Resources

Below are some basic protocols for working with human pluripotent stem cells (last updated July-2022). For any questions on training services or if extra detail is required, please contact hpscf@ohri.ca.

• Preparing Matrigel Plates

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

1. Thaw matrigel bottle at 4°C. Check the certificate of analysis on that lot to find its protein concentration.

2. Add enough cold DMEM/F12 media to the thawed matrigel to reach a final concentration of 5mg/mL.

3. Make 1mL aliquots of the prepared matrigel from step 2 in pre-chilled 15mL falcon tubes.

4. Freeze and store all aliquots at -20°C.

5. To make matrigel plates, remove one aliquot of matrigel (1mL) from -20°C and add 10mL of cold DMEM/F12. Mix well until the pellet thaws (without creating bubbles, and always keep the solution cold).

6. Transfer to a 50mL tube, then add 20mL of cold DMEM/F12 (1mL of matrigel aliquot is diluted in 30mL of DMEM/F12), and mix well.

7. Plate (1mL/well for 6 well plate, 0.5mL/well for 12 well plate, 0.25mL/well for 24 well plate). Make sure that the solution covers the entire surface area by shaking the plate gently. Do not re-freeze any leftover matrigel.

8. If using the plate immediately, let the plate sit at room temperature for 1 hour (or 30 minutes at 37°C), and observe matrigel under the microscope. Matrigel should be well dispersed and not "clumpy".

9. If using plates at another time, wrap the edge of the plate with parafilm and store at 4°C for up to 2 weeks.

Matrigel – BD/Fisher, cat#CB-40230

DMEM/F12 – Life Technologies, cat#11330-057

• Thawing ES or iPS cells (per cryo-vial)

1. Take a matrigel plate out of 4°C and warm at room temperature for one hour, or make a fresh matrigel plate (see preparing matrigel plate protocol).

2. Warm 4mL of Pluripotent Stem Cell (PSC) media in a 15mL falcon tube.

3. Remove cells from the liquid nitrogen tank and swirl in a 37°C bath until only a small ice chunk is left. Vial should thaw in 1-2 minutes. This step must be done quickly.

4. Ethanol cell tube and falcon media tube and place in the hood.

5. Use a 1mL wide mouth tip to slowly add cells to 4mL of pre-warmed media (avoid mixing cell suspension).

6. Spin at 130 rcf for 5 minutes.

7. Remove supernatant.

8. Add 2mL of PSC media, and with a wide mouth tip break up clumps gently. Transfer media to one well of a 6-well plate and add 2μL of ROCK inhibitor (Y27632, final concentration of 10μM). Plate into one matrigel coated well (of a 6-well plate).

9. Rock cells gently to evenly distribute cells, and place in a hypoxic incubator (5%O2, 10% CO2). Avoid plate disturbance for 24 hours post-seeding.
   NOTE: It is very important to avoid excessive breaking down of clumps or aggressive pipetting. This can significantly reduce the survival rate. The cells should remain in chunks of 100-300 cells at the time of seeding. While being gentle, try working quickly once cells are thawed to minimize the time they are in contact with cryoprotectant.

10. Remove media after 24 hours and add 2mL of PSC media (for a 6 well, 1mL for 12 well, and 0.5mL for 24 well plates). See Harvesting and Caring for hESC/iPSC protocol.

• PSC Media

mTeSR Plus from Stem Cell Technologies (cat# 5825). Please follow the supplier’s recommendations for storage.

• What is ROCK Inhibitor Y27632?

ROCK Inhibitor Y27632 is a selective inhibitor of the Rho-associated kinase p160 ROCK. 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. Note that Rock inhibitor aliquots are sensitive to light and repetitive freeze-thaw cycles. Make sure to use these aliquots within the supplier’s recommended shelf life.

• Harvesting and Caring for hESC/iPSC

1. The day after cells were thawed take a look at them under the microscope to determine the survival rate. NOTE: it is normal to observe a high number of unattached cells. As long as some cells are attached, colonies can arise from them within 3 - 7 days.

2. Remove media from the wells and pipette 2 mL (for a 6-well plate, 1mL for 12 wells, and 0.5mL for 24 wells plate) of fresh and warm PSC media per well. Return the plate to the incubator.

3. Cells are fed until 60-70% confluent (follow supplier recommendations).

4. As of day 2, cells should be observed and cleaned of any differentiated cells that might be growing.

5. To clean cells, use the picking hood and scrape off differentiated cells with a pipette tip.

6. Once cells are cleaned, change media as done in the above steps.

(See Freezing hESC/iPSC or Passing hESC/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. Lastly, seeded cells have a better survival rate when using a hypoxic incubator.

Normoxic: 37°C, 21% O2, 5% CO2

Hypoxic: 37°C, 5%O2, 10% CO2