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

Human primary hepatocytes were cultivated in a microfluidic bioreactor and in Petri dishes for 13 days. mRNA kinetics in biochips showed an increase in the levels of CYP2B6, CYP2C19, CYP2C8, CYP3A4, CYP1A2, CYP2D6, HNF4a, SULT1A1, UGT1A1 mRNA related genes when compared with post extraction levels. In addition, comparison with Petri dishes showed higher levels of CYP2B6, CYP2C19, CYP2C8, CYP3A4, CYP1A2, CYP2D6 related genes at the end of culture. Functional assays illustrated a higher urea and albumin production over the period of culture in biochips. Bioreactor drug metabolism (midazolam and phenacetin) was not superior to the Petri dish after 2 days of culture. The CYP3A4 midazolam metabolism was maintained in biochips after 13 days of culture, whereas it was almost undetectable in Petri dishes. This led to a 5000-fold higher value of the metabolic ratio in the biochips. CYP1A2 phenacetin metabolism was found to be higher in biochips after 5, 9 and 13 days of culture. Thus, a 100-fold higher metabolic ratio of APAP in biochips was measured after 13 days of perfusion. These results demonstrated functional primary human hepatocyte culture in the bioreactor in a long-term culture. Copyright © 2016 John Wiley & Sons, Ltd.

Multiparametric temporal analysis of the Caco-2/TC7 demonstrated functional and differentiated monolayers as early as 14 days of culture.

Reducing the differentiation period for obtaining an in vitro intestinal barrier model is required to reduce the duration and cost for drug screening assays. In this frame, the Caco-2/TC7 subclone differentiation state was investigated from day 0 (D0) to day 32 (D32). As such, the expression of 45 genes (including cell junction, cell polarization, cell functionality, drug transport and metabolism genes) was followed throughout the 32 days. In parallel, the monolayer polarization and the formation of the cellular junctions were characterized by the immuno-staining of occludin, claudin-1 and actin proteins. The cell monolayer permeability was analyzed via transepithelial electric resistance measurements and paracellular transport of Lucifer Yellow. The P-gp efflux efficiency was assessed by rhodamine 123 transport. Alkaline phosphate activity was quantified to assess the cell differentiation. Three stages of differentiation were observed using the clustering of principal component analysis of the RTqPCR data and the overall assays. From D0 to D10, cells were in a proliferation stage and under-differentiated; from D14 to D21 a stable differentiation stage was reached; from D25 to D32 the epithelium seemed to enter into a post-differentiated stage. This study demonstrates that Caco-2/TC7 cells are functional and ready for use in drug screening permeability assays from 14 days in culture when compared with conventional 21 days for Caco-2 cells. In addition, this study provides a refined set of data allowing temporal and multi scale investigations, due to the intracellular kinetics and mRNA levels that can be correlated with membrane protein kinetics and functional extracellular activities. Therefore, shorter time in culture combined with a better knowledge of the cells during the time in culture will in turn help to improve the quality and cost of Caco-2/TC7 assays for drug development.

Investigation of omeprazole and phenacetin first-pass metabolism in humans using a microscale bioreactor and pharmacokinetic models.

A new in vitro microfluidic platform (integrated insert dynamic microfluidic platform, IIDMP) allowing the co-culture of intestinal Caco-2 TC7 cells and of human primary hepatocytes was used to test the absorption and first-pass metabolism of two drugs: phenacetin and omeprazole. The metabolism of these drugs by CYP1A2, CYP2C19 and CYP3A4 was evaluated by the calculation of bioavailabilities and of intrinsic clearances using a pharmacokinetic (PK) model. To demonstrate the usefulness of the device and of the PK model, predictions were compared with in vitro and in vivo results from the literature. Based on the IIDMP experiments, hepatic in vivo clearances of phenacetin and omeprazole in the IIDMP were predicted to be 3.10 ± 0.36 and 1.46 ± 0.25 ml/min/kg body weight, respectively. This appeared lower than the in vivo observed data with values ranging between 11.9-19.6 and 5.8-7.5 ml/min/kg body weight, respectively. Then the calculated hepatic and intestinal clearances led to predicting an oral bioavailability of 0.85 and 0.77 for phenacetin and omeprazole versus 0.92 and 0.78 using separate data from the simple monoculture of Caco-2 TC7 cells and hepatocytes in Petri dishes. When compared with the in vivo data, the results of oral bioavailability were overestimated (0.37 and 0.71, respectively). The feasibility of co-culture in a device allowing the integration of intestinal absorption, intestinal metabolism and hepatic metabolism in a single model was demonstrated. Nevertheless, further experiments with other drugs are needed to extend knowledge of the device to predict oral bioavailability and intestinal first-pass metabolism.

First pass intestinal and liver metabolism of paracetamol in a microfluidic platform coupled with a mathematical modeling as a means of evaluating ADME processes in humans.

We developed a microfluidic platform to investigate paracetamol intestinal and liver first pass metabolism. This approach was coupled with a mathematical model to estimate intrinsic in vitro parameters and to predict in vivo processes. The kinetic modeling estimated the paracetamol and paracetamol sulfate permeabilities, the sulfate and glucuronide effluxes in the intestine compartment. Based on a gut model, we estimated intrinsic intestinal clearance of between 26 and 77 L/h for paracetamol in humans, a permeability of 10 L/h, and a gut availability between 0.17 and 0.53 (compared to 0.95-1 in vivo). The role played by the liver in paracetamol metabolism was estimated via in vitro intrinsic clearances of 7.6, 13.6, and 11.5 µL/min/10(6) cells for HepG2/C3a, rat primary hepatocytes, and human primary hepatocytes, respectively. Based on a parallel tube model to describe the liver, the paracetamol hepatic clearance, and the paracetamol hepatic availability in humans were estimated at 6.5 mL/min/kg of bodyweight (BDW) and 0.7, respectively (when compared to 5 mL/min/kg of BDW and 0.77 to 0.88 for in vivo values, respectively). The drug availability was predicted ranging between 0.24 and 0.41 (0.88 in vivo). The overall approach provided a first step in an integrated strategy combining in silico/in vitro methods based on microfluidic for evaluating drug absorption, distribution and metabolism processes.

Development of a new microfluidic platform integrating co-cultures of intestinal and liver cell lines.

We developed a new biological model to mimic the organ-organ interactions between the intestine and the liver. We coupled polycarbonate cell culture inserts and microfluidic biochips in an integrated fluidic platform allowing dynamic co-cultures (called IIDMP for Integrated Insert in a Dynamic Microfluidic Platform). The intestinal compartment was simulated using Caco-2 TC7 cells and the liver one by HepG2 C3A. We showed that Caco-2 TC7 viability, barrier integrity and functionality (assessed by paracellular and active transport), were not altered during co-cultures in the bioreactor in comparison with the conventional insert Petri cultures. In parallel, the viability and metabolism of the HepG2 C3A cells were maintained in the microfluidic biochips. Then, as proof of concept, we used the bioreactor to follow the transport of phenacetin through the intestinal barrier and its metabolism into paracetamol by the CYP1A of the HepG2 C3A cells. Our results demonstrated the performance of this bioreactor with cell co-cultures compared to static co-culture controls in which weak biotransformation into paracetamol was detected. Our study illustrated the interest of such a bioreactor combining the advantages of a cell culture barrier and of liver microfluidic cultures in a common framework for in vitro studies.

Metabolic characterization of primary rat hepatocytes cultivated in parallel microfluidic biochips.

The functionality of primary rat hepatocytes was assessed in an Integrated Dynamic Cell Cultures in Microsystem (IDCCM) device. We characterized the hepatocytes over 96 h of culture and evaluated the impact of dynamic cell culture on their viability, inducibility, and metabolic activity. Reverse Transcription quantitative Polymerase Chain Reaction (RTqPCR) was performed on selected genes: liver transcription factors (HNF4α and CEBP), nuclear receptors sensitive to xenobiotics (AhR, PXR, CAR, and FXR), cytochromes P450 (CYPs) (1A2, 3A2, 3A23/3A1, 7A1, 2B1, 2C6, 2C, 2D1, 2D2, and 2E1), phase II metabolism enzymes (GSTA2, SULT1A1, and UGT1A6), ABC transporters (ABCB1b and ABCC2), and oxidative stress related enzymes (HMOX1 and NQO1). Microperfused-cultured hepatocytes remained viable and differentiated with in vivo-like phenotype and genotype. In contrast with postadhesion gene levels, the first 48 h of perfusion enhanced the expression of xenosensors and their target CYPs. Furthermore, CYP3A1, CYP2B1, GSTA2, SULT1A1, UGT1A1, ABCB1b, and ABCC2 were upregulated in IDCCM and reached above postextraction levels all along the duration of culture. Metabolic activities were also confirmed with the detection of metabolism rate and induced mRNAs after exposure to several inducers: 3-methylcholanthrene, caffeine, phenacetin, paracetamol,, and midazolam. Finally, this metabolic characterization confirms that IDCCM is able to maintain rat hepatocytes functions to investigate drug metabolism.