Enhanced liver functions of hepatocytes cocultured with NIH 3T3 in the alginate/galactosylated chitosan scaffold.
Formation of primary hepatocyte spheroids in the hydrogel scaffold is a promising approach for enhancing liver-specific functions in liver tissue engineering as well as for developing bioartificial liver (BAL) devices. In the present study, a highly porous hydrogel scaffold composed of alginate (AL) and galactosylated chitosan (GC) as a synthetic extracellular matrix (ECM) for hepatocytes was fabricated with 150-200 microm pore size in diameter. Cell adhesion onto AL/GC and AL/chitosan film was 72.7 and 45% at 1 wt% of GC (or chitosan) to AL content whereas cell adhesion onto AL film was 28.5%. The optimal concentration of GC in AL/GC sponge was 1 wt% to AL content by the measurement of albumin secretion. Cell viabilities performed on AL and AL/GC sponges were 72.2+/-3.6 and 81.3+/-3.5% of control, respectively, after 10 days incubation. Hepatocytes were aggregated to form multicellular spheroids in AL/GC sponge with diameter enlarged up to about 100 microm, 36 h postseeding, whereas most of them in the AL sponge remained as single cells and only a few cells began to form aggregates. Intercellular molecules such as connexin32 and E-cadherin genes related with cell-cell contact were expressed in hepatocytes within AL/GC sponge at 36 h after incubation, but not in AL sponge. Treatment with a gap junctional intercellular communication (GJIC) inhibitor, 18beta-glycyrrhetinic acid, resulted in a 1.5-fold marked decrease in albumin secretion levels in AL/GC sponge. Specially, coculture of hepatocytes in AL and AL/GC sponges with NIH3T3 in a transwell insert resulted in enhanced increase of liver-specific functions, such as albumin secretion rates, ammonia elimination rates, and ethoxyresorufin-O-deethylase activity by cytochrome P4501A1, compared to those in hepatocyte monoculture. The results suggest that formation of hepatocyte spheroids in coculture system enhances liver-specific functions for the AL/GC sponge as a new synthetic ECM to design developed BAL devices.
Seo
Seog-Jin
SJ
School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, South Korea.
Kim
In-Yong
IY
Choi
Yun-Jaie
YJ
Akaike
Toshihiro
T
Cho
Chong-Su
CS
D000464:Alginates / Q000648:ultrastructure; D000818:Animals; D001672:Biocompatible Materials; D002448:Cell Adhesion / Q000502:physiology; D002478:Cells, Cultured; D048271:Chitosan; D018920:Coculture Techniques; D005690:Galactose; D020723:Glucuronic Acid; D022781:Hepatocytes / Q000502:physiology* / Q000648:ultrastructure; D006603:Hexuronic Acids; D008111:Liver Function Tests; D008297:Male; D008422:Materials Testing; D051379:Mice; D008813:Mice, Inbred ICR; D008858:Microscopy, Phase-Contrast; D041681:NIH 3T3 Cells
D000464:Alginates; D001672:Biocompatible Materials; D006603:Hexuronic Acids; D020723:Glucuronic Acid; D048271:Chitosan; D005690:Galactose