This week, a first vial of iPSC derived from hair follicles was thawed, using the same conditions as the last culture method, i.e. coating with Vitronectin and Essential 8 Flex as medium.
According to literature, the average doubling time of this cell line is 3.3 days and they take a long time to recover after cryopreservation. Does anyone have experience with stem cells derived from hair follicles and can confirm this?
This particular vial was frozen in 2018, five years ago, which may affect survival after thawing.
Photos of the cell culture are included for reference.
Moreover, I am adding the sole multimedia material available online documenting the development of this particular induced pluripotent stem cell (iPSC) type: https://www.youtube.com/watch?v=Dw18Aqh-gZo
Thank you in advance.
There is no reasonable time limit for storage of cells in liquid nitrogen, assuming that they do in fact stay submerged in liquid nitrogen the entire time.
In the real world, their viability is far more likely to be limited by protocol lapses that cause them to warm up and re-freeze frequently. For example: Folks don't label the top of the vial, so they have to take the box out, rummage through it while all the other samples warm up. Folks think they're smart and store the samples on purpose in the vapor, not in the liquid, which warms up every time someone opens the lid of the tank. Folks forget to check the LN2 level, run out altogether, and everything warms up. Then they fill it back up and don't tell anyone it ever happened. I've seen it all.
That's the kind of thing that far more likely limits the life span of your frozen cells. And it's difficult to translated that in terms of time passed -- but I guess 5 years have a whole lot of room for that stuff to happen.
To me it rather looks like an issue of cell density to be honest. Probably too few cells were frozen, or thawing onto culture vessels with a too big growth area for the low number of cells. Try thawing onto smaller culture vessels.
The storage in LN should not be an issue after 5 years, given they were kept frozen all the time (as explained already).
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