Cryopreservation of rat and mouse hepatocytes. I. Comparative viability studies

We have compared the viability of cultures of cryopreserved (CP) rat and mouse hepatocytes with fresh cells with respect to their attachment efficiency, uptake of neutral red (NR), 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carbo xyanilide xyanilide inner salt (XTT) metabolism, and ability to synthesize new proteins. Total recovery of rat hepatocytes after cryopreservation was 42.1 +/- 18.0%, with high viability (82.3 +/- 7.8%, as determined by trypan blue exclusion). These cells had significantly lower attachment efficiencies than fresh cells over 72 hr in culture. Viability of CP rat hepatocyte cultures was lower than fresh cell cultures, but was constant throughout 72 hr (approximately 68%). Total recovery of CP mouse hepatocytes (postthaw viability = 85.4 +/- 6.8%) was 53.6 +/- 14.7%. CP and fresh mouse hepatocyte cultures had similar attachment efficiencies and viabilities. NR uptake by CP rat hepatocyte cultures was significantly higher than in fresh cells at 48 and 72 hr after plating (400% and 810% of fresh cells, respectively). In contrast, CP mouse hepatocytes, which did not detach significantly in culture, took up NR to the same extent as fresh cells. The rate of NR uptake into rat and mouse hepatocytes, cultured for 24 hr, was unaltered by cryo-preservation. XTT metabolism by hepatocytes from either species was not affected by cryopreservation. Protein synthesis over 72 hr, as measured by incorporation of [3H]leucine, was lower in CP cultures than in fresh cells (CP rat hepatocyte protein synthetic activity was 32.3 +/- 6.8% of fresh, and CP mouse hepatocyte protein synthetic activity was 49.0 +/- 10.1% of fresh). Protein synthesis did not alter over 72 hr culture.