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.

• 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

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

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.

• 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).

• 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.

• 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.

*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.