Gut-liver microphysiological systems revealed potential crosstalk mechanism modulating drug metabolism.

The small intestine and liver play important role in determining oral drug's fate. Both organs are also interconnected through enterohepatic circulation, which imply there are crosstalk through circulating factors such as signaling molecules or metabolites that may affect drug metabolism. Coculture of hepatocytes and intestinal cells have shown to increase hepatic drug metabolism, yet its crosstalk mechanism is still unclear. In this study, we aim to elucidate such crosstalk by coculturing primary human hepatocytes harvested from chimeric mouse (PXB-cells) and iPSc-derived intestinal cells in a microphysiological systems (MPS). Perfusion and direct oxygenation from the MPS were chosen and confirmed to be suitable features that enhanced PXB-cells albumin secretion, cytochrome P450 (CYP) enzymes activity while also maintaining barrier integrity of iPSc-derived intestine cells. Results from RNA-sequencing showed significant upregulation in gene ontology terms related to fatty acids metabolism in PXB-cells. One of such fatty acids, arachidonic acid, enhanced several CYP enzyme activity in similar manner as coculture. From the current evidences, it is speculated that the release of bile acids from PXB-cells acted as stimuli for iPSc-derived intestine cells to release lipoprotein which was ultimately taken by PXB-cells and enhanced CYP activity.

New in vitro screening system to detect drug-induced liver injury using a culture plate with low drug sorption and high oxygen permeability.

Drug-induced liver injury (DILI) is a major factor underlying drug withdrawal from the market. Therefore, it is important to predict DILI during the early phase of drug discovery. Metabolic activation and mitochondrial toxicity are good indicators of the potential for DILI. However, hepatocyte function, including drug-metabolizing enzyme activity and mitochondrial function, reportedly decreases under conventional culture conditions; therefore, these conditions fail to precisely detect metabolic activation and mitochondrial toxicity-induced cell death. To resolve this issue, we employed a newly developed cell culture plate with high oxygen permeability and low drug sorption (4-polymethyl-1-pentene [PMP] plate). Under PMP plate conditions, cytochrome P450 (CYP) activity and mitochondrial function were increased in primary rat hepatocytes. Following l-buthionine-sulfoximine-induced glutathione depletion, acetaminophen-induced cell death significantly increased under PMP plate conditions. Additionally, 1-aminobenzotriazole reduced cell death. Moreover, mitochondrial toxicity due to mitochondrial complex inhibitors (ketoconazole, metformin, and phenformin) increased under PMP plate conditions. In summary, PMP plate conditions could improve CYP activity and mitochondrial function in primary rat hepatocytes and potentially detect metabolic activation and mitochondrial toxicity.

Optimization of physical microenvironment to maintain the quiescence of human induced pluripotent stem cell-derived hepatic stellate cells.

Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis by producing excessive extracellular matrix (ECM) following chronic inflammation. However, studying HSC function has been challenging due to the limited availability of primary human quiescent HSCs (qHSCs) in vitro, and the fact that primary qHSCs quickly activate when cultured on plastic plates. Advances in stem cell technology have allowed for the generation of qHSCs from human induced pluripotent stem cells (hiPSCs) with the potential to provide an unlimited source of cells. However, differentiated quiescent-like HSCs (iqHSCs) also activate spontaneously on conventional plastic plates. In this study, we generated iqHSCs from hiPSCs and developed a culture method to maintain such iqHSCs in a lowly activated state for up to 5 days by optimizing their physical culture microenvironment. We observed that three-dimensional (3D) culture of iqHSCs in soft type 1 collagen hydrogels significantly inhibited their spontaneous activation in vitro while maintaining their ability to convert to activated state. Activation of iqHSC was successfully modeled by stimulating them with the fibrotic cytokine TGFß1. Hence, our culture method can be used to generate HSCs with functions comparable to those in a healthy liver, facilitating the development of accurate in vitro liver models for identifying novel therapeutic agents.

Accurate Evaluation of Hepatocyte Metabolisms on a Noble Oxygen-Permeable Material With Low Sorption Characteristics.

In the pharmaceutical industry, primary cultured hepatocytes is a standard tool used to assess hepatic metabolisms and toxicity in vitro. Drawbacks, however, include their functional deterioration upon isolation, mostly due to the lack of a physiological environment. Polydimethylsiloxane (PDMS) has been reported to improve the function of isolated hepatocytes by its high oxygen permeability when used as a material of microphysiological systems (MPS). However, its high chemical sorption property has impeded its practical use in drug development. In this study, we evaluated a new culture material, 4-polymethyl-1-pentene polymer (PMP), in comparison with PDMS and conventional tissue culture polystyrene (TCPS). First, we confirmed the high oxygen permeability and low sorption of PMP, and these properties were comparable with PDMS and TCPS, respectively. Moreover, using primary rat hepatocytes, we demonstrated maintained high levels of liver function at least for 1 week on PMP, with its low chemical sorption and high oxygen permeability being key factors in both revealing the potential of primary cultured hepatocytes and in performing an accurate evaluation of hepatic metabolisms. Taken together, we conclude that PMP is a superior alternative to both PDMS and TCPS, and a promising material for a variety of drug testing systems.