STABILITY AND OPTIMIZATION OF CANALICULAR FUNCTION IN HEPATOCYTE COUPLETS

An enriched preparation of rat hepatocyte couplets was obtained by col lagenase perfusion and subsequent elutriation (> 85 per cent couplets and triplets; viability of over 95 per cent). Canalicular secretory ac tivity (the ability to accumulate cholyl-lysyl-fluorescein, CLF) was f irst apparent after 2 h of culture at 37-degrees-C and was present in over 80 per cent of the total population after 5-6 h. This remained al most constant for at least 4 h in both elutriated and directly plated cells. Initial storage of freshly prepared couplets at 4-degrees-C for up to 6 h prior to incubation had no adverse effect upon secretory fu nction. Reduction of canalicular secretory activity occurred at a conc entration of the hepatotoxic agent menadione (IC50 17 mum) that was lo wer than that required to induce mild plasma-membrane blebbing (IC50 4 3 mum). This study has optimized and characterized the canalicular sec retory effectiveness and stability of an enriched preparation of hepat ocyte couplets, and established the feasibility of studies of toxic ag ents on hepatobiliary function in a heterogeneous population of hepato cytes. In this preparation other biochemical parameters can be assesse d, thus complementing previous techniques using individual couplets.