Matrix-induced liver cell aggregates (MILCA) for bioartificial liver use.
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; 5 x 10(9) cells/cartridge, recirculation of 350 ml of plasma at 400 ml/min). In these studies, lidocaine (20 micrograms/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.
Kong
L B
LB
Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
Chen
S
S
Demetriou
A A
AA
Rozga
J
J
D000464:Alginates; D000596:Amino Acids / Q000097:blood; D000641:Ammonia / Q000097:blood; D000818:Animals; D001187:Artificial Organs; D001663:Bilirubin / Q000097:blood; D001709:Biotechnology; D002449:Cell Aggregation / Q000502:physiology*; D002470:Cell Survival; D017690:Cell Transplantation; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D017364:Collagenases / Q000378:metabolism; D003374:Coumarins / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004492:Edetic Acid / Q000378:metabolism; D005260:Female; D005782:Gels; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007773:Lactates / Q000097:blood; D019344:Lactic Acid; D008099:Liver / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D008863:Microspheres; D013552:Swine; D014468:Umbelliferones / Q000378:metabolism
D000464:Alginates; D000596:Amino Acids; D003374:Coumarins; D005782:Gels; D006603:Hexuronic Acids; D007773:Lactates; D014468:Umbelliferones; C017299:7-ethoxycoumarin; D019344:Lactic Acid; C031477:7-hydroxycoumarin; D000641:Ammonia; D020723:Glucuronic Acid; D003094:Collagen; D003577:Cytochrome P-450 Enzyme System; D004492:Edetic Acid; D007770:L-Lactate Dehydrogenase; D017364:Collagenases; D001663:Bilirubin