Investigation of expression and activity levels of primary rat hepatocyte detoxication genes under various flow rates and cell densities in microfluidic biochips

Abstract

We investigated the behavior of primary rat hepatocytes in biochips using a microfluidic platform (the integrated dynamic cell culture microchip). We studied the effects of cell inoculation densities (0.2-0.5 × 10(6) cells/biochip) and perfusion flow rates (10, 25, and 40 µL/min) during 72 h of perfusion. No effects were observed on hepatocyte morphology, but the levels of mRNA and CYP1A2 activity were found to be dependent on the initial cell densities and flow rates. The dataset made it possible to extract a best estimated range of parameters in which the rat hepatocytes appeared the most functional in the biochips. Namely, at 0.25 × 10(6) inoculated cells cultivated at 25 µL/min for 72 h, we demonstrated better induction of the expression of all the genes analyzed in comparison with other cell densities and flow rates. More precisely, when primary rat hepatocytes were cultivated at these conditions, the time-lapse analysis demonstrated an over expression of CYP3A1, CYP2B1, ABCC1b and ABCC2 in the biochips when compared to the postextraction levels. Furthermore, the AHR, CYP1A2, GSTA2, SULT1A1, and UGT1A6 levels remained higher than 50% of the postextraction values whereas values of HNF4α, CEBP, and PXR remained higher than 20% during the duration of the culture process. Nevertheless, an important reduction in mRNA levels was found for the xenosensors CAR and FXR, and the related CYP (CYP2E1, CYP7A1, CYP3A2, and CYP2D2). CYP1A2 functionality was illustrated by 700 ± 100 pmol/h/10(6) cells resorufin production. This study highlighted the functionality in optimized conditions of primary rat hepatocytes in parallelized microfluidic cultures and their potential for drug screening applications.

Bioreactor technologies to support liver function in vitro

“…The feasibility of carrying out transcriptomic, proteomic, metabolic and toxicologic characterization with this arrangement, was illustrated and showed that HepG2/C3a phenotypic responses including albumin secretion, sensitivity to acetaminophen toxicity, and relative expression of CYP450 and transport genes appeared more physiologically close to primary hepatocytes in the microfluidic compared to static petri dish culture, a result that may in part due to the quasi-3D aggregate structures that form in regions of the recessed structures [74,79,80][81–84]. Initial studies analyzing primary rat [85,86] and human [74,87] cells in short term culture (4 days) indicate that these cells were less likely to form aggregates, and optimization of operating conditions for these cells is evolving. The results assessing drug metabolism with cryopreserved human cells are instructive in two key ways, first showing that these cells attach and survive in both the recessed features as well as the top of the PDMS exposed directly to flow.…”

Lab on a chip-based hepatic sinusoidal system simulator for optimal primary hepatocyte culture

“…In addition, many studies have reported that mRNA expression of genes involved in xenobiotic metabolism and transport, including CYP1A1, 1 A2, 2B6, 2C9, 3 A4, multidrug resistance protein 1, MRP2, and UDP-glucuronosyltransferase, was upregulated by medium flow (Baudoin et al 2014;Dash et al 2013;Vinci et al 2011). In our study, hepatocytes exposed to flow conditions expressed higher levels of hepatocyte-specific markers, such as CYP1A2, MRP1, and UGT1A5, over 13 days.…”

A novel toxicogenomics-based approach to categorize (non-)genotoxic carcinogens

“…As additional in vitro system, one could consider to use rat primary hepatocytes. These cells have been demonstrated to retain high mRNA levels of CAR and other xenobiotic receptors, as well as Phase I and Phase II genes (Baudoin et al 2014). This may improve the detection of CAR activators, which were missed using PMH and mESC.…”