(347b) The Design of Three-Dimensional Liver Mimics Using Nano-Scale Polyelectrolyte Scaffolds

Interactions between hepatocytes (parenchymal) and nonparenchymal (endothelial, Kupffer and stellate) liver cells are essential for the development and maintenance of hepatocyte specific functions. Hepatic tissue engineering is considered a promising approach for treating liver diseases, and in the testing of drugs and toxins. The design of tissue-engineered livers and liver-support devices can be accelerated if model hepatic units are available to systematically test their response to a range of stimuli. There is currently no generally applicable methodology to design hepatic culture systems where primary hepatocytes and liver sinusoidal endothelial cells (two of the major cell types found in the liver) are arranged in a stratified structure. We report the assembly of 3D liver mimics comprised of primary rat hepatocytes, liver sinusoidal endothelial cells (LSECs) and an intermediate nanoscale polyelectrolyte (PE) scaffold. The intermediate nanoscale PE scaffold was assembled using the layer by layer deposition of polyelectrolytes. This self-assembly involves the sequential deposition of cationic and anionic polyelectrolytes where multilayers are formed due to electrostatic interactions. In our studies, the PE scaffold is deposited above a live layer of hepatocytes. Chitosan served as the cationic polyelectrolyte and hyaluronic acid as the anionic polyelectrolyte.

Hepatocyte-PE-LSEC cellular constructs were assembled and maintained up to ten days in culture. A wide range of bilayer numbers and LSECs were tested. The thickness and viscoelastic properties of the polymeric scaffold play a significant role in the ability to construct and maintain the 3D architecture. These physical properties were monitored under hydrated conditions using QCM-D measurements. Among the conditions tested, hepatocytes cultured with 15 chitosan/hylauronic acid layers and 5,000 LSECs was found to be the most promising culture condition. These 3D constructs exhibited two-fold higher albumin secretion and stable urea production. In comparison hepatocyte-LSEC cultures in the absence of the PE scaffold, and hepatocyte monolayers, exhibited a reverse trend. The phenotype of LSECs was maintained in the 3D liver mimics and verified by the uptake of ac-LdL. The actin cytoskeleton in the presence of PE scaffolds was localized in the peripheral regions of the cells, similar to hepatocytes cultured in a collagen sandwich. Well defined bile canaliculi were also observed in the hepatocyte-PE-LSEC liver mimics. Ongoing investigations include the measurements of detoxification kinetics in the 3D hepatic architectures.