improving viability of pelleted cells
Posted 14 March 2013 - 07:34 AM
Posted 14 March 2013 - 08:40 AM
Cell viability will be dependent upon temperature. When we grow cells under normal conditions this is at 37oC. When we subculture the cells they are at ambient temperature (approx 21oC) but the PBS, Trypsin and Media are all warmed to 37oC in a waterbath prior to adding to the cells. Any centrifugation is done again at ambient temperature.
Viable Cells can be kept for very short periods at 4oC (i.e. 5-10 minutes), but as you have found they will lose viability quickly over many hours. Also cells tend not to like being kept at a very high density i.e. like in a pellet.
Unfortunately you do not have an incubator which you can keep the cells in for as many hours as you require.
The only thing I can suggest is that you keep your endothelial cells at a lower concentration at ambient temperature. You can do this very easily by using a non TC treated flask which will not allow the suspended endothelial cells to adhere.
The other problem you have is that you are keeping the cells in HBSS. Again there are no supplements for the cells i.e. Gluatamine, Foetal Calf Serum, Minerals or Vitamins. Again you could supplement your HBSS with some of these?
I hope this gives you something to go on
Posted 15 March 2013 - 05:47 AM
I agree with Uncle Rhombus. Try ambient temperature.
What if instead of storing cells in lower counts, you suspended them in more medium? If volume does matter, then I guess it is worth trying to resuspend the pellet right after centrifugation (and then mix the cell suspension again prior usage). I would preferably use culture medium (would it matter to your experiments?)
Since you haven't got an incubator, I would suggest trying to keep cells in a waterbath at 37C. In case that you do not have a waterbath, then you could use "ice-packs" pre-warmed at 37C (in a waterbath in the lab).Try of course to keep sterility. I have successfully used pre-warmed "ice-packs" and my cells were all happy and alive ( they were placed in pre-warmed ice-packs for around 12 hours)
As far as lymphocytes (whole blood samples) and biopsies (placed in culture medium) are concerned, many times MD's store them at 4C until they reach the lab. From my experience, such samples grow just fine if we culture them within the next 48h.
Nevertheless, I guess 37C would be best.
Hope I helped