Hello,
Recently I thawed couple of cell lines, RPMI8226 and JJN3 and they both didn't do well during my first passage.
I thawed the vials in 37 deg water bath for couple of minutes. Transferred the cells with media into a conical tube. Centrifuged the tube at the lowest speed for 5mins. Aspyrated the supernatant and resuspended the cells in 10mL media in T-75 flask for 3 days.
I use the following media:
Gibco RPMI 1640 with L-glutamine + Penstrep + Glutamine + 10% FBS
My percent live cells was very low when I checked them 3 days later. I am thinking of using 20% FBS next time I thaw cells. Is there anything else I can do differently to keep the cells alive?
Thank you!
You mention thawing the vial at 37 degrees "for couple of minutes". Be careful how long you let the cells warm up in the presence of the DMSO that's in the freezing media. DMSO can be toxic to cells (I believe the cut-off is thought to be around 0.1% - the freezing media is usually well above that, 10% or so). So, that could be one issue.
We hold the vial in the water bath at 37 degrees and swirl it. The instant it is liquid, it immediately gets diluted in media.
I'd also check the process by which your cells are being frozen. If they stay in the DMSO freezing media for too long at room temp, it can also affect the viability of the cells. If they are frozen incorrectly, nothing you do during the thawing process will help.
When you the the cells in water bath pay attention to the it to like 75% or less not more than that and try to add about one ml of fresh medium drop by drop and slowly to the cells before transferring to centrifuge tube to let the cells adjusted to the normal temperature. You mention that after 3 days you have some cells alive this could due to the storage of cells you need to storage cells with DMSO and FBS at -80 for at least few hours or one day before storage at liquid nitrogen this will help cells not to broken down if you store them immediately at liquid nitrogen.
zainab
- Badges
- Science topic
- Similar topics
- Biological Science
- Cell Biology
- Culture Cells