Engineered Liver Platforms for Different Phases of Drug Development

Abstract

Drug-induced liver injury (DILI) remains a leading cause of drug withdrawal from human clinical trials or the marketplace. Owing to species-specific differences in liver pathways, predicting human-relevant DILI using in vitro human liver models is crucial. Microfabrication tools allow precise control over the cellular microenvironment towards stabilizing liver functions for weeks. These tools are used to engineer human liver models with different complexities and throughput using cell lines, primary cells, and stem cell-derived hepatocytes. Including multiple human liver cell types can mimic cell-cell interactions in specific types of DILI. Finally, organ-on-a-chip models demonstrate how drug metabolism in the liver affects multi-organ toxicities. In this review we survey engineered human liver platforms within the needs of different phases of drug development.

Keywords: drug-induced liver injury; liver-on-a-chip; microfabrication; micropatterned co-cultures; primary human hepatocytes.

Figure 1. The drug development pipeline

Starting with a set of between 10,000 and 20,000 compounds created via combinatorial chemistry, early stages of the pipeline greatly reduce the number of potential compounds for further development. Eventually only one drug gets launched into the marketplace after $3-5B and 12-15 years of going through the pipeline. The discovery/screening, lead optimization and ADME/Tox (absorption, distribution, metabolism, excretion, and toxicity), and preclinical animal testing are the phases where engineered human liver models can make the greatest impact to reduce and in some cases replace the usage of animals and prevent harm to patients in clinical trials and in the marketplace.

Figure 2. High-throughput hepatic systems

(A) 532-well micropillar and microwell plate combination that can contain as little as 60 nL in each well. Hepatic cells (i.e. THLE-2) suspended in a Matrigel™ droplet are spotted on each micropillar, which are then placed in a microwell containing recombinant adenoviruses. The adenoviruses are engineered to transduce the cells with different combinations of drug metabolism genes towards determining enzymes involved in the observed toxicity of a drug. Adapted from [11] with permission from Nature Publishing Group. (B) Hanging-drop method for generating liver spheroids of controlled diameters. Briefly, a cell mixture is seeded into the wells of a specialized 96-well plate that allows the spheroid to form and mature in a hanging drop. The spheroids are then transferred to another culture plate for drug dosing. Adapted from [13] with permission from Springer.

Figure 3. Micropatterned co-cultures

(A) Tissue culture polystyrene (or glass) can be uniformly coated with extracellular matrix protein (ECM) such as collagen and protected with a polydimethylsiloxane (PDMS) stamp. Exposed areas of ECM are ablated under oxygen plasma, leaving micropatterned ECM islands that match the geometry of the PDMS stamp. Hepatocytes selectively attach to ECM islands, and nonparenchymal cells (NPCs) fill in the surrounding area. (B) An industry standard 96-well plate showing uniform hepatocyte islands micropatterned using the process in panel A. The NPCs used in this example are 3T3-J2 murine embryonic fibroblasts surrounding the primary human hepatocyte colonies. Reprinted from [6] with permission from Sage Publications. More recently, MPCCs have been adapted to a 384-well plate format by the Bhatia group at the Massachusetts Institute of Technology.

Figure 4. Liver-on-a-chip platforms

(A) Liver-on-a-chip platforms are often manufactured with soft lithography techniques, whereby a design is transferred to SU-8 photoresist using ultraviolet (UV) light through a high-resolution mask such as a transparency. Polydimethylsiloxane (PDMS) is then poured over the patterned SU-8 to construct the microfluidic channel. After curing and boring the inlet and outlet ports, the PDMS is oxidized and bonded to the device substrate. (B) A liver-on-a-chip platform featuring HepG2/C3A cells is established on a silicon chip sandwiched between plexiglass layers. (C) Using a physiologically-based pharmacokinetic (PBPK) model, the fluidic circuit is designed to mimic the interactions between the liver and gastrointestinal (GI) compartments. (D) The liver on-a-chip platform is connected with a GI platform consisting of a co-culture of Caco-2 and HT29-MTX cells. Reprinted from [65] with permission from the Royal Society of Chemistry.

Figure I. Parenchymal and nonparenchymal cell types of the liver

Kupffer macrophages (KMs), hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs), and biliary epithelial cells are the major nonparenchymal cells (NPCs) in the liver. By secreting the indicated molecules, these NPCs impact the phenotype of primary human hepatocytes (PHHs). Percentages represent the relative number of each cell type in the human liver. Adapted from [6].