(278f) High-Throughput Drug Screening Using 3D Micro Organotypic Liver Models

Authors: 
Orbach, S., Virginia Tech
Rajagopalan, P., Virginia Tech
There are thousands of chemicals whose effects on human and animal populations are unknown or only partially understood. Specifically, the combinatorial effects of these chemicals to the liver are unknown. Traditional in vitro and in vivo studies, while informative, can be laborious and time-consuming. High-throughput screening (HTS) methodologies can rapidly test individual and cocktails of chemicals across a wide range of concentrations. This approach can identify chemical mixtures that are toxic to the liver. To date, HTS assays on liver cells have primarily been conducted on 2D monolayers or co-cultures. These analyses are useful, but do not capture the complex interplay that exists between hepatic cells and their in vivomicroenvironment.

When the principal liver cell types, hepatocytes, are cultured in standard monolayers, they begin to lose their liver-specific functions within 24 hours. Moreover, these models fail to recapitulate the native stratified architecture of the liver. We have designed 3D liver models that contain all hepatic cell types and maintain the cellular compositions over extended time periods. The combination of HTS methodologies and these 3D liver models has the potential to transform current testing of pharmaceuticals and environmental toxicants.

We have assembled a novel micro liver organotypic culture model assembled in a 96-well plate (denoted as mOCM) that can be used to rapidly screen individual and mixtures of chemicals. A novel aspect of the mOCMs is the use of automated procedures. An automatic cell dispenser was used to coat the substrates with collagen, seed the hepatic cell types, change medium, administer chemicals, and conduct assays within the 96-well plates. The mOCMs contained a biopolymeric membrane composed of collagen (type 1) and hyaluronic acid that separated the hepatocytes from the liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs). This membrane mimicked the height and composition of the Space of Disse that separates the hepatocytes from the LSECs and KCs in vivo.Acetaminophen (APAP), troglitazone and perfluorooctanoic acid (PFOA) were administered to the mOCMs across a range of concentrations (sub-lethal to highly toxic). Hepatocyte monolayer (HM) and collagen sandwich (CS) cultures served as 2D comparisons. The mOCMs exhibited up to 43 ± 6%, 33 ± 8% and 69 ± 3% decreases in viability when treated with 10 mM APAP, 50 mM troglitazone and 0.5 mM PFOA, respectively. When these concentrations were increased to 40 mM APAP, 200 mM troglitazone and 2 mM PFOA, cell death increased up to 79 ± 2%, 67 ± 12% and 84 ± 4%. In contrast, the HM and CS cultures only exhibited significant cell death at the highest concentrations of PFOA and APAP. The increased sensitivity of the mOCMs compared to the HM and CS cultures clearly demonstrate that these cultures maintain biotransformation capabilities of these chemicals.

Changes in viability, necrosis and apoptosis were simultaneously measured in the cultures using a high-throughput assay (ApoTox-GloTM Triplex Assay). This HTS assay simultaneously provided information on cell death, apoptosis and necrosis by simply measuring the luminescence and fluorescence from each culture. In response to 40 mM APAP, there was a 6.9-fold increase in necrosis relative to controls in the mOCMs. The corresponding change was only 1.6-fold in HM and CS cultures. In response to 200 mM troglitazone, necrosis increased by 2.9, 6.4 and 16.1-fold in the HM, CS and mOCMs, respectively. Apoptosis increased up to 3.0-fold and 2.1-fold in the 2D cultures and mOCMs, respectively, relative to controls. In vivo, the expected mechanism of cell death for both APAP and troglitazone is necrosis. Therefore, these results indicate that not only are the mOCMs more sensitive than traditional 2D cultures, but the mechanism of cell death is more representative of the hepatic response in vivo.

Ongoing efforts are focused on the use of HTS assays to investigate changes in hepatic cellular functions, protein expression and functional markers in the presence of individual and cocktails of chemicals. By seamlessly combining HTS methodologies with a liver mOCM we seek to provide a platform for medium to high throughput testing of toxicants.