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.
Highlights
The liver is the largest organ in the body and performs vital functions related to digestion, metabolism and the detoxification of xenobiotics [1]
We observed that the mixture of hepatocytes and hepatic stellate cells (HSCs) and the hepatocytes themselves, aggregated in the center of the concave microwell flow chip to form small spheroids (Figure 2a)
These results showed the effect of the concave microwell arrays on spheroid formation and indicated that concave microwell arrays more effectively generate homogeneous heterospheres from co-cultured hepatocytes and HSCs
Summary
The liver is the largest organ in the body and performs vital functions related to digestion, metabolism and the detoxification of xenobiotics [1]. The healthy liver is composed of a parenchymal compartment, hepatocytes and a non-parenchymal compartment comprising endothelial cells, Kupffer cells and hepatic stellate cells (HSCs) [2]. The hepatocytes constitute approximately 80% of the human liver, and are involved in protein synthesis and storage, carbohydrate transformation, cholesterol synthesis, and detoxification [3,4,5]. HSCs are known to be involved in the regeneration of the liver and the production of mediators, such as hepatocyte growth factor (HGF), insulin-like growth factor (IGF) and transforming growth factor β1(TGF-β1) [8,9,10]. For in-depth studies of the liver under in vitro conditions, new cell culture models, which allow 3-D micro cell–cell formation and cell–cell interactions affecting the modulation of hepatocytes and HSCs, and facilitate the investigation of metabolic functions, are required
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