Mans Minekus, Phillipe Marteau, Robert Havenaar and Jos HJ Huis in
A multicompartmental in vitro model has been described, which simulates the dynamic events occurring within the lumen of the gastrointestinal tract of man and monogastric animals. The accuracy of the model for reproducing in vivo data on gastrointestinal transit, pH, bile salt concentrations and the absorption of glucose was tested. The in vivo conditions simulated in the model were based on studies in healthy human volunteers. Mathematical modelling of gastric and ileal delivery with power exponential equations was used'for the computer control of meal transit. The model appeared to reproduce accurately the pre-set data on meal transit, pH and bile salt concentrations in the different gastrointestinal compartments. Glucose absorption from the small intestine was almost complete. This model reproduces very closely the dynamic conditions based on the in vivo situation in monogastric animals and man. Therefore, the model can be an important tool in studying the fate of ingested components (for example, food, microorganisms and medicines) during gastrointestinal transit and, consequently, may contribute to the replacement of studies using laboratory animals.
Development of a Size-dependent Aerosol Deposition Model Utilising Human Airway Epithelial Cells for Evaluating Aerosol Drug Delivery
Daniel Cooney, Masha Kazantseva and Anthony J. Hickey
Aerosol delivery to the airways of the human respiratory tract, followed by absorption, constitutes an alternative route of administration for compounds unsuitable for delivery by conventional oral and parenteral routes. The target for aerosol drug delivery is the airways epithelium, i.e. tracheal, bronchial, bronchiolar and alveolar cells, which become the site of drug deposition. These epithelial layers also serve as a barrier to the penetration of inhaled material. An in vitro model for aerosol deposition and transport across epithelia in the human airways may be a good predictor of in vivo disposition. The present preliminary studies begin an investigation that blends the dynamics of aerosol delivery and the basis of an in vitro simulated lung model to evaluate the transport properties of a series of molecular weight marker compounds across human-derived bronchiolar epithelial cell monolayers. An Andersen viable cascade impactor was used as a delivery apparatus for the deposition of size-segregated particles onto monolayers of small airway epithelial cells and Calu-3 cells. It was shown that these cell layers can withstand placement in the impactor, and that permeability can be tested subsequent to removal from the impactor.
An Evaluation of a Novel Chick Cardiomyocyte Micromass Culture Assay with Two Teratogens/Embryotoxins Associated with Heart Defects
Helena S. Hurst, Richard H. Clothier and Margaret Pratten
This study was aimed at determining whether the chick cardiomyocyte micromass (MM) system could be employed to predict the teratogenicity/embryotoxicity of exogenous chemicals. Two documented teratogens/embryotoxins, sodium valproate (the sodium salt of valproic acid; VPA) and all-trans retinoic acid (tRA), were used in the initial phase of the study. White Leghorn 5-day-old embryo hearts were dissociated to produce a cardiomyocyte suspension in Dulbecco’s Modified Eagle’s Medium. Cultures were incubated at 37°C in 5% CO2 in air, and observations were made every 24 hours over 5 days, for the detection of beating. Culture viability was assessed by using the resazurin reduction assay for determining culture activity and the kenacid blue assay for determining cell number. It was found that tRA significantly reduced cell activity and beating, whilst not affecting total cell number. VPA up to 500μM induced no cytotoxicity in the MM cardiomyocyte cultures, whilst all the VPA concentrations tested reduced beating. The results demonstrate the potential of the chick cardiomyocyte MM culture assay to identify teratogens/embryotoxins that alter functionality, which may result in a teratogenic outcome, whilst not causing cytotoxicity (direct embryotoxicity). This could form part of a screen for developmental toxicity related to cardiac function, whilst limb cultures and brain cultures based on the same system could be relevant to teratogenic effects on those tissues.
Mathieu Vinken, Elke Decrock, Elke De Vuyst, Luc Leybaert, Tamara Vanhaecke and Vera Rogiers
This study was set up to critically evaluate a commonly-used in vitro model of hepatocellular apoptotic cell death, in which freshly isolated hepatocytes, cultured in a monolayer configuration, are exposed to a combination of Fas ligand and cycloheximide for six hours. A set of well-acknowledged cell death markers was addressed: a) cell morphology was studied by light microscopy; b) apoptotic and necrotic cell populations were quantified by in situ staining with Annexin-V, Hoechst 33342 and propidium iodide (PI); c) apoptotic and necrotic activities were monitored by probing caspase 3-like activity and measuring the extracellular leakage of lactate dehydrogenase (LDH), respectively; and d) the expression of apoptosis regulators was investigated by immunoblotting. The initiation of apoptosis was evidenced by the activation of caspase 8 and caspase 9, and increased Annexin-V reactivity. Progression through the apoptotic process was confirmed by the activation of caspase 3 and Bid, the enhanced expression of Bax, and the occurrence of nuclear fragmentation. Late transition to a necrotic appearance was demonstrated by an increased number of PI-positive cells and augmented extracellular release of LDH. Thus, the in vitro model allows the study of the entire course of Fas-mediated hepatocellular apoptotic cell death, which is not possible in vivo. This experimental system can serve a broad range of in vitro pharmaco-toxicological purposes, thereby directly assisting in the reduction of animal experimentation.
An In Vitro Model for Studying Neutrophil Activation During Cardiopulmonary Bypass by Using a Polymerase Chain Reaction Thermocycler
Min Tang, Xiao-Gang Zhao, Y. John Gu and Chang-Zhi Chen
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Development of In Vitro Models for a Better Understanding of the Early Pathogenesis of Batrachochytrium dendrobatidis Infections in Amphibians
Pascale Van Rooij, An Martel, Melanie Brutyn, Sofie Maes, Koen Chiers, Lieven Van Waeyenberghe, Siska Croubels, Freddy Haesebrouck1 and Frank Pasmans
Batrachochytrium dendrobatidis, the causal agent of chytridiomycosis, is implicated in the global decline of amphibians. This chytrid fungus invades keratinised epithelial cells, and infection is mainly associated with epidermal hyperplasia and hyperkeratosis. Since little is known about the pathogenesis of chytridiomycosis, this study was designed to optimise the conditions under which primary keratinocytes and epidermal explants of amphibian skin could be maintained ex vivo for several days. The usefulness of the following set-ups for pathogenesis studies was investigated: a) cultures of primary keratinocytes; b) stripped epidermal (SE) explants; c) full-thickness epidermal (FTE) explants on Matrigel™; d) FTE explants in cell culture inserts; and e) FTE explants in Ussing chambers. SE explants proved most suitable for short-term studies, since adherence of fluorescently-labelled zoospores to the superficial epidermis could be observed within one hour of infection. FTE explants in an Ussing chamber set-up are most suitable for the study of the later developmental stages of B. dendrobatidis in amphibian skin up to 5 days post-infection. These models provide a good alternative for in vivo experiments, and reduce the number of experimental animals needed.
An Alternative Gas-phase In Vitro Exposure System for Toxicity Testing: The Interaction Between Nitrous Oxide and A549 Cells
Antonietta Stellavato, Marcella Cammarota, Nadia Miraglia, Angela Simonelli and Mariateresa Giuliano
An original in vitro approach was adopted to expose cells to volatile agents. The anaesthetic nitrous oxide (N2O) was chosen as the model agent, and type II pneumocyte-like cells (A549 cells) were used as the target to represent the lungs. A time-lapse microscopy station was equipped with a manual gas mixer that allowed the generation of a mixture of N2O/air/CO2 in the gas phase, to provide a uniform distribution of the volatile agent. The dissolution of N2O in the culture medium was monitored by gas chromatography–electron capture detection. Biochemical alterations, in terms of homocysteine accumulation, demonstrated that intracellular methionine synthase had been inactivated by N2O absorbed by the cells, a process that also occurs in vivo. Toll-like receptors, which are key molecules in inflammatory lung diseases, were also investigated at the molecular level. Our experiments indicated that biochemical and molecular alterations occurred in the cells, even under conditions where neither morphologic changes nor consistent alterations in cell proliferation were evident. This in vitro exposure system can be efficiently adopted for looking at the repeat-dose effects of volatile agents on respiratory tissues. Moreover, it could be of further benefit for identifying the wide range of specific cell targets, and for monitoring relevant endpoints in the cellular and molecular processes that occur during exposure to volatile compounds.