Flow-Based Three-Dimensional Co-Culture Model for Long-Term Hepatotoxicity Prediction

Abstract

We developed concave microwell arrays to establish a size-controllable 3-D co-culture liver model for in vitro drug toxicity testing, to predict hepatotoxicity. The interaction of hepatocytes with hepatic stellate cells (HSCs) was investigated by co-culturing primary 3-D hepatocyte spheroids and HSCs (heterosphere), using 3-D liver-on-a-chip. The effect of HSCs was investigated during spheroid formation; they were involved in controlling the organization of spheroidal aggregates and the formation of tight cell–cell contacts. Scanning electron microscopy (SEM) images showed that co-cultured spheroids with smoother surfaces in the flow chip aggregated more tightly and rapidly, compared to mono-cultured spheroids, until 13 days. Metabolic function analysis revealed that heterospheres secreted 40% more albumin and urea than hepatospheres on day 13. Additionally, an acetaminophen (AAP) and isoniazid (INH) concentration-dependent increase in CYP3A4 expression was detected in the 3-D cultures, and an increase in Lactate dehydrogenase (LDH) release after AAP and INH treatment was observed. CYP1A2, Mrp1 and UGT1A5 mRNA expression levels in the heterospheres and hepatospheres were evaluated from days 3 to 13. To examine the potential for toxicity testing in the flow-conditioned culture of the heterospheres, we evaluated cytotoxicity using the endpoint LDH release in the heterospheres and hepatospheres. IC50 values for AAP and INH after 24 h of exposure were calculated from the dose–response curves of the compounds. Flow-conditioned heterosphere culture results suggest that it may be suitable for long-term culture and cytotoxicity testing. Thus, our co-culture system closely resembles the in vivo environment and allows long-term in vitro hepatotoxicity prediction.

Liver microphysiological platforms for drug metabolism applications

“…The liver plays a principal role in the digestion, metabolism and detoxification of xenobiotic compounds 36 . Drug‐induced liver injury (DILI), which leads to liver failure and drug attrition, 37 is the major cause of the withdrawal of approved drug compounds from the market 38 .…”

“…The heterospheres formed as a result of a co‐culture demonstrated higher CYP activity than hepatospheres upon treatment with acetaminophen, and the experiments with repeated exposure of the spheroids to acetaminophen and isoniazid show more stable IC 50 values in heterospheres. These results suggest that the system is an optimized tool for drug toxicity studies, and due to improved metabolic competence is suitable for investigating drug‐drug interactions (DDIs) 36 …”

“…The liver plays a principal role in the digestion, metabolism and detoxification of xenobiotic compounds. 36 Drug‐induced liver injury (DILI), which leads to liver failure and drug attrition, 37 is the major cause of the withdrawal of approved drug compounds from the market. 38 Therefore, the need for developing an in vitro model with higher reproducibility of in vivo liver environment is staggering, and microfluidic liver‐on‐a‐chip (LOC) platforms have gained popularity in the research in the domain of drug toxicity assessment.…”

Bioengineered Liver Models for Investigating Disease Pathogenesis and Regenerative Medicine

“…However, spheroids remain difficult to form with > 50% of PHH donors, 34 potentially due to variable ECM secretion rates across cells from different donors. This issue can be mitigated with the incorporation of liver and nonliver NPCs with hepatocytes toward improving spheroid formation efficiency, 38,39 potentially due to augmented ECM deposition from the NPCs. Additionally, the coculturing of hepatocytes with liver NPCs facilitates the study of how multiple liver cells affect disease progression, highlighted in later sections.…”

Metabolic Alterations in Spheroid-Cultured Hepatic Stellate Cells

“…Several studies have reported the use of spheroids to study hepatic cells, including cultures of hepatocytes only, as well as coculture studies with HSCs and Kupffer cells. One study reported the creation of a 3D hepatocyte/HSC coculture spheroid using low-adhesion poly-DL-lactide [9]. Another study reported that the coculture system suitably mimicked the in vivo environment, showing utility for the prediction of in vivo hepatotoxicity [10].…”