TITLE: Basic maintenance protocol for human induced pluripotent stem cell (hIPSCs)
AUTHORS: William J Buchser, Mallory Wright, purva patel, jwaligor, ckremitz

[DESCRIPTION]
Human Induced pluripotent stem cell (hIPSCs) maintenance protocol. This includes thawing, passaging, performing a media change, single cell dissociating, and freezing hIPSCs.

[STEPS]
SECTION: Thawing iPSCs
1. Quickly thaw the frozen vial in a 37 °C water bath 2 min
 


SECTION: Thawing iPSCs
2. Using a 10 ml serological pipette, add 9 mL of media into 15 ml conical tube. 

SECTION: Thawing iPSCs
3. Add the contents of the cryovial into the 15 ml conical tube with a 5 ml serological pipette
 

SECTION: Thawing iPSCs
4. Spin down conical tube 1200 rpm, 5 min 

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


SECTION: Thawing iPSCs
6. Add the whole cell suspension  into one well of a 6-well plate coated with matrigel  

SECTION: Passaging IPSCs
7. Ideal confluency for passaging IPSCs is about ~60%. This would be about 1 million cells in a 6-well plate.
 
SECTION: Passaging IPSCs
8. Aspirate the media off the wells from the plate
SECTION: Passaging IPSCs
9. Rinse each well or flask with DPBS -/- (1ml per well of a 6 well, 3 ml per T-25)
SECTION: Passaging IPSCs
10. Aspirate the DPBS -/-. You can keep this in the hood at room temperature for storage.
SECTION: Passaging IPSCs
11. 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 1 min at Room temperature
 



SECTION: Passaging IPSCs
12. Fully aspirate ReLeSR and leave the plate in the incubator for5 min
 


SECTION: Passaging IPSCs
13. 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)
SECTION: Passaging IPSCs
14. Use the same serological pipette to rinse the wells 3-4 times to capture all the cells.
SECTION: Passaging IPSCs
15. Use the same pipette to transfer cells to a 15 ml conical tube. Triturate gently without bubbles once or twice with a P-1000. 
SECTION: Passaging IPSCs
16. 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).
 

SECTION: Passaging IPSCs
17. Place wells back into incubator 
SECTION: Performing a media change
18. 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
 

SECTION: Single-cell dissociating iPSC
19. 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).
SECTION: Single-cell dissociating iPSC
21. Put in the incubator until you see cells detaching (2 minutes to 5 minutes MAX), move the flask around to aid in that process.
SECTION: Single-cell dissociating iPSC
20.  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).
SECTION: Single-cell dissociating iPSC
22. After cells detach, add 2x the original volume minimum of fresh media in the vessel (to stop enzymatic reactions)
SECTION: Single-cell dissociating iPSC
23. Take cells and put them in a 15ml conical tube. Spin down at200 rpm, 5 min (200g/RCF). 

SECTION: Single-cell dissociating iPSC
24. 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.
 

SECTION: Freezing iPSCs
25. Aspirate the media and rinse with 1 ml DPBS-/- per well. Add 1mL of ReLeSR to each well. Aspirate ReLeSR after 1-minute incubation
SECTION: Freezing iPSCs
26. Add 1mL of ReLeSR to each well. Aspirate ReLeSR after 1-minute incubation
SECTION: Freezing iPSCs
27. Allow the vessel to sit for 7 min in the incubator. Checking every minute to ensure gaps are forming in the clusters.

SECTION: Freezing iPSCs
28. Aspirate 1mL of Cryostor CS10 (keep Cryostor on ice) for each well using a 5mL serological pipette.
SECTION: Freezing iPSCs
29. 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