Viability and function of cryopreserved rat hepatocyte monolayers as a function of cryopreservation media composition

To improve post-thaw attachment efficiency in culture exhibited by hepatocytes frozen in suspension, we have frozen rat hepatocytes as monolayers on collagen substrates and compared post-thaw viability and function of these cryopreserved monolayers with non-cryopreserved control cultures. To measure viability, carboxyfluorescein diacetate (CFDA) de-acetylation by intracellular esterases in viable cells was visualised under a confocal laser scanning microscope (CLSM). Function was assessed by the ability to metabolise kaempherol into two glucuronide metabolites by HPLC (Oliveira and Watson, 2000). Hepatocytes were prepared from male Sprague Dawley rats by collagenase perfusion (viability 80-90%). Petri dishes (60 mm) coated with 30 g/cm2 type I collagen isolated from rat tail tendons were seeded with 3×106 viable cells in 2 ml Chee's medium (CM) containing 5% v/v foetal calf serum (FCS). Monolayer 24 h cultures of hepatocytes were frozen for 24 h at −70 °C and thawed according to Watts and Grant (1996), except that the cryopreservation (CP) medium was 10% DMSO in CM with 0-90% FCS present (see Fig. 1). Control cultures (24 h) were also treated with CP media, but not frozen. Media were changed in post-thaw and control cultures every 24 h thereafter. For viability assessment, 96 h control and post-thaw cultures were stained with 25 M CFDA for 20 min in the dark at 4 °C and imaged under CLSM. For functional assessment, 96 h cultures were incubated for 1 h with 100 M kaempherol in Krebs Hepes buffer and the metabolites analysed by HPLC