Human in vitro physiological models studying disease and drug treatment effects are urgently needed as more relevant tools to identify new drug targets and therapies. We have developed a human microfluidic two-organ-chip model to study pancreatic islet–liver cross-talk based on insulin and glucose regulation. We have established a robust co-culture of human pancreatic islet microtissues and liver spheroids maintaining functional responses up to 15 days in an insulin-free medium. Functional coupling, demonstrated by insulin released from the islet microtissues in response to a glucose load applied in glucose tolerance tests on different days, promoted glucose uptake by the liver spheroids. Co-cultures maintained postprandial glucose concentrations in the circulation whereas glucose levels remained elevated in both single cultures. Thus, insulin secreted into the circulation stimulated glucose uptake by the liver spheroids, while the latter, in the absence of insulin, did not consume glucose as efficiently. As the glucose concentration fell, insulin secretion subsided, demonstrating a functional feedback loop between the liver and the insulin-secreting islet microtissues. Finally, inter-laboratory validation verified robustness and reproducibility. Further development of this model using tools inducing impaired glucose regulation should provide a unique in vitro system emulating human type 2 diabetes mellitus.

ABSTRACT:HepaRG cells, a newly developed human hepatoma cell line, differentiate into hepatocyte-like morphology by treatment with dimethyl sulfoxide (DMSO). The expression of cytochrome P450 (P450) enzymes, transporter proteins, and transcription factors was stable in differentiated HepaRG cells over a period of 6 weeks when cultured with DMSO. Compared with human hepatocytes, expression of P450 in HepaRG cells was in general lower with the exception for a considerably higher expression of CYP3A4 and CYP7A1. The expression of P450s generally decreased when DMSO was removed from the medium, whereas transporters and liver-specific factors were unaffected. The relative mRNA content of drug-metabolizing P450s displayed the highest resemblance between human hepatocytes and differentiated HepaRG cells 1 day after removal of DMSO from the medium. The metabolism of midazolam, naloxone, and clozapine in HepaRG cells was similar to human hepatocytes, indicating the function of CYP3A4, CYP1A2, and UDP-glucuronosyltransferase enzymes. However, the metabolism of 7-ethoxycoumarin and dextromethorphan was low, confirming low levels of CYP2E1 and CYP2D6 in HepaRG cells. The P450 probe substrates indicate a decrease in CYP1A2, CYP2B6, CYP2C9, and CYP3A4 activities in HepaRG cells 1 day after removal of DMSO from the medium. The activities were then relatively stable in DMSO-free medium for up to 14 days. Based on the stable expression of liver-specific functions over a long period in culture, the relative mRNA content of drug-metabolizing P450s, and metabolic properties, HepaRG cells provide a valuable in vitro model for human drug metabolism studies.Recently, a new human hepatoma cell line, HepaRG, was derived from a hepatocellular carcinoma. Seeded at low density HepaRG cells emerge as two cell types when they reach confluence. One is flattened, retains a clear cytoplasm, and surrounds the other. The second forms clusters of granular epithelial cells resembling hepatocytes. Addition of 2% dimethyl sulfoxide (DMSO) and 50 M hydrocortisone hemisuccinate to the culture medium induces differentiation of the hepatocyte-like cells into more granular cells, closely resembling typical adult primary hepatocytes with one or two nuclei and bile canaliculi-like structures. The hepatocyte-like cells represent around 50 to 55% of the total cell population (Cerec et al., 2007). The HepaRG cells express a large panel of liver-specific genes including several cytochrome P450 (P450) enzymes such as CYP1A2, CYP2B6, CYP2C9, CYP2E1, and CYP3A4, which is in contrast to other hepatoma cell lines such as HepG2. The activity of P450s was demonstrated by using several probe substrates (Aninat et al., 2006).The levels of P450s in HepaRG cells are dependent on the duration of confluence and for most of them on the presence of DMSO in the culturing medium. The HepaRG cell line was recently found to be a valuable human-relevant in vitro model for investigating P450 induction properties of drug compounds (Kanebratt and Andersson, 2008). The expression and ...

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT• We have suggested that 4b-hydroxycholesterol may be used as an endogenous marker of CYP3A activity.• Recently, we found unexpectedly that the plasma concentration of 4b-hydroxycholesterol continued to increase for several weeks after complete induction of CYP3A4/5 by carbamazepine.• In the present study we investigated the time course of elimination of 4b-hydroxycholesterol from the circulation following CYP3A induction with rifampicin. WHAT THIS STUDY ADDS• 4b-Hydroxycholesterol is eliminated very slowly from the circulation with an apparent half-life of 17 days.• The long half-life results in a low variation in plasma concentration with time, but excludes 4b-hydroxycholesterol as a marker for rapid changes in CYP3A activity. AIMSThe oxysterol 4b-hydroxycholesterol has been suggested as a marker for CYP3A4/5 activity. We have previously shown that plasma 4b-hydroxycholesterol continues to increase for several weeks after maximal induction of CYP3A4/5 by carbamazepine at the dose given. In the present study we aimed to determine the time course of the decrease in plasma 4b-hydroxycholesterol after termination of induction of CYP3A4/5 by rifampicin. An additional aim was to determine the variation in plasma level of 4b-hydroxycholesterol with time in 12 untreated healthy volunteers. METHODSTwenty-four healthy subjects were allocated into three study groups of equal sizes. The volunteers were treated with rifampicin (either 20 mg day -1 , 100 mg day -1 or 500 mg day -1 ) for 2 weeks. Blood samples were taken before, during and after rifampicin treatment. In another group of 12 untreated volunteers blood samples were collected at different time points in order to determine the intraindividual variations in plasma 4b-hydroxycholesterol concentrations. Plasma levels of 4b-hydroxycholesterol were determined by isotope-dilution gas chromatography-mass spectrometry. RESULTSRifampicin treatment increased plasma 4b-hydroxycholesterol levels. After termination of rifampicin treatment plasma levels of 4b-hydroxycholesterol decreased slowly with an apparent half-life of 17 days. The intraindividual variation in plasma levels of 4b-hydroxycholesterol in untreated subjects was low, with coefficients of variation of between 4.8 and 13.2% over a period of 3 months. CONCLUSIONSAfter termination of induction of CYP3A4/5, plasma 4b-hydroxycholesterol levels decreased slowly during 8 weeks. The half-life of elimination (17 days) resembled that of cholesterol rather than other oxysterols. The long half-life results in stable plasma concentrations with time.