Hepatocytes maintain greater fluorescent bile acid accumulation and greater sensitivity to drug-induced cell death in three-dimensional matrix culture

Abstract

Primary hepatocytes undergo phenotypic dedifferentiation upon isolation from liver that typically includes down regulation of uptake transporters and up regulation of efflux transporters. Culturing cells between layers of collagen in a three‐dimensional (3D) “sandwich” is reported to restore hepatic phenotype. This report examines how 3D culturing affects accumulation of fluorophores, the cytotoxic response to bile acids and drugs, and whether cell to cell differences in fluorescent anion accumulation correlate with differences in cytotoxicity. Hepatocytes were found to accumulate fluorescent bile acid (FBA) at significantly higher levels than the related fluorophores, carboxyfluorescein diacetate, (4.4‐fold), carboxyfluorescein succinimidyl ester (4.8‐fold), and fluorescein (30‐fold). In 2D culture, FBA accumulation decreased to background levels by 32 h, Hoechst nuclear accumulation strongly decreased, and nuclear diameter increased, indicative of an efflux phenotype. In 3D culture, FBA accumulation was maintained through 168 h but at 1/3 the original intensity. Cell to cell differences in accumulated FBA did not correlate with levels of liver zonal markers L‐FBAP (zone 1) or glutamine synthetase (zone 3). Cytotoxic response to hydrophobic bile acids, acetaminophen, and phalloidin was maintained in 3D culture, and cells with higher FBA accumulation showed 12–18% higher toxicity than the total population toward hydrophobic bile acids (P < 0.05). Long‐term imaging showed oscillations in the accumulation of FBA over periods of hours. Overall, the studies suggest that high accumulation of FBA can indicate the sensitivity of cultured hepatocytes to hydrophobic bile acids and other toxins.

Long‐term human primary hepatocyte cultures in a microfluidic liver biochip show maintenance of mRNA levels and higher drug metabolism compared with Petri cultures

“…Several hypotheses may reflect this result. It may be due to (i) a reduction of the cell viability, (ii) either a reduction of the esterase activity and weaker functionality of those enzymes (iii) or a weaker transport of the compound by the hepatic transporter in the cells due to the differentiation [23,24]. In the present study, additional viability using the trypan blue assay and the continuous basal metabolism check points (glucose, albumin, urea analyses over time) showed that the reduction of calcein signals was not due to cell death (although viability was low in biochips on day 5, similar results were found on days 9 and 13).…”

Vascularization of connective tissue in Organ-on-Chip

“…The model was capable to metabolize carboxydichlorofluorescein diacetate (CDFDA) by hepatic esterase(C.-J. Lin et al 2016) and its bioproduct, a fluorescent compound called carboxydichlorofluorescein (CDF) accumulated in the bile and was excreted into the bile canaliculi similarly to in vivo (Nakao et al 2011) (Murray, Han, and Wolkoff 2014). This result is a good example that demonstrates how replicating the bioarchitecture of tissue can restore organ in vivo functions, including metabolic pathways, maintaining cellular phenotype, tissue polarity, bio-synthetic and transportation ability of tissues(F. M. Chen and Liu 2016).…”