Evaluation of seven drug metabolisms and clearances by cryopreserved human primary hepatocytes cultivated in microfluidic biochips.

We present characterization of the metabolic performance of human cryopreserved hepatocytes cultivated in a platform of parallelized microfluidic biochips. The RTqPCR analysis revealed that the mRNA levels of the cytochromes P450 (CYP 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4) were reduced after the adhesion period (when compared to the post-thawing step). The microfluidic perfusion played a part in stabilizing and partially recovering the levels of the HNF4α, PXR, OAPT2, CYP 1A2, 2B6, 2C19 and 3A4 mRNA on contrary to non-perfused cultures. Fluorescein diacetate staining and P-gp mRNA level illustrated the hepatocytes' polarity in the biochips. Drug metabolism was assessed using midazolam, tolbutamide, caffeine, omeprazole, dextromethorphan, acetaminophen and repaglinide as probes. Metabolite detection and quantification revealed that CYP1A2 (via the detection of paraxanthine), CYP3A4 (via 1-OH-midazolam, and omeprazole sulfone detection), CYP2C8 (via hydroxyl-repaglinide detection), CYP2C19 (via hydroxy-omeprazole detection) and CYP2D6 (via dextrorphan detection) were functional in our microfluidic configurations. Furthermore, the RTqPCR analysis showed that the drugs acted as inductors leading to overexpression of mRNA levels when compared to post-thawing values (such as for HNF4α, PXR and CYP3A4 by dextromethorpahn and omeprazole). Finally, intrinsic in vitro biochip clearances were extracted using a PBPK model for predictions. The biochip predictions were compared to literature in vitro data and in vivo situations.

Metabolomics-on-a-chip and predictive systems toxicology in microfluidic bioartificial organs.

The world faces complex challenges for chemical hazard assessment. Microfluidic bioartificial organs enable the spatial and temporal control of cell growth and biochemistry, critical for organ-specific metabolic functions and particularly relevant to testing the metabolic dose-response signatures associated with both pharmaceutical and environmental toxicity. Here we present an approach combining a microfluidic system with (1)H NMR-based metabolomic footprinting, as a high-throughput small-molecule screening approach. We characterized the toxicity of several molecules: ammonia (NH(3)), an environmental pollutant leading to metabolic acidosis and liver and kidney toxicity; dimethylsulfoxide (DMSO), a free radical-scavenging solvent; and N-acetyl-para-aminophenol (APAP, or paracetamol), a hepatotoxic analgesic drug. We report organ-specific NH(3) dose-dependent metabolic responses in several microfluidic bioartificial organs (liver, kidney, and cocultures), as well as predictive (99% accuracy for NH(3) and 94% for APAP) compound-specific signatures. Our integration of microtechnology, cell culture in microfluidic biochips, and metabolic profiling opens the development of so-called "metabolomics-on-a-chip" assays in pharmaceutical and environmental toxicology.

Selective control of liver and kidney cells migration during organotypic cocultures inside fibronectin-coated rectangular silicone microchannels.

In this work, the behaviors of embryonic liver and kidney explants were studied inside rectangular polydimethylsiloxane (PDMS) microchannels. The organs were cultured under monoculture and coculture conditions on PDMS coated with or without fibronectin. The results demonstrated that the migration of cells from both organs is dependent on culture conditions and thus can be selectively controlled. In liver monocultures without fibronectin, cell migration in the microchannels resulted in the formation of a dense 3D tissue. Fibronectin reduced liver cell migration and enhanced the emergence of cells demonstrating typical hepatocyte phenotypes at the vicinity of the explant. The migration rate in liver-liver cocultures, with and without fibronectin, was roughly twice the rate of cells under monoculture conditions. In cocultures, both livers merged to form a large tissue in which the two initial organs could not be identified. In kidney monocultures, with and without fibronectin, we did not observe any migration inside the microchannels. Contrary to liver cells, kidney cell migration was triggered when both fibronectin coating and coculture with liver or another kidney explant were used. The migration was more largely observed in coculture with liver when compared to kidney-kidney cocultures. In the case of liver-kidney coculture with fibronectin, the progression of the kidney cells inside the microchannels appears as a displacement of the entire kidney explant in the direction of the liver. The liver cells did not move in those cases. After contact, we observed a complete merging of both liver and kidney explants. In contrast, for liver-kidney cocultures without fibronectin, only the liver moved toward the kidney.

Development of a renal microchip for in vitro distal tubule models.

Current developments in tissue engineering and microtechnology fields have allowed the proposal of pertinent tools, microchips, to investigate in vitro toxicity. In the framework of the proposed REACH European directive and the 3R recommendations, the purpose of these microtools is to mimic organs in vitro to refine in vitro culture models and to ultimately reduce animal testing. The microchip consists of functional living cell microchambers interconnected by a microfluidic network that allows continuous cell feeding and waste removal controls by fluid microflow. To validate this approach, Madin Darby Canine Kidney (MDCK) cells were cultivated inside a polydimethylsiloxane microchip. To assess the cell proliferation and feeding, the number of inoculated cells varied from 5 to 10 x 10(5) cells/microchip (corresponding roughly to 2.5 to 5 x 10(5) cells/cm2) and from four flow rates 0, 10, 25, and 50 microL/min were tested. Morphological observations have shown successful cell attachment and proliferation inside the microchips. The best flow rate appears to be 10 microL/min with which the cell population was multiplied by about 2.2 +/- 0.1 after 4 days of culture, including 3 days of perfusion (in comparison to 1.7 +/- 0.2 at 25 microL/min). At 10 microL/min flow rate, maximal cell population reached about 2.1 +/- 0.2 x 10(6) (corresponding to 7 +/- 0.7 x 10(7) cells/cm(3)). The viability, assessed by trypan blue and lactate deshydrogenase measurements, was found to be above 90% in all experiments. At 10 microL/min, glucose monitoring indicated a cell consumption of 16 +/- 2 microg/h/10(6) cells, whereas the glutamine metabolism was demonstrated with the production of NH3 by the cells about 0.8 +/- 0.4 micromol/day/10(6) cells. Augmentation of the flow rate appeared to increase the glucose consumption and the NH3 production by about 1.5- to 2-fold, in agreement with the tendencies reported in the literature. As a basic chronic toxicity assessment in the microchips, 5 mM and 10 mM ammonium chloride loadings, supplemented in the culture media, at 0, 10, and 25 micaroL/min flow rates were performed. At 10 microL/min, a reduction of 35% of the growth ratio with 5 mM and of 50% at 10 mM was found, whereas at 25 microL/min, a reduction of 10% with 5 mM and of 30% at 10 mM was obtained. Ammonium chloride contributed to increase the glucose consumption and to reduce the NH3 production. The microchip advantages, high surface/volume ratio, and dynamic loadings, coupled with the concordance between the present and literature results dealing with ammonia/ammonium effects on MDCK illustrate the potential of our microchip for wider in vitro chronic toxicity investigations.

Trends in the development of microfluidic cell biochips for in vitro hepatotoxicity.

Current developments in the technological fields of liver tissue engineering, bioengineering, biomechanics, microfabrication and microfluidics have lead to highly complex and pertinent new tools called "cell biochips" for in vitro toxicology. The purpose of "cell biochips" is to mimic organ tissues in vitro in order to partially reduce the amount of in vivo testing. These "cell biochips" consist of microchambers containing engineered tissue and living cell cultures interconnected by a microfluidic network, which allows the control of microfluidic flows for dynamic cultures, by continuous feeding of nutrients to cultured cells and waste removal. Cell biochips also allow the control of physiological contact times of diluted molecules with the tissues and cells, for rapid testing of sample preparations or specific addressing. Cell biochips can be situated between in vitro and in vivo testing. These types of systems can enhance functionality of cells by mimicking the tissue architecture complexities when compared to in vitro analysis but at the same time present a more rapid and simple process when compared to in vivo testing procedures. In this paper, we first introduce the concepts of microfluidic and biochip systems based on recent progress in microfabrication techniques used to mimic liver tissue in vitro. This includes progress and understanding in biomaterials science (cell culture substrate), biomechanics (dynamic cultures conditions) and biology (tissue engineering). The development of new "cell biochips" for chronic toxicology analysis of engineered tissues can be achieved through the combination of these research domains. Combining these advanced research domains, we then present "cell biochips" that allow liver chronic toxicity analysis in vitro on engineered tissues. An extension of the "cell biochip" idea has also allowed "organ interactions on chip", which can be considered as a first step towards the replacement of animal testing using a combined liver/lung organ model.

Guidance of liver and kidney organotypic cultures inside rectangular silicone microchannels.

We have studied the effect of rectangular polydimethylsiloxane (PDMS) microchannels on the behavior of embryonic liver and kidney explants maintained in contact with these microchannels. The microchannel widths were varied from 35 to 300 microm and depth from 45 to 135 microm. The growth of these tissue types were compared to the development on flat silicone and plastic control material. At seeding, due to the viscoelastic properties of both organs, "capillary-like filling" was observed inside the narrowest microchannels. In those cases, the tissues grew to a confluent layer joining the microchannels with no cell migration and proliferation inside the microchannels. In the largest microchannels, only a weak migration was observed and the cellular behavior appears quite similar to that of PDMS flat culture conditions. In intermediate geometries, we observed different tissue growth progressed inside those microchannels with an average growth properties inside the microchannels when compared to other sizes. The liver tissues velocity of up to 72 microm/day resulting to form a dense three-dimensional multicellular 'liver-like tissue'. Scanning electron microscopy (SEM) observations demonstrated that the tissue was organized like an epithelial layer with round cells embedded in an extracellular matrix. Liver cell mobility may result primarily from the activity of the marginal cells, whereas the sub-marginal cells appeared passively dragged. Parenchymal organization demonstrating differentiated states was also observed. Kidney grew mainly on the microchannel walls and the tissues never appeared dense and organized as the liver ones.

Evaluation of a liver microfluidic biochip to predict in vivo clearances of seven drugs in rats.

We investigated metabolic clearances of phenacetin, midazolam, propranolol, paracetamol, tolbutamide, caffeine, and dextromethorphan by primary rat hepatocytes cultivated in microfluidic biochips. The levels of mRNA of the HNF4α, PXR, AHR, CYP3A1, and CYP1A2 genes were enhanced in the biochip cultures when compared with postextraction levels. We measured a high and rapid adsorption on the biochip walls and inside the circuit for dextromethorphan and midazolam, a moderate adsorption for phenacetin and propranolol, and a low adsorption for caffeine, tolbutamide, and paracetamol. Drug biotransformations were demonstrated by the formations of specific metabolites such as paraxanthyne (caffeine), paracetamol (phenacetin), 1-OH midazolam (midazolam), paracetamol sulfate (paracetamol and phenacetin), and dextrorphan (dextromethorphan). We used a pharmacokinetic model to estimate the adsorption and in vitro intrinsic drug clearance values. We calculated in vitro intrinsic clearance values of 0.5, 3, 12.5, 83, 100, 160, and 900 µL/min per 10(6) cells for the tolbutamide, caffeine, paracetamol, dextromethorphan, phenacetin, midazolam, and propranolol, respectively. A second model describing the liver as a well-stirred compartment predicted in vivo hepatic clearances of 0.1, 13.8, 30, 44.1, 61, 72, 85, and 61 mL/min per kg of body mass for the tolbutamide, caffeine, paracetamol, midazolam, dextromethorphan, phenacetin, and propranolol, respectively. These values appeared consistent with previously reported data.

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

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.

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.