TITLE: Basic Maintenance Protocol for Human Induced Pluripotent Stem Cells (hiPSCs) Introduction
AUTHORS: William J Buchser, Mallory Wright, purva patel, Jason Waligorski, Colin Kremitzki

[DESCRIPTION]
This protocol details the maintenance of human induced pluripotent stem cells (hiPSCs), covering thawing, passaging, media changes, single-cell dissociation, and cryopreservation. It begins with thawing hiPSCs, followed by plating on an extracellular matrix (Matrigel) and regular media changes every 1-2 days. Cells are passaged upon reaching 60-70% confluency. Finally, the protocol includes guidelines for freezing hiPSCs for long-term storage. This streamlined approach ensures effective culture and management of hiPSCs.

[STEPS]
SECTION: Thawing iPSCs
1. Quickly thaw the frozen vial of iPSCs in a water bath at 37°C. Gently agitate the vial in the water bath until the cells are just thawed (usually takes about 1-2 minutes).
 


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

SECTION: Thawing iPSCs
3. Using a 5 mL serological pipette, transfer the contents of the thawed cryovial into the 15 mL conical tube containing the media.


SECTION: Thawing iPSCs
5. After centrifugation, carefully aspirate the supernatant without disturbing the cell pellet.

SECTION: Thawing iPSCs
6. Resuspend the cell pellet in 1 mL of mTeSR media containing ROCKi (Y-27632):
ROCKi: Use Y-27632 (STEMCELL Technologies Inc., Catalog #72304) at a final concentration of 1 µL per 1 mL of media



SECTION: Thawing iPSCs
7. Transfer the resuspended cells to the well containing 2 mL of mTeSR media in the 6-well plate.

Perform the transfer with minimal trituration to avoid excessive cell clumping or damage
 
 
  
 
 
SECTION: Passaging IPSCs
8. Ideal confluency for passaging iPSCs is approximately 60%, which corresponds to about 1 million cells per well in a 6-well plate.

Do not allow cells to reach 90% confluence. Pass cells more frequently if needed, such as on Monday and Friday. Some cells may be cultured at different concentrations to ensure availability on various days. Passage sooner if separate colonies begin to merge.


 
SECTION: Passaging IPSCs
9. Aspirate the media from each well or flask.
SECTION: Passaging IPSCs
10. Rinse each well or flask with DPBS -/- to remove residual media and non-adherent cells:
6-well plate: Add 1 mL of DPBS -/- per well.
T-25 flask: Add 3 mL of DPBS -/-.
SECTION: Passaging IPSCs
11. Aspirate the DPBS -/- from each well or flask.
SECTION: Passaging IPSCs
12. Add 1 mL of ReLeSR to each well of the 6-well plate or 3 mL of ReLeSR to the T-25 flask. Start your timer and allow ReLeSR to act on the cells for 1 minute.
 



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


SECTION: Passaging IPSCs
14. Remove the plate/flask from the incubator.

Use a 5 mL serological pipette to add 1 mL of mTeSR plus media to each well of the 6-well plate or 3 mL of mTeSR plus media to the T-25 flask.

Note: This step inactivates the ReLeSR
SECTION: Passaging IPSCs
15. Use the same serological pipette to rinse the well once, if needed, to collect the aggregates. Avoid being aggressive to prevent breaking down the clusters. 

Do not use a P-1000 tip for aspiration, as its fine bore can reduce aggregate size.
SECTION: Passaging IPSCs
16. Transfer the cell suspension from the wells/flask to a 15 mL conical tube. 

Gently triturate the cell suspension once with a P-1000 pipette, taking care to avoid bubbles. One trituration only!
SECTION: Passaging IPSCs
17. Transfer the desired amount of cells (typically a 1:10 split) into a new Matrigel-coated well. Ensure that the Matrigel is removed from the new well and that the well is pre-warmed with 2 ml of mTeSR media before adding the cells.

Plate the resuspended cells at a 1:10 dilution in each well of a 6-well plate.
For example, if you are plating into a 6-well plate and have a total of 1 mL of cell suspension, add 100 µL of the cell suspension per well.

Use the distributed technique: Aspirate 100 µL with a P-1000 pipette, go to a corner of the plate, and put the tip just under the surface. Watch the liquid level and dispense about ⅕ of the pipette tip's volume, count to 3, then keep the tip submerged, move to the other corners, and finally to the center of the plate, repeating the process.

Lid the plate and immediately gently tap on two sides in a left/right motion and a front/back motion to distribute the cells (avoid circular motion as it leads to poor distribution).

 
 
 
SECTION: Performing a Media Change for iPSCs
18. Ideally, you want to bring media to RT before use, however if you are under time constraints, using cold media is okay unless you are dealing with a problematic cell line.

 Do NOT warm media in a water bath at 37 C. To bring to RT, simply take out of the fridge for 30min - 1 hr.

Daily Media Change: Aspirate old media from wells or flasks and add 2mL of fresh mTeSR per well.


SECTION: Freezing iPSCs
19. 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
20. Add 1mL of ReLeSR to each well. Aspirate ReLeSR after 1-minute incubation
SECTION: Freezing iPSCs
21. Allow the vessel to sit for 5-7 minutes in the incubator. Checking every minute to ensure gaps are forming in the clusters.

SECTION: Freezing iPSCs
22. Aspirate 1mL of Cryostor CS10 (keep Cryostor on ice) for each well using a 5mL serological pipette.
SECTION: Freezing iPSCs
23. 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
SECTION: Thawing iPSCs
4. Spin down the contents of the conical tube at 200 x g for 5 minutes. While the cells are centrifuging, add 2 mL of mTeSR media+ ROCKi to one well of a 6-well plate