Basic maintenance protocol for human induced pluripotent stem cell (hIPSCs)

Quickly thaw the frozen vial in a 37°C water bath 0h 2m 0s

Using a 10 ml serological pipette, add 9mL of media into 15 ml conical tube.

Add the contents of the cryovial into the 15 ml conical tube with a 5 ml serological pipette

Spin down conical tube 1200rpm

When done spinning, aspirate supernatant very carefully and resuspend the pellet in 1 ml of mTeSR plus media with Rocki (Y-27632)

Add the whole cell suspension into one well of a 6-well plate coated with matrigel

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Ideal confluency for passaging IPSCs is about ~60%. This would be about 1 million cells in a 6-well plate.

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Aspirate the media off the wells from the plate

Rinse each well or flask with DPBS -/- (1ml per well of a 6 well, 3 ml per T-25)

Aspirate the DPBS -/-. You can keep this in the hood at room temperature for storage.

Add 1 ml ReLeSR* per well of 6-well plates or 3 ml ReLeSR for a T-25. Start your timer and leave it on the cells for 0h 1m 0s at Room temperature

Fully aspirate ReLeSR and leave the plate in the incubator for0h 5m 0s

Remove the plate/flask from the incubator, and use a 5ml serological to add 1 ml of mTeSR plus media in each well of the 6-well, or 3ml of that media in a T-25 (to inactivate the ReLeSR)

Use the same serological pipette to rinse the wells 3-4 times to capture all the cells.

Use the same pipette to transfer cells to a 15 ml conical tube. Triturate gently without bubbles once or twice with a P-1000.

Transfer cells to a 15 ml conical tube. Triturate gently without bubbles once or twice with a P-1000. Transfer the desired amount of cells (typically a 1:10 split) into a new matrigel coated well (make sure you remove matrigel media from the new well and leave the well in 2 ml mTeSR media before you add cells).

Place wells back into incubator

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Change the media daily for the iPSCs. Aspirate media off, rinse with DPBS-/-, and then add 2 ml mTeSR plus per well of a 6-well plate and 6ml of media per T-25 flask

Aspirate media and Rinse with DPBS-/-. Add 1X TrypLE, 1ml per 6-well, 2ml per T-25, 3ml per T-75 (can use as low as 0.75X TrypLE according to GESC, dilute in 0.5mM EDTA pH 8).

Add 1X TrypLE, 1ml per 6-well, 2ml per T-25, 3ml per T-75 (can use as low as 0.75X TrypLE according to GESC, dilute in 0.5mM EDTA pH 8).

Put in the incubator until you see cells detaching (2 minutes to 5 minutes MAX), move the flask around to aid in that process.

After cells detach, add 2x the original volume minimum of fresh media in the vessel (to stop enzymatic reactions)

Take cells and put them in a 15ml conical tube. Spin down at200rpm (200g/RCF).

Take off the supernatant, and resuspend the cell pellet in the media. Media per well in a 6-well is 2ml, 12-well is 1ml, T-25 is 5ml, T-75 is 20 mL.

Aspirate the media and rinse with 1 ml DPBS-/- per well. Add 1mL of ReLeSR to each well. Aspirate ReLeSR after 1-minute incubation

Add 1mL of ReLeSR to each well. Aspirate ReLeSR after 1-minute incubation

Allow the vessel to sit for 0h 7m 0s in the incubator. Checking every minute to ensure gaps are forming in the clusters.

Aspirate 1mL of Cryostor CS10 (keep Cryostor on ice) for each well using a 5mL serological pipette.

Dispense 1mL of Cryostor CS10 into each well. Each well is rinsed 2-3 times, then the resuspension is transferred to a vial (keep Cryostor on ice) using the 5mL serological bore to scrape/press on the surface of each well