Extending the viability of human precision-cut intestinal slice model for drug metabolism studies.

Human Precision-cut intestinal slices (hPCIS) are used to study intestinal physiology, pathophysiology, drug efficacy, toxicology, kinetics, and metabolism. However, the use of this ex vivo model is restricted to approximately a 24 h timeframe because of declining viability of the hPCIS during traditional culture. We hypothesized that we could extend the hPCIS viability by using organoid medium. Therefore, we cultured hPCIS for up to 72 h in organoid media [expansion medium (Emed) and differentiation medium (Dmed)]. After incubation, we assessed culture-induced changes on viability markers, specific cell type markers and we assessed the metabolic activity of enterocytes by measuring midazolam metabolite formation. We show that the adenosine triphosphate (ATP)/protein ratio of Emed-cultured hPCIS and morphology of both Emed- and Dmed-cultured hPCIS was improved compared to WME-cultured hPCIS. Emed-cultured hPCIS showed an increased expression of proliferation and stem cell markers, whereas Dmed-cultured hPCIS showed an increased expression of proliferation and enterocyte markers, along with increased midazolam metabolism. Using the Emed, the viability of hPCIS could be extended for up to 72 h, and proliferating stem cells remained preserved. Using Dmed, hPCS also remained viable for up to 72 h, and specifically rescued the metabolizing enterocytes during culture. In conclusion, by using two different organoid culture media, we could extend the hPCIS viability for up to 72 h of incubation and specifically steer stem cells or enterocytes towards their original function, metabolism, and proliferation, potentially allowing pharmacokinetic and toxicology studies beyond the 24 h timeframe.

Development of a Novel Ex-vivo 3D Model to Screen Amoebicidal Activity on Infected Tissue.

Amoebiasis is a parasitic disease that causes thousands of deaths every year, its adverse effects and resistance to conventional treatments have led to the search of new treatment options, as well as the development of novel screening methods. In this work, we implemented a 3D model of intestine and liver slices from hamsters that were infected ex vivo with virulent E. histolytica trophozoites. Results show preserved histology in both uninfected tissues as well as ulcerations, destruction of the epithelial cells, and inflammatory reaction in intestine slices and formation of micro abscesses, and the presence of amoebae in the sinusoidal spaces and in the interior of central veins in liver slices. The three chemically synthetized compounds T-001, T-011, and T-016, which act as amoebicides in vitro, were active in both infected tissues, as they decreased the number of trophozoites, and provoked death by disintegration of the amoeba, similar to metronidazole. However, compound T-011 induced signs of cytotoxicity to liver slices. Our results suggest that ex vivo cultures of precision-cut intestinal and liver slices represent a reliable 3D approach to evaluate novel amoebicidal compounds, and to simultaneously detect their toxicity, while reducing the number of experimental animals commonly required by other model systems.

Induction of phase I and II drug metabolism in rat small intestine and colon in vitro.

The aim of this study was to evaluate drug metabolism in rat small intestinal and colon precision-cut slices during 24 h of incubation and the applicability of these slices for enzyme induction studies. Various parameters were evaluated: intracellular levels of ATP (general viability marker), alkaline phosphatase activity (specific epithelial marker), villin expression (specific epithelial marker), and metabolic rates of 7-ethoxycoumarin (CYP1A), testosterone (CYP3A and CYP2B), and 7-hydroxycoumarin (glucuronide and sulfate conjugation) conversions. ATP and villin remained constant up to, respectively, 5 and 8 h in small intestine and up to 24 h in colon. The metabolic rate remained constant in small intestinal slices up to 8 h and decreased afterward to 24 to 92%, depending on the substrate studied. The inducibility of metabolism in small intestinal and colon slices was tested with several inducers at various concentrations and incubation times. The following inducers were used: 3-methylcholanthrene, beta-naphthoflavone, indirubin, and tert-butylhydroquinone (aryl hydrocarbon receptor ligands), dexamethasone (glucocorticoid receptor/pregnane X receptor ligand) and phenobarbital (constitutive androstane receptor ligand). After incubation with inducers, metabolic rates were evaluated with 7-ethoxycoumarin and testosterone (phase I) and 7-hydroxycoumarin (phase II) as substrate. All inducers elevated the metabolic rates consistent with the available published in vivo induction data. Induction of enzyme activity was already detectable after 5 h (small intestine) and after 8 h (colon) for 3-methylcholanthrene and beta-naphthoflavone and was clearly detectable for all tested inducers after 24 h (up to 20-fold compared with noninduced controls). In conclusion, small intestinal and colon precision-cut slices are useful for metabolism and enzyme induction studies.

Prediction of whole-body metabolic clearance of drugs through the combined use of slices from rat liver, lung, kidney, small intestine and colon.

1: The aim was to investigate whether precision-cut rat tissue slices could be used to predict metabolic drug clearance in vivo. To obtain a complete picture, slices not only from liver, but also from lung, kidney, small intestine and colon were included. 2: The metabolic clearances of 7-ethoxycoumarin, 7-hydroxycoumarin, testosterone, methyltestosterone and warfarin were determined by measuring the disappearance of these compounds during incubation with slices prepared from liver, lung, kidney, small intestine and colon. 3: The total in vitro metabolic clearance was determined by adding the individual in vitro organ clearances from the slices. Prediction based on the in vitro clearance was within an order of magnitude to the corresponding in vivo values. Interestingly, the relative contribution of extrahepatic metabolic clearance of the studied compounds to total clearance was remarkably high, ranging from 35 to 72% of the total metabolic clearance. 4: It is concluded that the model of multi-organ precision-cut slices is a useful in vitro tool for prediction of in vivo metabolic clearance. In addition, it provides information about the relative contribution of the liver, lung, kidney, small intestine and colon to the total metabolic clearance.

Cryopreservation of precision-cut tissue slices for application in drug metabolism research.

Cryopreservation of tissue slices greatly facilitates their use in drug metabolism research, leading to efficient use of human organ material and a decrease of laboratory animal use. In the present review, various mechanisms of cryopreservation such as equilibrium slow freezing, rapid freezing and vitrification, and their application to cryopreservation of tissue slices are discussed as well as the viability parameters often used to evaluate the success of cryopreservation. Equilibrium freezing prevents intracellular ice formation by inducing cellular dehydration, but (large) ice crystals are still formed in the interstitial space of the slices. Upon rapid freezing, (small) intra- and extracellular ice crystals are formed which slices from some tissues can resist. Vitrification prevents the formation of both intra- and extracellular ice crystals while an amorphous glass is formed of the slice liquid constituents. To vitrify, however, high molarity solutions of cryoprotectants are required that may be toxic to the slices. The use of mixtures of high molarity of cryoprotectants overcomes this problem. We conclude that vitrification is the approach that most likely will lead to the development of universal cryopreservation methods for tissue slices of various organs from various animal species. In the future this may lead to the formation of a tissue slice bank from which slices can be derived at any desirable time point for in vitro experimentation.

Incubation at 37 degrees C prior to cryopreservation decreases viability of liver slices after cryopreservation by rapid freezing.

Precision-cut liver slices are to some extent resistant to ice formation induced by rapid freezing. Susceptibility to rapid freezing damage has been shown to be (partly) dependent on intrinsic properties of cells. In the present study an attempt was made to decrease the susceptibility of rat liver slices for rapid freezing damage: the slices were pre-incubated at 37 degrees C under oxygen, prior to cryopreservation to recover from low ATP levels, impaired ion regulation and cell swelling induced by their preparation. It was shown that, unexpectedly, recovery of cellular homeostasis prior to the cryopreservation procedure by the 37 degrees C pre-incubation markedly decreased viability of rapidly frozen slices (in which ice was formed), but not of vitrified slices (in which no ice was formed), in a time- and temperature-dependent manner. UW was found to protect slices from this 'warm pre-incubation phenomenon.' Apparently, pre-incubation prior to freezing causes certain cellular alterations that render slices more susceptible to rapid freezing damage.

Comparison of in vitro preparations for semi-quantitative prediction of in vivo drug metabolism.

Various in vitro preparations were compared with respect to their ability to mimic in vivo metabolism. For this purpose, S9-liver homogenate, microsomes, cryopreserved hepatocytes, cryopreserved liver slices and fresh liver, lung, kidney, and intestinal slices were incubated with three drugs in development, which are metabolized in vivo by a wide range of biotransformation pathways. Metabolites were identified and quantified with liquid chromatography-mass spectometry/UV from the in vitro incubations and compared with metabolite patterns in feces, urine, and bile of dosed rats. In vitro systems with intact liver cells produced the same metabolites as the rat in vivo and are a valuable tool to study drug metabolism. Phase I metabolites were almost all conjugated in intact cells, whereas S9-homogenate only conjugated by sulfation and N-acetylation. Microsomes and S9-homogenate are useful to study phase I metabolism but not for the prediction of in vivo metabolism. Extra-hepatic organ slices did not form any metabolites that were not produced by liver cells, but the relative amounts of the various metabolites differed considerably. Small intestinal slices were more active than liver slices in the formation of the N-glucuronide of compound C, which is the major metabolite in vivo. When the relative contribution of liver and small intestinal slices to the metabolism of this compound was taken into account, it appeared that the in vivo metabolite pattern could be well predicted. Results indicate that for adequate prediction of in vivo metabolism, fresh or cryopreserved liver slices or hepatocytes in combination with slices of the small intestines should be used.