Hi everyone, I have recently started working with iPSC's, specifically Kolf1.2J and WTC11 lines, and am having issues with reviving the cells where they do not attach post thaw and plating.
For cryopreservation, cells were dissociated with accutase 1:3 in PBS for 1 min or versene (gentle cell disociation reagent from stemcelltech) for 5 min, washed with PBS, scraped in 1 ml of KSR media with 10% DMSO, transferred to Mr Frosty, transferred to -80C overnight, transferred to liquid Nitrogen the next day. When thawing cells, they're thawed fast at RT, put in 15 ml conical with 9ml of warm PBS, centrifuged 200g for 5 min RT, resuspended in stemflex media with 10um Rock inhibitor, and plated on synthemax coated wells.
I have revived other people's cryostocks and have not had issues, which suggests that the issue lies with my freezing process. Any suggestions or things to try would be greatly appreciated!
Hello Calvin Wong
You may have to make certain modifications in your cryopreservation protocol. Your thawing protocol seems okay.
During cryopreservation, is it necessary to scrape the cells? Don’t the cells detach from the substratum when treated with accutase/versene? If you must get the cells in suspension after detachment, you may tap the flask at the sides so that the loosely attached cells move into suspension.
After obtaining the cell suspension, centrifuge the cells at 1000rpm for 5-7 mins and discard the supernatant. Keep the cell pellet on ice. Meanwhile prepare the freezing medium required to freeze the cells. The freezing medium should consist of 90%FBS and 10% DMSO. FBS with its high protein content, protects cells against shear forces and gives the medium a desirable osmotic environment with a physiological viscosity. You may use less KSR instead (e.g., 25% instead of 90%) as an alternative to FBS. Prepare the freezing media beforehand and place on ice before use.
After having placed the cells in 1ml freezing media in freezing cryovials, you may transfer to Mr. Frosty, then transfer to -80 degree C overnight, and later transfer to liquid Nitrogen the next day.
Best,
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