
  • PBS Dulbecco's with Ca2+ & Mg2+ (D-PBS +/+)
  • PBS Dulbecco's w/o Ca2+ & Mg2+ (D-PBS –/–)
  • Cell culture vessels, tissue-culture-treated polystyrene surface

Protocol  

Transferring fresh or frozen cultures to the DEF-CS system

Preparation

  • Coating of cell culture vessels
    1. Dilute the required volume of DEF-CS COAT-1 in D-PBS +/+ before use. Make a 1:10 dilution.
    2. Mix the diluted DEF-CS COAT-1 solution gently and thoroughly by pipetting up and down.
    3. Add an appropriate volume of diluted DEF-CS COAT-1 solution to a cell culture vessel (use 0.1 ml/cm2), making sure that the entire surface is covered.
    4. Incubate the cell culture vessel at 37°C ± 1°C, 5% CO2, and >90% humidity for a minimum of 20 min, or at room temperature (15–25°C) for 0.5–3 hr.
    5. Aspirate the DEF-CS COAT-1 solution from the cell culture vessel immediately before use.
  • Preparing supplemented DEF-CS medium
    1. Prepare the appropriate volume of supplemented DEF-CS medium by adding DEF-CS GF-1 (dilute 1:333), GF-2 (dilute 1:1,000), and GF-3 (dilute 1:1,000) to DEF-CS Basal Medium.
    2. Prepare fresh medium on the day of intended use and warm it to 37°C ± 1°C immediately before use. Discard any leftover warm medium.
    3. Warm all other necessary reagents to room temperature (15–25°C) before use.

Transferring fresh hiPS cells from other feeder-free, monolayer culture systems

Fresh cultures should be transferred on days when they would normally be passaged. It is important to count the cells and use the recommended seeding density of 5–7 x 104 cells/cm2.

  1. Check cells under a microscope; photo document as necessary.
  2. Aspirate the medium from the cell culture vessel and wash the cell layer once with D-PBS –/–.
  3. Add 20 μl/cm2 of TrypLE Select Enzyme (1X) to the cell culture vessel and incubate for 5 min, or until the cell layer has detached. Detachment can be aided by swirling the cell culture vessel or by tapping the side of the cell culture vessel firmly but gently.
  4. Resuspend the cells in prewarmed, supplemented DEF-CS medium (40 μl/cm2) and pipet up and down several times to ensure a single-cell suspension. (The cells will aggregate if left too long in TrypLE Select Enzyme).
  5. Centrifuge the cells at 200g for 2–5 min.
  6. Count the cells in a hemocytometer or in a cell counter that has been optimized for the cell type.
  7. Add the appropriate volumes of cell suspension and additional medium (if necessary) to the newly coated cell culture vessel to obtain 5.0–7.0 x 104 cells/cm2. The seeding volume of supplemented DEF-CS medium should be 0.15–0.25 ml/cm2.
  8. Tilt the vessel backwards and forwards gently to ensure that the cell suspension is dispersed evenly over the surface, then place in an incubator at 37°C ± 1°C, 5% CO2, and >90% humidity.

Transferring cryopreserved hiPS cells from other feeder-free, monolayer culture systems

We recommend a seeding density at thawing of 1.5–2.5 x 105 cells/cm2.

  1. Thaw the cells according to your preferred protocol.
  2. Transfer the cells to a newly coated cell culture vessel with prewarmed, supplemented DEF-CS medium.
  3. Tilt the vessel backwards and forwards gently to ensure that the cell suspension is dispersed evenly over the surface, then gently place in an incubator at 37°C ± 1°C, 5% CO2, and >90% humidity.

Scaling up

The single-cell passaging method employed by the DEF-CS culture system causes iPS cells to initially assume a distinct morphology and sparser distribution relative to cells cultured using colony-based passaging methods. However, as the cells proliferate and form denser populations, morphologies commonly associated with undifferentiated stem cells (e.g., high nucleus-to-cytoplasm ratio, clearly defined borders, and prominent nucleoli) emerge.

  • It may take 2–5 passages to adapt a cell line to the DEF-CS culture system. 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 few passages.
  • Use a 1:10 dilution of DEF-CS COAT-1:D-PBS +/+ for the first few passages to provide extra support during the adaptation process.
  • To prevent cell loss during scale-up, we recommend not counting cells at passage when the total number of cells is quite low.
  • If the hiPS cells were sparsely seeded or thawed in aggregates, they will grow as colonies on COAT-1. As a general rule, when passaging hiPS cells that are growing as colonies, the area covered by the cells at passage should not be less than 20% of the area of the destination vessel.
  • For passages involving cells growing in a homogeneous monolayer (normal DEF-CS culture system characteristics), cells are ready for passage when they have acquired the morphology displayed in Figures 3 and 4 in the Cellartis DEF-CS 500 Culture System User Manual. However, if cells remain sparsely distributed after seven days in culture, a passage is still recommended. The area of the destination vessel should be 3–6 times the area of the current vessel.
  • Once cells have been scaled up to a T-25 flask, they should be cultured according to the Cellartis DEF-CS 500 Culture System User Manual.

Related Products

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

License Statement

ID Number  
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.

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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

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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. 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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