I am working with rat hepatocytes isolation. I use a 2 step collagenase perfusion for it. However,often the viability is not good-only around 70 %. So, I am wondering is there any significant difference when experiment is conducted with 70 percent viable(or sometimes even less viable) cells to that conducted with 80 percent or more in terms of cell signaling? If there is then could anyone elaborate a little about the respective.
Thank you.
Getting viability of cells over 80%is not always easy. Lot of people stuggle with it.70% it can be used but is not ideal. If you doing suspension you cant wash dead cells but if you plate them you can wash dead ones. The trouble is using less than 80% viability you may be selecting a population.
There are three critical steps: collagenase when to stop at right time, ph and perfusion rate ideal 5-5.5
I don’t have particular experience with hepatocytes, but I have performed several primary cultures by enzymatic digestions. I believe the problem with viability over 70% is that the method that is used to measure it is tripan blue. I believe that method is not very sensible and viabilities below 70% mean very low viability. 7aad is a more sensible method to measure it, however, the best method is plating efficiency. If you are able to grow your cells after enzymatic digestion or at least see them attached, means your cells are fine. If only few cells attach means digestion has affected your cells.
Hope it helps!
Joaquim Pombo Thank you for your reply. Actually, I wanted to know the reason why it is not ideal- Could the response from the cells be different,if yes then why? I ususally observe the liver until it gets mushy and it takes about 6 min of collagenase perfusion at a rate of 30 ml/min.I think the pH is fine as I make HBSS solution around 7.3(expecting it to rise a little while warming it). Isin't the perfusion rate of 5-5.5 ml/min too low. I work with rats so maybe it might differ from mice.
Diego Raffo Thank you for your reply. I seed at a density of 1 million cells per 35 mm dish.I think the cells are attaching well. I am just concerned that would there be any difference in response from cells incase of isolation with 80% viability to that with 70 % or less viability, after treating them with certain inducers?
That is hard to tell, cells will be stressed after digestion, but eventually they will recover. In my opinion, if you see that cells grow as usual and have adequate morphology without sings of stress you should continue with your experiments, it´s very hard to obtain cells from tissue without stressing them.
At this moment I am working with MOLT-4 cell line. When cell viability is even slightly decreased I got different results. E.g., if I start my experiment on monday after a weekend or if I start my experiment next day after partially exchanging the medium for a fresh one, my cytotoxicity assays look different, some mixtures of my anti-tumor reagents are stronger then the other ones, when I do assay using cell culture from monday, and results are reversed, when I use cell culture from Tuesdey. However, I have worked on many cell lines, and primary cell cultures, where decreased viability could make results slightly less pronounced, but they were still similar enough. So, it depends.
Thank you professor Patrycja Koszałka for sharing some insightful information.I guess cancer cells are a bit unpredictable than primary cells.Now I guess that lower viability could be one of the indicators (which may or maynot be necessarily true) that the experiment did not went as it should have,so the response from the cells could be unpredictable and hence undesirable to go through with subsequent experiment.
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