improving viability of pelleted cells - (Mar/14/2013 )
We are investigating cell delivery techniques in rats. The cells we are using are rat aortic endothelial cells and they must be trypsinized, washed and pelleted in HBSS in the lab then brought to the animal facility on ice until the rats are prepared surgically for the delivery. The cells are quickly re-suspended before delivery and they are luciferase expressing. We noticed that the first couple of rats have great luciferase activity but as the surgeries progress the luciferase signal drops accordingly. It appears that the cells begin to lose viability while sitting on ice over the 4 hours or so it takes to perform all the surgeries. We have no access to a cell culture incubator in the surgery suite but we do have a 4 degree refrigerator. Will keeping the pelleted cells at 4 degrees improve their vaibility over 4 hours or is there something we can do to keep them alive for longer?
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
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