Oct 18, 2024
Version 4
Basic Maintenance Protocol for Human Induced Pluripotent Stem Cells (hiPSCs) Introduction V.4
- 1Washington University, Saint Louis
- WashU FIVE @ MGI
Protocol Citation: William Buchser, Mallory Wright, Purva Patel, Jason Waligorski, Colin Kremitzki 2024. Basic Maintenance Protocol for Human Induced Pluripotent Stem Cells (hiPSCs) Introduction. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbx1q1lpk/v4Version created by Mallory Wright
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 18, 2024
Last Modified: October 18, 2024
Protocol Integer ID: 110313
Keywords: pluripotent stem cell, human induced pluripotent stem cell, induced pluripotent stem cell, freezing hipsc, thawing hipsc, cryopreservation, management of hipsc, cell, covering thawing
Abstract
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.
Materials
ROCKiSTEMCELL Technologies Inc.Catalog #72304 -20 °C
Note
- iPSCs like being in clusters. Single cell iPSCs tend to die. ROCKi prevents that from happening
- Need a 10mM stock: dilute 5mg of the powder in 1.56 ml nuclease free water. Aliquot into smaller quantities. Store in -20C.
- RevitaCell is a good alternative, either can be used
ReLeSRSTEMCELL Technologies Inc.Catalog #05872 Room temperature
TrypLE Select (1X) no phenyl redThermo Fisher ScientificCatalog #12563-011 Room temperature
Matrigel Matrix hESC qualifiedCorningCatalog #354277 20 °C
DMEM/F12 (1:1)ThermofisherCatalog #11320033 4 °C
CryoStor CS10-100mlSTEMCELL Technologies Inc.Catalog #07930 4 °C
DBPS -/- (without calcium / magnesium)Thermo Fisher ScientificCatalog #14190144 Room temperature
Antibiotic/Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062 -20 °C
Thawing iPSCs
9m
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).
Note
Swirl the tube as the ice melts until only a tiny amount of ice is in the vial, and then remove it from the water bath.
Using a 10 mL serological pipette, add 9 mL of media into a 15 mL conical tube.
2m
Using a 5 mL serological pipette, transfer the contents of the thawed cryovial into the 15 mL conical tube containing the media.
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
After centrifugation, carefully aspirate the supernatant without disturbing the cell pellet.
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
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
Note: When adding the cells to the 6-well plate, shake the plate in a square motion—up and down, and side to side—to ensure even distribution of the clusters.
Cells immediately after thawing; high density due to some cell death.
Passaging IPSCs
6m
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.
Example image of ideal confluency for passaging at ~ 60%. This would be about 1 million cells in a 6-well plate.
Aspirate the media from each well or flask.
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 -/-.
Aspirate the DPBS -/- from each well or flask.
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.
Note
ReLeSR is an enzyme-free reagent for the dissociation of cells as aggregates without manual selection of differentiated areas or scraping to remove cell aggregates
Fully aspirate the ReLeSR and leave the plate in the incubator for 5 minutes (ideally). Note: You may need to optimize this timing for your specific cell type.
Note
If cells were recently treated in ROCKi, leave cells for 5-10 more minutes). After the incubation period, you may observe gap formations in the colonies.
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
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.
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!
Note: If the aggregate size is too large after plating a well, triturate with a P1000 pipette one more time to reduce the aggregate size
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.
Note: Lid the plate and immediately tap gently on two sides or move the plate in a square motion—up and down, and side to side—to evenly distribute the clusters. Avoid circular motions, as they can lead to poor distribution.
What the transferred cells look like immediately after plating (this is low density, see first image for a higher density version and add more cells)
Performing a Media Change for iPSCs
Media Preparation:
Ideally, bring the media to room temperature (RT) before use.
Important: Do NOT warm mTeSR media in a water bath at 37°C. To bring it to RT, simply remove it from the fridge and let it sit for 30 minutes to 1 hour.
Daily Media Change:
Aspirate the old media from wells or flasks and add 2 mL of fresh mTeSR per well. While cells can go more than one day without a media change, we have not tested that approach.
Freezing iPSCs
7m
Aspirate the media and rinse with 1 ml DPBS-/- per well.
Add 1mL of ReLeSR to each well. Aspirate ReLeSR after 1-minute incubation
Allow the vessel to sit in the incubator for the optimized time for your cells (usually 5-7 minutes), checking occasionally 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 1-2 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