Biomedical Engineering Reference
In-Depth Information
cells, and recently endothelial cells (ECs) have been studied as
a nonparenchymal cell that may extend the biological function
of hepatocytes [64-68]. Our laboratory has developed a novel
hepatocyte-EC coculture system, which enables cultured hepatocytes
to preserve their function long term, using cell sheet engineering
[69]. A bovine carotid artery-derived EC sheet and a rat hepatocyte
sheet can be heterogeneously layered, and the double-layered cell
sheet can be cultured in vitro. Clear ECM deposition is detected in
the space between the EC sheet and the hepatocyte sheet. At six
days' cultivation the hepatocytes in the layered EC sheet/hepatocyte
sheet group maintain their round-shaped nuclei with numerous
healthy mitochondria, and many well-developed bile canaliculi
networks are formed among the hepatocytes, while the hepatocytes
in the hepatocyte sheet alone show marked degradation due to its
unusual cell shape and insufficient mitochondria within the cells,
as well as few bile canaliculi networks among the hepatocytes.
Importantly, the bile canaliculi in the EC sheet/hepatocyte sheet
group are expanded, which indicates active bile secretion into
the bile canaliculi. While the hepatocyte monolayer shows rapid
reduction of albumin and urea secretions, in the heterogeneous
layered sheet the albumin secretion level is highly preserved for
at least 28 days and urea synthesis is also significantly higher
than the cultivation of hepatocytes alone. The expression levels of
hepatocyte-specific genes, including albumin, hepatocyte nucleus
factor 4, multidrug resistance-associated protein 2, claudin-3, and
glucose-6-phoshatase, are significantly higher in the heterogeneous
cell sheet compared to the hepatocyte sheet alone.
In vivo transplantation of hepatocyte sheets has already been
performed using mouse models [20]. After the development of a
vascularized cavity within a subcutaneous space created by implant-
ing a basic fibroblast growth factor-releasing device, hepatocyte
sheets were then transplanted into the site. The engineered-tissue
graft shows long-term persistence (at least 235 days), having sev-
eral characteristics of liver-specific function within the subcutane-
ous space. Hepatocyte sheets can be easily layered, and transplanta-
tion of layered hepatocyte sheets shows superior functional tissue
volume compared to that of a single hepatocyte sheet or suspended
hepatocytes in a Matrigel ECM. In addition, hepatocyte sheets have
already been fabricated by using human liver-derived hepatocytes,
indicating the potential for clinical applicability. Hepatocyte sheet
