From last week when i culture HEK293 and AAV293 cells from cyropreserve tubes, i faced the problem of cell floating when i observed it on next day. Although i add mycoplasma inhibitor and carry all the procedure with great care to avoid any contamination. My media contain 10% FBS and PS.
Any good suggestions.... ??????
Thanks.
I have not worked with AAV293 cells but it sounds like your cells did not survive the freeze-thaw. Are you certain the cells were frozen back appropriately and never thawed prior to waking them up? Was DMSO used or a cryomedia? Was the cell density in a good range?
I would try increasing the FBS percentage to 20-40% until the cells adhere and begin to replicate normally.
Dear Philip Zakas, we used DMSO and we checked the cell density, its almost 80% and more before cryopreservation. At the same quantity we cryopreserve the cells before and it works.
Some time when we thaw the cells than its grow well, however when we do passaging for the same cells after reaching the appropriate confluence, than we face the problem of floating cells.
So It would be better to increase the % of FBS during thawing or at passaging step? or at both ?
What % DMSO and what density in terms of cells per mL when frozen?
Once the cells are thawed, transfer the cells directly to a plate or flask with warm media with 20-40% FBS. Avoid centrifugation in case the g-forces are damaging the cells. Once you see cells adhering and replicating, replace the media to remove the floating cells. The residual DMSO from the cryovial should not be an issue in the flask since it will be significantly diluted out.
We use 10% DMSO and the density of cell per 100mm plate was 80-90%.
Thanks Philip Zakas for your humble feedback. I will follow your suggestions, i hope it will work.
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