Developing Human In Vitro Systems
“Even though the ultimate effect of a drug may be toxicity to a certain organ, there are internal organ intra-actions going on. Almost all compounds are metabolized by the liver; the lack of hepatic metabolism has been one of the major barriers that in vitro systems have needed to overcome to replace in vivo animal testing.
If you tested a drug only on one type of cell, such as kidney, neuron, or heart cells, which do not metabolize drugs well in the absence of the liver, then it is possible to either overstate or understate toxicity. We need to be able to test new chemical entities in a physiologically relevant human in vitro system to provide a much clearer picture on human drug toxicity, with the inclusion of hepatic metabolism as one of the key components,” explained Albert Li, president and CEO, APSciences and In Vitro ADMET Laboratories.
The patented Integrated Discrete Multiple Organ Culture plate (IdMOC™) provides a method to model in vivo multiple-organ interaction in vitro. The standard-sized plate consists of multiple inner wells within a larger interconnecting chamber. Multiple cell types are individually cultured in the inner wells as physically separated, discrete entities, and the chamber is filled and integrated with a single, universal medium, flooding the wells and allowing well-to-well communication by diffusion.
A basic assay established proof of concept that exposure to hepatocytes is necessary for correct quantification of drug metabolism. Hepatocytes and reporter cells, cells without strong metabolism capabilities such as 3T3 cells, were co-cultured in different wells and exposed to cyclophosamide, which is only toxic after metabolism. As more wells of hepatocytes were added to the IdMOC plate, toxicity increased, demonstrating that toxicity was dependent on the amount of hepatic metabolism.
Metabolism-dependent immunotoxicity was also shown using cyclophosphamide, which was found to be relatively noncytotoxic to splenocytes but was cytotoxic to splenocytes in the presence of hepatocytes.
Assembling the Human Liver on a Chip
“To date, there is no in vitro system that simulates the liver’s complexity from in vivo sinusoid-like structures to perfusion-mimicking physiologic conditions of the blood flow within the sinusoidal network. With the expectation that culturing hepatocytes under in vivo-like conditions will result in in vivo-like cell functionality, and resultant response to drug stimuli, we established the HepaChip® concept, a liver-cell culturing approach that combines a plug-and-play microfluidic device with liver-like artificial sinusoids,” explained Jan Böttger, University of Leipzig.
The microscope-slide-sized HepaChip features a system of branching microchannels and cell culture chambers with parallel cell-assembly ridges located in the middle of the chamber, approximately the dimensions of sinusoidal structures in the human liver. Built-in flow barriers reduce flow velocity and shear forces.
To create the artificial sinusoids, positive dielectrophoresis, via application of an AC electric field, directs hepatocytes and liver endothelial cells toward the assembly ridges. The surfaces of the cell chambers are modified to enable optimal cell adhesion that imitates the natural liver.
Proof of concept was shown using cryopreserved human hepatocytes and human liver endothelial cells and also with primary mouse hepatocytes and human liver endothelial cells. Slender lucid spaces between hepatocytes indicated the formation of bile canaliculi, an indicator of reestablished cell polarity, a function typically not well established in standard two-dimensional liver-cell cultures. HepaChip cultures also demonstrate improved liver-specific metabolic functions, in particular the secretion and biotransformation of albumin and urea by phase I cytochromes, P450 3A4 and 1A2, and phase II enzymes, UGT and SULT.