Characterisation of a canine epithelial cell line for modelling the intestinal barrier

Michelle J. Farquhar, Emma McCluskey, Ruth Staunton, Kevin R. Hughes and Jennifer C. Coltherd

Little is known about how food interacts with the intestinal epithelium during the digestion process. However, it is known that ingredients in food can modulate the intestinal barrier, and have the potential to disrupt homeostasis of the gut. Here, we characterise a conditionally immortalised canine intestinal epithelial cell (cIEC) line for use in in vitro assays, to assess the effect of food ingredients on intestinal barrier function, permeability, cell health, and inflammation. Microscopy and flow cytometry confirmed that cIECs had a phenotype consistent with those of epithelial origin, and were able to differentiate to mature enterocytes. The cIECs also formed a monolayer when grown on Transwell® inserts, producing functional tight junctions between the cells. In contrast to the human-derived Caco-2 cell line, transepithelial electrical resistance (TEER) was increased in cIECs in response to two different raw ingredients. The exposure of cIECs to known inflammatory stimuli and raw ingredients induced the nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB). This work demonstrates the value of a unique cIEC in vitro model to study the effects of food ingredients on canine intestinal function and health, and supports continued efforts to reduce and refine the use of animals in scientific research.

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In Vitro Models of the Blood–Brain Barrier

Per Garberg

In this literature review on in vitro models of the blood–brain barrier (BBB), it is concluded that there is a need to identify a unified in vitro model for the BBB. The best evaluated model at present is based on the use of primary cultures of bovine brain endothelial cells. Primary cell cultures are usually shown to retain several BBB characteristics, but are time-consuming and difficult to establish. To make a unified in vitro model for the BBB more generally available, it is strongly suggested that such a model should be based on the use of an established cell line. To identify the best in vitro model, an evaluation of the most promising immortalised BBB-derived endothelial cell lines, as well as other established cell lines presently used as BBB models, is highly recommended. An evaluation of possible species variation is also important, in order to establish the most relevant species to be used. Furthermore, it is also suggested that the specific properties of in vitro BBB models, as compared to models for the “intestinal barrier”, for example, should be evaluated. Finally, it is recommended that an evaluation of available computer models is performed, to further improve early predictions for drug candidates with regard to BBB permeability.
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An Inter-laboratory Study to Evaluate the Effects of Medium Composition on the Differentiation and Barrier Function of Caco-2 Cell Lines

Flavia Zucco, Anne-Françoise Batto, Giovanna Bises, Jean Chambaz, Arianna Chiusolo, Rosa Consalvo, Heide Cross, Gianni Dal Negro, Isabella de Angelis, Gérard Fabre, François Guillou, Sebastian Hoffman, Loïc Laplanche, Etienne Morel, Martine Pinçon-Raymond, Pilar Prieto, Laura Turco, Giulia Ranaldi, Monique Rousset, Yula Sambuy, Maria Laura Scarino, François Torreilles and Annalaura Stammati

Differentiated human intestinal Caco-2 cells are frequently used in toxicology and pharmacology as in vitro models for studies on intestinal barrier functions. Since several discrepancies exist among the different lines and clones of Caco-2 cells, comparison of the results obtained and optimisation of models for use for regulatory purposes are particularly difficult, especially with respect to culture conditions and morphological and biochemical parameters. An inter-laboratory study has been performed on the parental cell line and on three clonal Caco-2 cell lines, with the aim of standardising the culture conditions and identifying the best cell line with respect to parameters relevant to barrier integrity, namely, trans-epithelial electrical resistance (TEER) and mannitol passage, and of epithelial differentiation (alkaline phosphatase activity). Comparison of the cell lines maintained in traditional serum-supplemented culture medium or in defined medium, containing insulin, transferrin, selenium and lipids, showed that parameter performance was better and more reproducible with the traditional medium. The maintenance of the cell lines for 15 days in culture was found to be sufficient for the development of barrier properties, but not for full epithelial differentiation. Caco-2/TC7 cells performed better than the other three cell lines, both in terms of reproducibility and performance, exhibiting low TEER and mannitol passage, and high alkaline phosphatase
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The Bovine Corneal Opacity and Permeability Test in Routine Ocular Irritation Testing and Its Improvement Within the Limits of OECD Test Guideline 437

Arnhild Schrage, Susanne N. Kolle, Maria C. Rey Moreno, Kimberly Norman, Hans Raabe, Rodger Curren, Bennard van Ravenzwaay and Robert Landsiedel

Data on eye irritation are generally needed for the hazard identification of chemicals. As the Bovine Corneal Opacity and Permeability (BCOP) test has been accepted by many regulatory agencies for the identification of corrosive and severe ocular irritants since September 2009 (OECD Test Guideline 437, TG 437), we evaluated this alternative method for routine testing at BASF. We demonstrated our technical proficiency by testing the reference standards recommended in TG 437, and 21 additional materials with published BCOP and in vivo data. Our results matched the published in vitro data very well, but with some intentionally selected false negatives (FNs) and false positives (FPs), the concordance was 77% (24/31), with
FN and FP rates of 20% (2/10) and 24% (5/21), respectively. In addition, we tested 21 in-house materials, demonstrating the utility of the BCOP assay for our own test material panel. Histopathological assessment of the corneas by light microscopy was also conducted, as this was suggested as a means of improving the identification of FNs. The histopathology corrected the classification of some FNs, but also increased the number of FPs. Parallel to the test method evaluation, we compared three new opacitometer models with the current standard device. We recommend the use of an opacitometer developed in our BASF laboratory, which has certified components and electronic data storage, resulting in what we consider to be excellent sensitivity, stability and reproducibility.
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