Induced pluripotent stem (iPS) cells originate from adult cells that have been reprogrammed with key transcription factors to exhibit pluripotency. Fibroblasts are a popular source of adult cells for reprogramming. Once the reprogramming factors have been delivered to fibroblasts, the cells can be transferred to the DEF-CS system to maximize the number of emerging colonies and for 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.

• 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

Preparation

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 Cellartis DEF-CS Medium for Maintenance by adding GF-1 (dilute 1:333) and GF-2 (dilute 1:1,000) to 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 Basal Medium.

Coating cell culture vessels

1. Dilute the required volume of COAT-1 in D-PBS +/+ before use. Make a 1:5 dilution at Day 7, 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).

Protocol overview

Protocol for the reprogramming of fibroblasts 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 fibroblasts 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 fibroblasts (Day 0)

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

Daily media changes (Days 1, 2, 3, 4, 5, and 6)

Prepare the appropriate volume of fibroblast medium (2 ml/well of a 6-well plate) and warm it to 37°C ± 1°C before use. On Day 1, you can perform the media change 24 hr ± 2 hr post-transduction.

• Media change 100% of the volume
  
  1. Carefully discard all of the media in the wells.
  2. Carefully add 2 ml of fibroblast medium per well.
  3. Place the cells in an incubator at 37°C ± 1°C, 5% CO₂, and >90% humidity.

Replate transduced fibroblasts onto coated 6-well plates (Day 7)

On Day 7 post-transduction, the transduced fibroblasts should be transferred to 6-well plates coated with COAT-1. Do not switch to Cellartis DEF-CS medium at this time. Plate the transduced fibroblasts using the fibroblast medium specific to your transduction method of choice.

• Preparation
  
  1. Warm the fibroblast medium to 37°C ± 1°C. Coat the appropriate number of wells of the 6-well plate(s) with COAT-1 diluted 1:5.
  2. Check the cells under a microscope; photo document as necessary.
  3. Aspirate all of the media from cell culture vessel and gently wash the cell layer with D-PBS –/–.
  4. Add 500 µl/well (of a 6-well plate) of TrypLE Select (room temperature) to the cells. Make sure that the entire surface of the well is covered. Incubate for 3 min, or until the cells have detached.
  5. Resuspend the cells in 2 ml/well (of a 6-well plate) of prewarmed fibroblast medium and transfer all cells from each well to a single centrifuge tube.
  6. Centrifuge the cells at 200g for 4 min.
  7. Carefully aspirate the media and resuspend the cells in an appropriate amount of fibroblast medium.
  8. Count the cells and seed them at 0.2–1.0 x 10⁴ cells/cm² into 6-well plate(s) coated with COAT-1. Use 2 ml of fibroblast medium per well. We recommend using two different seeding densities.
  9. 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.

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)
  
  1. Carefully discard all of the medium in the wells.
  2. Carefully add 2 ml 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 9–28)

• 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 medium in the wells.
  3. Carefully add 2 ml of Cellartis DEF-CS Medium for Maintenance to each 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 21–28 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 the 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. Working 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 I 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 the media from the cell culture vessel and gently wash the cell layer with D-PBS –/–.
  3. Add the appropriate volume (Table I) of TrypLE Select (room temperature) to the wells. 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 I) 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 I. Suggested schedule for scaling up of reprogrammed clones

*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.