Protocols for working with induced pluripotent stem cells (iPSC).

Thawing iPS cells (per cryo-vial)

  1. Warm 4ml of PSC media + gent in a 15ml falcon tube
  2. Remove cells from freezer and swirl in 37 degree water bath until only a small ice chunk is left
  3. Ethanol cell tube and falcon media tube and place in the hood.
  4. Use a 1ml wide mouth tip to slowly add cells to 4ml of pre-warmed media
  5. Spin at 850rpm for 5min
  6. Remove supernatant
  7. Add 2ml of PSC media+gent and with a wide mouth tip break up clumps. Transfer media to one well of a 6 well plate add 2ul of ROCK inhibitor (Y27632, final concentration of 10uM)Plate into one matrigel coated well (of a 6 well plate)
  8. Remove media after 24 hours and add 2ml of PSC media + gent (for a 6 well, 1ml for 12 well and 0.5ml for 24 well plate). See Harvesting and Caring for iPSC protocol

What is ROCK Inhibitor Y27632?

ROCK Inhibitor Y27632 is a selective inhibitor of the Rho associated kinase p160ROCK. Treatment with ROCK Inhibitor Y27632 prevents dissociation induced apoptosis of human embryonic stem cells (hESC) and human induced pluripotent stem cells (iPSC), increasing the survival rate and maintaining pluripotency during subcultivation and thawing of hESCs and hiPSCs. ROCK Inhibitor Y27632 also has been shown to enhance the survival rate of stem cells during cryopreservation.

Preparing Matrigel

Note: All steps involving matrigel should be done as quickly as possible and stay as cold as possible.

  1. Take matrigel bottle (10ml) and add 10ml of cold DMEM/F12 media.
  2. Aliquot 1ml of matrigel in pre-chilled 15ml falcon tubes.
  3. Freeze all aliquots at -20C.
  4. To make matrigel plate, remove one aliquot of matrigel (1ml) from -20C and add 18ml of cold DMEM/F12. Let pellet thaw and once thawed mix well (without creating bubbles) and plate (1ml/well for 6 well plate, 0.5ml/well for 12 well plate, 0.25ml/well for 24 well plate)
  5. If using plate immediately, let plate sit at room temperature for 1 hour, observe matrigel under the microscope. Matrigel should be well dispersed and not "clumpy".
  6. Transfer matrigel to a second plate (this is now your MG2 plate). Wrap edge of plate with paraffin and store at 4 degrees.
  7. If using plate at another time, wrap edge of plate with paraffin and store at 4 degrees.

(material: Matrigel – BD/Fisher, cat#CB-40230 and DMEM/F12 – Life Technologies, cat#11330-057)

Harvesting and Caring for iPSC

Note: PSC media was determined to be more efficient in a hypoxic environment. We have also observed that less differentiation occurs when cells are grown in hypoxic incubators versus normoxic. Also when seeding cells there’s a better survival rate when using a hypoxic incubator. Our PSC media is made in house but PSC media can be purchased through Life Technologies or Stem Cell Technologies.

  1. Remove PSC media+gent from 4C and place in 37C water bath.
  2. The day after cells were thawed take a look at them under the microscope to determine survival rate. (If cells were frozen only with mFreSR the survival rate is appr. 10%, if cells were frozen using the Bio-Cool survival rate is appr. 60%).
  3. Remove media from the wells and pipette 2ml (for a 6 well, 1ml for 12 well and 0.5ml for 24 wells plate) of fresh media per well.
  4. Place plate in 37C incubator (normoxic or hypoxic)
  5. Cells are fed every day for the next 3-5 days until 80% confluent.
  6. As of day two cells should be observed and cleaned of any differentiated cells that might be growing.
  7. To clean cells use the picking hood and "scrap off" differentiated cells with a pipette tip.
  8. Once cells are cleaned changed media as done in above steps. (See Freezing iPSC or Passing iPSC)

Normoxic: 37C and 5% CO2

Hypoxic: 37c and 5% Nitrogen, 10% CO2

Passaging with EDTA solution

EDTA solution: Add 500ul of 0.5M EDTA (pH 8.0) into 500ml of DPBS (-/-). Add 0.9g of NaCl and adjust the osmolarity to 340 mOsm. Filter the solution to sterilize and store it at 4C for up to 6 months. We want the least amount of disturbance for the cells during dissociation therefore the EDTA solution is at the same osmolarity as the PSC media.

  1. Add 2ml of PSC media to a 6 well matrigel coated plate.
  2. Take the plate to be passaged and remove the media from the well and wash twice with 1ml of PBS(-/-).
  3. Add 1ml of the EDTA solution to the well and leave for 4min at room temperature. Don’t move the plate around as cells can start detaching.
  4. Once 4 min. is up remove EDTA solution and add 1ml of PSC media.
  5. Scrape cells and divide cells amongst the 6 wells of your plate containing PSC media (I’ve been taking 160ul into each well). Avoid breaking up the pieces in very small pieces. Try to keep large chunks. Preferably use a wide mouth pipette tip.
  6. Swirl and incubate at 37C.

Note: Once the cells have been scraped you want to transfer them to the new plate as soon as possible because the cells will reattach quickly.

The above protocol was taken from the following paper:

Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions Jeanette Beers,1 Daniel R. Gulbranson,2,3 Nicole George,4 Lauren I. Siniscalchi,1 Jeffrey Jones,4,5 James A. Thomson,2,3,6 and Guokai Chen1,2

Freezing iPS Cells

  1. Turn on Bio-Cool (Controlled Rate Freezer) and adjust SP temperature to -7C.
  2. Remove cells from incubator and observe confluency (80% confluent) and morphology under the microscope.
  3. If confluent, remove old media and wash twice with PBS(-/-) then add 1 ml of EDTA solution (see passaging with EDTA solution)
  4. Incubate at 37 degrees for 4 minutes.
  5. Aspirate off the EDTA solution and add 1 ml of mFreSR media (cat#05855, Stem Cell Technologies)
  6. Use cell scraper to score cells in equal sizes (5 horizontal scores and 5 vertical scores per well) then scrape all cells into a corner.
  7. Aliquot 1ml or resuspended cells per cryotube using a wide mouth tip.
  8. Place tubes in Bio-Cool and incubate for 10min.
  9. Get liquid nitrogen.
  10. After 10min. seed the cells by dipping a spatula into liquid nitrogen and touching the side of the cryo-vial for approximately 10 seconds or until you see a crystal form on the side of the cryo-vial.
  11. Start program 1 by pushing "PROG" button and going through the program by pushing the button again and you should see the rate of 0.5C/min. , then press "RUN".
  12. Once temperature reaches -65C the cryo-tubes can be transferred and stored in liquid nitrogen.


Another option is to place the cryo-tubes in a freezing container (Biocision-CoolCell) and store at -80C overnight. The next day store cryo-tubes in liquid nitrogen