Hi, I am isolating primary mouse hepatocyte. The problem I am facing is sometimes I am getting very low viable cells (80%). The problem is particularly occuring with one specific strain so this could be strain specific. But few other things that I would like to include. Whenever I am getting low cell viability although the liver gets pale in washing step and looks like digested in successive digestion step, but the liver does not inflate during the start of perfusion. Is it happening due to this? When I am getting good viable cells the liver generally inflate within seconds of perfusion. And also the liver looks clumpy even after it looks digested. But the clumpiness is not seen when I am getting good viable cells. Although I observed this thing in a particular strain I may not be right as well. If anything I am doing wrong. Please suggest.
Hi Kakali,
although it is best to have good inflation during your perfusion (this will usually lead to higher yields), it is not mandatory to produce highly viable cells from mouse liver. On the mice that you have low viability, are you manipulating the liver more (or more roughly) to try to get the cells out? I have found that this can result in a very significant decrease in cell viability. What strain are you working with that keeps giving you trouble? A lot of the strains from the collaborative cross have less than optimal perfusion/digestion results.
Hi Valerie,
Thank you so much for your suggestion. I am using sv129 strain. With WT mouse with same strain I usually get high viability but with KO colony viability is very low. So in particular it may be genotype specific. Even after a good inflation, wash and digestion my cell viability remained low for KO. If this is the problem with the genotype how to address this? I made sure to be as gentle as possible during isolation and subsequent plating.
Hi Kakali,
That can be very frustrating. If inflation is good and you are confident in the flush and perfusion (and are doing everything the same as with the WT), the only other suggestion I have is to use Percoll in your spins to increase your viability. Usually, I have problems with KO or CC mice giving low yields but not usually low viabilities unless there is a liver-specific issue in the strain or the KO is causing a liver induced issue. As long as you are being gentle with the dissociation (very important) and using all the same techniques, I would keep performing as usual but use a Percoll step. If you already are using Percoll, consider increasing the percentage up to 50%.