Matrix-Induced Liver Cell Aggregates (MILCA) for Bioartificial Liver Use

Abstract

Ex vivo reproduction of liver microstructure using isolated hepatocytes is critical for bioartificial liver use. We have developed a method of producing matrix-induced liver cell aggregates (MILCA) using a small number of collagen-coated beads as a nidus for formation of hepatocyte aggregates. Porcine hepatocytes were obtained by EDTA/collagenase digestion. Cell viability was assessed by trypan blue exclusion and LDH release. Cytochrome P-450 activity was determined at 4 and 24 hours by measuring the formation of 7-hydroxycoumarine (7-HC) from 7-ethoxycoumarine (7-EC). At 4 hours, the viability of MILCA was 92±2%, LDH release was 100+22 U/L and 7-HC formation was 140±34 nM/g cells. At 24 hours, MILCA viability remained greater than 90%, but 7-HC formation was lower than that of parallel control monolayer hepatocyte cultures (194±43 vs 481±78 nM/g cells; p<0.002). On transmission electron microscopy, MILCA ultrastructure resembled that of a normal liver (maintenance of cell polarity, gap junctions, bile canaliculi, intact organellae, glycogen granules). MILCA were subsequently inoculated into hollow-fiber bioreactors which were perfused for 6 hours with plasma recovered from patients with fulminant hepatic failure (n=6; 5x10⁹ cells/cartridge, recirculation of 350 ml of plasma at 400 ml/min). In these studies, lidocaine (20 μg/ml) was cleared in less than 3 hours and 7-HC production at 6 hours was 71+8 nM/g cells. Other MILCA effects noted in this system included lowering of plasma lactate, bilirubin and ammonia and increase in the level of several non-essential amino acids.