I am planning primary mouse hepatocyte isolations. I am concerned about the high sodium composition of buffers that have been recommended for this purpose. In particular, I am concerned because we will use CO2 to euthanize the animals immediately prior to isolating hepatocytes. I am wondering if anyone has used solutions like the Histidine-tryptophan-ketoglutarate (HTK) solution or University of Wisconsin (UW) solution, which contain low sodium, since these are used successfully with human liver transplantation. Does anyone make up their own low sodium solution for primary mouse hepatocyte isolations?
Article Role of pH in protection by low sodium against hypoxic injur...
Right Stephen. I agree that it is preferable for the liver to use anesthesia. Nevertheless since we are able to place a catheter within 60 seconds of CO2 euthanasia, and get viable hepatocytes, I would like to explore the question of using a low sodium buffer to increase hepatocyte numbers after CO2, which would be a useful strategy for any mice without committee approvals.
Well, in my previous group of work (I have now move to another lab/topic of research) we used to prepare home made UW (modified UW) and HTK solutions. In UW solution we have principally changed hydroxyethyl starch by PEG as this later is better for preservation of isolated cells and not as difficult to dissolve. You can consult our publications (for example Cell Transplantation 4(2): 245-251; 1995; and all the papers of Joaquín V. Rodríguez).
Anyway, I think it is not advisable to try to digest the liver with collagenase in these solutions as they are not designed to be used at 37°C (are usually harmful to cells if not at 0-4°C) and the activity of this enzyme will probably be affected by the composition of solutions (you have to add Calcium for proper activity). Perhaps you can try to lower sodium concentration of HBSS.
If you are worried about sodium concentrations, there have been some papers substituting choline chloride in a 1:1 substitution by weight. I experimented with it a bit a few years ago, and it had no adverse effects to the cells. Didn't have a chance to prove the benefits.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques