Extracorporeal Plasma Perfusion of Cultured Hepatocytes: Effect of Intermittent Perfusion on Hepatocyte Function and Morphology

Abstract

The most promising approaches to developing a temporary bioartificial liver support system involve incorporating cultured primary hepatocytes into an extracorporeal perfusion device. As a result, it is important to characterize both the phenotypic response of these cells during extracorporeal perfusion and the critical factors involved in maintaining differentiated cell function over extended periods of perfusion. In this study, hepatocytes cultured in a collagen sandwich configuration were connected to a rat via a hollow fiber plasma separator and perfused with plasma on line. Perfusions were either continuous for 48 hr or intermittent for up to 174 hr with 6 hr per day of extracorporeal plasma perfusion alternating with 18 hr of culture medium perfusion. During perfusion cell morphology was continuously monitored by time-lapse video microscopy. After the procedure, hepatocytes were returned to static culture and function was evaluated by measuring the rates of urea synthesis daily for 7 days. During plasma perfusion all hepatocytes accumulated cytoplasmic lipid droplets in a time dependent manner. Urea synthesis was maintained at initial levels for up to 20 hr of continuous plasma perfusion. However, urea synthesis rates were reduced by 31 and 52% after 30 and 48 hr of continuous plasma exposure, respectively. With intermittent perfusions, as well as with control cells perfused with culture medium only, urea synthesis rates did not decrease for at least 78 hr of total perfusion. There was no difference between the urea synthesis rates after 48 hr of cumulative plasma exposure time between cells subjected to continuous and intermittent plasma perfusion. These results suggest that cultured hepatocytes may be exposed to plasma for at least 20 hr with no significant reduction in liver-specific function. Furthermore, an intermittent plasma perfusion schedule can be used to divide the useful plasma perfusion time over several days with no adverse effects on cell function.