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User-generated protocol

Reprogramming peripheral blood mononuclear cells (PBMCs) using the Cellartis DEF-CS 500 Culture System

Introduction Materials required Protocol

Introduction  

Induced pluripotent stem (iPS) cells originate from adult cells that have been reprogrammed with key transcription factors to exhibit pluripotency. PBMCs are a popular source of adult cells for reprogramming due to routine blood collection methods and the presence of a wide variety of banked blood samples. Once the reprogramming factors have been delivered to PBMCs, the cells can be transferred to the DEF-CS system to maximize the number of emerging colonies and for the robust expansion into stable iPS cell lines.

This protocol has been developed using Sendai viruses for delivery of the transcription factors. Optimization may be necessary if using other delivery methods.

Materials required  

  • Cellartis DEF-CS 500 Culture System (Takara Bio, Cat. # Y30010)
    (includes COAT-1, Basal Medium, GF-1, GF-2, and GF-3)

  • Stem Cell Cutting Tools (Vitrolife, Cat. # 14601)

  • Transfer Pipettes (Vitrolife, Cat. # 14319)

  • PBS Dulbecco's with Ca2+ & Mg2+ (D-PBS +/+)

  • PBS Dulbecco's w/o Ca2+ & Mg2+ (D-PBS –/–)

  • TrypLE Select Enzyme (1X), no phenol red
    (Thermo Fisher Scientific, Cat. # 12563011)

  • Cell culture vessels, tissue-culture-treated polystyrene surface

Protocol  

Preparing medium and coating cell culture vessels

Detailed information about media preparation is available in the Cellartis DEF-CS 500 Culture System User Manual.

Maintenance medium for human iPS cells

Prepare an appropriate volume of supplemented Cellartis DEF-CS Medium for Maintenance by adding GF-1 (dilute 1:333) and GF-2 (dilute 1:1,000) to Cellartis DEF-CS Basal Medium.

Passaging medium for human iPS cells

Prepare an appropriate volume of Cellartis DEF-CS Medium for Passaging by adding GF-1 (dilute 1:333), GF-2 (dilute 1:1,000), and GF-3 (dilute 1:1,000) to Cellartis DEF-CS Basal Medium.

Coating cell culture vessels

  1. Dilute the required volume of COAT-1 in D-PBS +/+ before use. Make a 1:5 dilution on Day 3, at colony picking, and for the first passage during scale-up. Use a 1:20 dilution for subsequent passages.
  2. Mix the diluted COAT-1 solution gently and thoroughly by pipetting up and down.
  3. Add the appropriate volume of diluted COAT-1 solution to the cell culture vessel, making sure the entire surface is covered. Use the following volumes of COAT-1 solution: 50 µl/well of a 96-well plate, 200 µl/ well of a 48-well plate, 400 µl/well of a 24-well plate, 800 µl/well of a 12-well plate, or 1.5 ml/well of a 6-well plate.
  4. Place the cell culture vessel in an incubator for a minimum of 20 min at 37°C ± 1°C, 5% CO2, and >90% humidity, or for 0.5–3 hr at room temperature (RT, 15–25°C).
  5. Aspirate the COAT-1 solution from cell culture vessel just before use.

Protocol Overview

Reprogramming PBMCs timeline

Protocol for the reprogramming of PBMCs into iPS cells generates colonies that show typical monolayer growth when passaged in the Cellartis DEF-CS 500 Culture System. Panel A. Suggested schedule for reprogramming of PBMCs in the DEF-CS system. Panel B. Representative photos of the cells during reprogramming and after transfer into the DEF-CS system.

Reprogramming protocol

Transduce PBMCs (Day 0)

Deliver the reprogramming factors to your PBMCs using your method of choice.

Replate transduced PBMCs onto coated 6-well plates (Day 3)

On Day 3 after transduction, the transduced PBMCs should be transferred to 6‑well plates coated with COAT-1. Do not switch to Cellartis DEF-CS medium at this time. Continue culturing the transduced PBMCs using the PBMC medium specific to your transduction method of choice.

NOTE: Change pipette tips between wells to prevent cross-contamination.

  • Preparation
    Warm the PBMC medium to 37°C ± 1°C. Coat the appropriate number of wells in the 6-well plate(s) with COAT-1 diluted 1:5.

  • Passaging
    1. Transfer the cell suspension from each well to a separate sterile centrifuge tube.
    2. Rinse each well with 500 µl of the PBMC medium and transfer to the corresponding tube.
    3. Centrifuge the cells at 200g for 10 min.
    4. Discard the supernatants and resuspend each cell pellet in 500 µl of the PBMC medium.
    5. Count the cells in a hemocytometer or a cell counter (optimized for the specific cell type).
    6. Seed 2.5–5 x 104 cells/well in 6-well plate(s) coated with COAT-1. Use 2 ml of PBMC medium per well. We recommend using two different seeding densities.

PBMC media changes (Days 4 and 6)

For media changes on Days 4 and 6, do not switch to Cellartis DEF-CS medium. Continue using the same PBMC medium used on Day 3 (above).

NOTE: Change pipette tips between wells to prevent cross-contamination.

  • Preparation
    Warm the PBMC medium (1 ml/well of a 6-well plate) to 37°C ± 1°C.

  • Media change (50% of the volume)
    1. Carefully discard 1 ml of media per well.
    2. Carefully add 1 ml PBMC medium per well.
    3. Place the cells in an incubator at 37°C ± 1°C, 5% CO2, and >90% humidity.

Begin the transition to DEF-CS medium (Day 7)

  • Preparation
    Prepare the appropriate volume of supplemented Cellartis DEF-CS Medium for Maintenance (1 ml/well of a 6-well plate) and warm it to 37°C ± 1°C before use.
  • Media change (50% of the volume)
    1. Carefully discard 1 ml of PBMC medium per well.
    2. Carefully add 1 ml of supplemented Cellartis DEF-CS Medium for Maintenance per well.
    3. Place the cells in an incubator at 37°C ± 1°C, 5% CO2, and >90% humidity for 24 hr.

Complete the transition to DEF-CS medium (Day 8)

  • Preparation
    Prepare the appropriate volume of Cellartis DEF-CS Medium for Maintenance (2 ml/well of a 6-well plate) and warm it to 37°C ± 1°C before use.
  • Media change (100% of the volume) 24 hr ± 2 hr after performing the media change on Day 7
    1. Carefully discard all of the media in the wells.
    2. Carefully add 2 ml of Cellartis DEF-CS Medium for Maintenance.
    3. Place the cells in an incubator at 37°C ± 1°C, 5% CO2, and >90% humidity.

Daily DEF-CS media changes (Days 9–21)

  • Preparation
    Prepare the appropriate volume of Cellartis DEF-CS Medium for Maintenance (2 ml/well of a 6-well plate) and warm it to 37°C ± 1°C before use.
  • Media change (100% of the volume)
    1. Examine the cells under a microscope and check for colonies; photo document as necessary.
    2. Carefully discard all of the media in the wells.
    3. Carefully add 2 ml of Cellartis DEF-CS Medium for Maintenance per well.
    4. Place the cells in an incubator at 37°C ± 1°C, 5% CO2, and >90% humidity.

Colony picking (during the time span of Days 15–21 post-transduction)

When colonies are 1.5–3 mm in diameter, they are ready to be transferred/picked.

  • Preparation
    Prepare the appropriate volume of Cellartis DEF-CS Medium for Passaging (250 µl/well of a 48-well plate) and warm it to 37°C ± 1°C before use. Coat the appropriate number of wells (one well for each colony to be picked) with 200 μl/well of COAT-1 solution, diluted 1:5.
  • How to pick colonies
    1. Aspirate the COAT-1 solution from cell culture vessel and add 250 µl of Cellartis DEF-CS Medium for Passaging per well.
    2. Try to keep the plate at 37°C ± 1°C.
    3. Work under a dissection microscope, use a fresh Stem Cell Cutting Tool to microdissect a colony into 2–4 pieces.
    4. Use a fresh Transfer Pipette to transfer each piece into a separate well of the 48-well plate.
    5. Repeat Steps 3 and 4 until the desired number of colonies has been picked.
    6. Place the cells in an incubator at 37°C ± 1°C, 5% CO2, and >90% humidity for 48–54 hr. Do not move the plate during this time.

Scale-up using the Cellartis DEF-CS 500 Culture System

  • Changing media during scale-up
    Prepare the appropriate volume of Cellartis DEF-CS Medium for Maintenance and warm to 37°C ± 1°C immediately before use.

    1. Check cells under a microscope; photo document as necessary.
    2. Carefully aspirate the medium and pipet newly warmed medium into the cell culture vessel. Avoid flushing medium directly onto the cell layer.
    3. Place the cell culture vessel in an incubator at 37°C ± 1°C, 5% CO2, and >90% humidity.
  • Passaging during scale-up
    Prepare the appropriate volume of Cellartis DEF-CS Medium for Passaging and warm to 37°C ± 1°C immediately before use. Coat the appropriate number of wells with COAT-1 (1 well per clonal population, in the appropriate format; see Table 1 below). As a general rule, the area covered by the cells at passage should not be less than 20% of the area of the destination vessel. Passage single and/or small colonies to a new 48-well plate. If a larger area in the well is covered by cells, passage to a 24-well plate.

    1. Check the cells under a microscope; photo document as necessary.
    2. Aspirate media from cell culture vessel and gently wash the cell layer with D-PBS –/–.
    3. Add the appropriate volume (Table 1) of TrypLE Select (room temperature) to the cells. Make sure that the entire surface of the well is covered. Incubate for 5 min or until the cells have detached.
    4. Resuspend the cells in the appropriate volume (Table 1) of prewarmed Cellartis DEF-CS Medium for Passaging and transfer all cells from a well to a newly coated well.

      NOTE: Counting the cells is not recommended.
    5. Immediately after plating, hold each cell culture vessel in one hand and mix gently using a figure-eight motion, which distributes the cells evenly over the surface. Place in an incubator at 37°C ± 1°C, 5% CO2, and >90% humidity.

      NOTE: When the cells have been scaled up to one T-25 flask per clone, the lines should be cultured according to the Cellartis DEF-CS 500 Culture System User Manual.

Table 1. Suggested schedule for scaling up reprogrammed clones.

Passage number Starting format Passage interval* New format Dilution of COAT-1 Volume of diluted COAT-1 Volume of TrypLE Select Volume of Cellartis DEF-CS Medium for Passaging
Colony picking Colony in 1 well in 48-well plate 5–10 days 1 well in 48-well plate 1:5 200 μl/well in 48-well plate 50 μl/well in 48-well plate 250 μl/clone
1 (start scale-up) 1 well in 48-well plate 2–5 days 1 well in 24-well plate 1:5 400 µl/well in24-well plate 50 µl/well in 48-well plate 1 ml/clone
2 1 well in 24-well plate 2–5 days 1 well in 12-well plate 1:20 800 µl/well in 12-well plate 100 µl/well in 24-well plate 2 ml/clone
3 1 well in 12-well plate 2–5 days 1 well in 6-well plate 1:20 1.5 ml/well in 6-well plate 200 µl/well in 12-well plate 3 ml/clone
4 1 well in 6-well plate 2–5 days 1 T-25 flask 1:20 2.5 ml per T-25 flask 300 µl/well in 6-well plate 5 ml/clone


*If a clone grows fast and the culture is very dense at passaging, it is possible to expedite the scale-up by skipping some vessels—i.e., passaging from a well of a 24-well plate directly into a well of a 6-well plate.

Related Products

Cat. # Product Size Price License Quantity Details
Y30010 Cellartis® DEF-CS™ 500 Culture System 1 Kit USD $495.00

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.

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).

Back

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.

Back

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.

Back

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).

Back

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.

Back

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.

Back

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).


User-generated protocols

User-generated protocols

User-generated protocols are based on internal proof-of-concept experiments, customer collaborations, and published literature. In some cases, relevant results are discussed in our research news BioView blog articles. While we expect these protocols to be successful in your hands, they may not be fully reviewed or optimized. We encourage you to contact us or refer to the published literature for more information about these user-generated and -reported protocols. 

If you are looking for a product-specific, fully optimized User Manual or Protocol-At-A-Glance, please visit the product's product page, open the item's product details row in the price table, and click Documents. More detailed instructions for locating documents are available on our website FAQs page.

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What does it take to generate good science? Careful planning, dedicated researchers, and the right tools. At Takara Bio, we thoughtfully develop best-in-class products to tackle your most challenging research problems, and have an expert team of technical support professionals to help you along the way, all at superior value.

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Takara Bio USA, Inc. provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. As a member of the Takara Bio Group, TBUSA is part of a company that holds a leadership position in the global market and is committed to improving the human condition through biotechnology. Our mission is to develop high-quality innovative tools and services to accelerate discovery.

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES (EXCEPT AS SPECIFICALLY NOTED).

Clontech, TaKaRa, cellartis

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