Fish intestinal cultures for ecotoxicological studies: in vitro and primary culture models
Creators: name: Langan, Laura
Publisher: name: University of Plymouth
Year: 2017
Subjects: subject: In vitro
subject: Gastrointestinal tract
subject: Primary culture
subject: 3Rs (Reduce, Refine, Replace)
subject: Ecotoxicology
subject: Rainbow trout
subject: Three dimensional cultures
subject: PhD

description: Chapters published: Langan, L.M., Dodd, N.J.F., Owen, S.F., Purcell, W.M., Jackson, S.K. and Jha, A.N. 2015. Direct measurements of oxygen gradients in spheroid culture system using electron paramagnetic resonance oximetry. PLoS One, 11(2): e0149492 (Chapter 6) Langan, L.M., Harper, G.M., Owen, S.F., Purcell, W.M., Jackson, S.K. and Jha, A.N. 2017. The rainbow trout gastrointestinal cell line RTgutGC grown on Transwells: biomolecular response to copper. Ecotoxicology (Chapter 4)
description: Ecotoxicity testing of chemicals for environmental risk assessment is an area where a high number of vertebrates are used across a variety of industrial sectors. The application of the 3Rs in toxicity testing using fish address both the ethical and societal concerns around this issue in addition to the increasing legislative requests for the incorporation of animal alternatives. This thesis aims to highlight the potential of 3D cell culture models to "bridge the gap" between in vitro and in vivo screening procedures for testing of chemicals with the potential to persist or bioaccumulate thereby improving the predictive power of screening procedures. This thesis examines two alternative methods for their potential use as an intestinal based toxicokinetic tool for environmental risk assessment, utilising an in vitro fish cell line replacement tool (RTgutGC). In addition, for the first time a new intestinal primary cell culture based model was developed to address both intestine region specific response (pyloric, anterior, mid and posterior) and size related adaptability to toxins. Paramagnetic oximetry was used to measure oxygen content within 3D structures (spheroids) in order to better understand the microenvironment of these culture models. Using histology, immunohistochemistry, transepithelial electrical resistance (TEER), transmission electron microscopy (TEM), metabolic, fluorescence and gene expression assays, the comparability of this system to native intestinal response was established. Following exposure to carefully chosen environmental contaminants (Benzo[a]pyrene and Copper), the RTgutGC cell line demonstrated comparable responses to existing literature in terms of uptake, metabolism, DNA damage and the presence an equivalent saturable level. Primary enterocytes cultured on transwell inserts remained viable for upto six weeks, with permeability and metabolic activity comparable to native tissue (both in vitro and ex vivo). Taken in combination, these features of enterocytes represent a profile more closely representative of the intestine then the widely used "gut sac" method. With the potential advantages of incorporating complexity at differing levels (connective tissue layer, intestinal bacteria biome), the intestinal models described offer the potential to screen highly persistent toxins which may require prolonged incubation, in addition to the exploration of complex experimental designs which minimise animal usage (uptake, depuration, uptake). As a consequence, the models developed within this thesis significantly enrich the emerging fish based in vitro testing strategies.