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.

Keywords: co-culture; hepatic stellate cell; hepatotoxicity; microfluidic; spheroid.

Figure 1

Fabrication of a concave microwell array polydimethylsiloxane (PDMS) plate. (i) Schematic diagram of concave microwell array made using the conventional soft-lithography procedure and meniscus of the PDMS pre-polymer. (ii) Experimental setup. Micropipette tips were used as inlet reservoirs. The osmotic pump was dipped into the polyethylene glycol (PEG) solution to generate the flow rate of the microsystem; the coiled tube was used as an outlet reservoir. (iii) Schematic process of cell seeding and docking within concave microwell structures. After gently aspirating cells that were not docked, the remaining cells docked within concave microwells.

Figure 2

(a) Light microscopic images of spheroids formed in concave microwells. (b) Viability of cells in the hepatospheres cultured for (i) 7 and (ii) 14 days; heterospheres cultured for (iii) 7 and (iv) 14 days in the concave flow chip. Scale bar = 500 µm.

Figure 3

Scanning electron microscopy (SEM) images of heterospheres cultured for (a) 7 and (b) 14 days; hepatospheres cultured for (c) 7 and (d) 14 days. Scale bar = 10 μm.

Figure 4

(a) Immunostaining for serum albumin (green) in the (i) hepatospheres and (ii) heterospheres cultured for 2 weeks. The nuclei were stained with DAPI (blue). Scale bar = 100 μm. (b) Analysis of the function of heterospheres and hepatospheres, measured in terms of the secretion of (i) albumin and (ii) urea. Data are represented as the mean ± standard deviation (SD) of five independent experiments. * p < 0.05 for hepatosphere vs. heterosphere.

Figure 5

Time-course of marker expression for specific hepatocytes. The quantification of the relative gene expression of (a) CYP1A2, (b) Mrp1, and (c) UGT1A5. Data are represented as the mean ± standard error (SE) of three independent experiments. * p < 0.05.

Figure 6

(a) Luminescence assay of cytochrome P450 activity in the hepatospheres and heterospheres after AAP treatment. Cytochrome P450 enzyme activity was detected according to the luminescence emitted upon addition of a detection reagent; the degree of luminescence emitted was measured using a luminometer. (b) The assessment of cell death using the LDH assay. The hepatospheres and heterospheres were treated with AAP and INH for 24 h. Data are depicted as the percentage of the total LDH amount. All values are showed as the mean ± SE of four independent experiments. * p < 0.05 (c) IC₅₀ Determination curve.