Apr 28, 2025
Human Pluripotent Stem Cell Culture
- Anita Adami1
- 1Laboratory of Molecular Neurogenetics, Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, BMC A11, Lund University, 221 84 Lund, Sweden.
External link: https://doi.org/10.1016/j.xgen.2025.100979
Protocol Citation: Anita Adami 2025. Human Pluripotent Stem Cell Culture. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg334pqg25/v1
Manuscript citation:
Adami A, Garza R, Gerdes P, Johansson PA, Dorazehi F, Koutounidou S, Castilla-Vallmanya L, Atacho DA, Sharma Y, Johansson JG, Tam O, Kirkeby A, Barker RA, Hammell MG, Douse CH, Jakobsson J (2025) LINE-1 retrotransposons mediate cis-acting transcriptional control in human pluripotent stem cells and regulate early brain development. Cell Genomics 5(10). doi: 10.1016/j.xgen.2025.100979
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: April 24, 2025
Last Modified: April 28, 2025
Protocol Integer ID: 152744
Keywords: ASAPCRN, human pluripotent stem cell culture this protocol, human pluripotent stem cell culture, pluripotent stem cell, induced pluripotent stem cell, stem cell, thawing
Funders Acknowledgements:
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-000520
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-025170
Abstract
This protocol described how to perform routine maintenance of human induced pluripotent stem cells (iPSCs), including freezing and thawing.
Troubleshooting
Coating Plates with Laminin-521
Coat your plates with Laminin-521 (0.7 μg/cm²; Biolamina) diluted in DPBS +/+ at 37°C for at least 2 hours before use or at 4°C overnight.
Thaw Frozen Cells
Take your vials of frozen cells and keep them on dry ice until ready.
Prepare one 15 ml tube with wash buffer (9.5 ml DMEM/F-12 + 0.5 ml KSR) per vial.
Put the vials in 37°C water bath. Check the vial periodically. Take vial out when there is only a small chunk of ice left.
Spray the vial with 70% EtOH. Wipe dry. In the hood, open the vial and take the cells out of the vial with a P1000 into your tubes with wash media.
Pellet cells in the conical tube at 400g for 5 min. Take off supernatant. Resuspend the cell pellet in appropriate amount of iPS media supplemented with Rock inhibitor (StemMACS iPS-Brew XF, 0.5% penicillin/streptomycin and 10 μM of Y27632).
Plate the cells onto a Laminin-521 coated plate and put the plate back into the incubator.
Regular Culture of Human Pluripotent Stem Cells
Feed cells with pre-warmed iPS media every day (StemMACS iPS-Brew XF with 0.5% penicillin/streptomycin).
Split the cells when they are 70-90% confluent.
Take off media and rinse with 1 ml PBS -/-.
Add 500 μl of Accutase and incubate at 37°C for 7-10 minutes.
Monitor cell detaching under the microscope.
Harvest your cell suspension into a 15 ml tube with pre-warmed wash buffer.
Pellet cells in the conical tube at 400g for 5 min. Take off supernatant. Resuspend the cell pellet in appropriate amount of iPS media supplemented with Rock inhibitor (StemMACS iPS-Brew XF, 0.5% penicillin/streptomycin and 10 μM of Y27632).
Put the new plates in the incubator.
Freezing Cells
Split cells following the same steps as in the section above.
Count your cells with a cell counter and transfer the desired amount to a new 15 ml tube (we normally freeze 300,000 cells per vial) and spin at 400xg for 5 min.
Resuspend the pellet in the appropriate volume of Cryostor (1 ml per vial) and immediately transfer to the labeled cryotubes.
Put vials into Mr. Frosty and place the Mr. Frosty in a -80°C freezer as quickly as possible.
The next day, transfer all the vials to -150°C freezer or liquid nitrogen for long-term storage.