I have stored (in -20C) some mouse livers which were flash frozen in liquid nitrogen immediately after harvesting. Will a week in -20C affect P450 activity significantly?
Hi Subhasree,
Replying to your very practical question, its easier to mention that in many labs, mice were killed rapidly, decapitated to collect livers frozen in liquid nitrogen, and stored in −80 °C rather than in −20 °C. However, if following the beneath protective procedure, even keeping murine liver samples in −20 °C would be OK. Namely, in our and other labs the freezing method and the amount of cryoprotectant (18% DMSO) was found crucial for the preservation of monooxygenases (CYP) metabolism in mouse species. In fact, it was demonstrated that the increase of DMSO concentration from 12% to 18% efficiently preserved the rates of CYP activities in liver slices. The improvement by higher DMSO concentration can be explained by improved inhibition of ice crystal formation. Remarkably, it was found that not only phase I but also phase II metabolism was well preserved in thawed slices of murine livers, but that some metabolic rates were even increased after cryopreservation, such as for testosterone and 7-hydroxycoumarin (7-HC). The reason of this is unknown but it may be due to the increased substrate availability in the damaged slice.
Best wishes,
Ilya
Dear Dr. Nag,
Would you mind to reply to my answer (which might help you with how to store murine livers at −20 °C)?
Sincerely,
Dr. Ilya Tsyrlov
Sorry Dr. Tsyrolov for my tardiness. So, we had already harvested the livers and stored them at -20 for a couple of days (4 days for the earliest collected samples) and 2 days for the last ones. I have just prepared microsomes from one of the groups and we are running some gels presently to evaluate 450 activity (paricularly CYP1A1/B1). I will know where we stand in a couple of days. I think your cryopreservation step preventing formation of ice crystals makes a lot of sense. The next batch of murine livers that I work on , I would like to evaluate one cryoprotected group against another, surely. And thank you again for the detailed answer. I appreciate it very much.
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