Abstract

The success of cryopreservation of isolated hepatocytes with existing methodologies is assessed with respect to the retentivity of cell integrity/viability (defined by trypan blue) and metabolic activities upon thawing in comparison to those of freshly prepared cells. But the ability of the cryopreserved cells to transport xenobiotics relative to that of freshly prepared cells has not been investigated. In this study, we optimized our previous methodology for cryopreservation and evaluated the metabolism and transport of thawed hepatocytes. Half of the freshly, isolated rat hepatocytes prepared by collagenase perfusion were immediately used for studies of transport of [¹⁴C]taurocholate, [³H]estrone sulfate and [³H]estradiol 17β-d-glucuronide (1 μM) and metabolism of 7-hydroxy-4-(trifluoromethyl)-coumarin (100 μM), (3,4-difluorobenzyloxy)-5,5-dimethyl-4-(4-methylsulfonylphenyl)-(5H)-furan-2-one (250 μM), bufuralol (100 μM), and tolbutamide (100 μM), probes for UDP-glucuronyl transferase (UGT) and CYP3A, CYP2D, and CYP2C, respectively. The remaining half was cryopreserved using an optimized, programmed-freezing protocol, which was developed to minimize the prolonged release of latent heat during freezing. With the exception of the UGT probe, no significant difference (P > 0.05) was found in both metabolism and transport with freshly isolated versus cryopreserved hepatocytes upon thawing. In conclusion, we have demonstrated for the first time that thawed rat hepatocytes cryopreserved by a programmed-freezing protocol retain drug transport activities.