Improving a fish intestinal barrier model by combining two rainbow trout cell lines: epithelial RTgutGC and fibroblastic RTgutF

Carolin Drieschner,Philippe Renaud,Michael Burkard,Hannah Schug,Kristin Schirmer,Niels C Bols,Nguyen T K Vo

doi:10.1007/s10616-019-00327-0

Abstract

An in vitro model of the fish intestine is of interest for research and application in diverse fields such as fish physiology, aquaculture and chemical risk assessment. The recently developed epithelial barrier model of the fish intestine relies on the RTgutGC cell line from rainbow trout (Oncorhynchus mykiss), cultured in inserts on permeable membranes. Our aim was to extend the current system by introducing intestinal fibroblasts as supportive layer in order to reconstruct the epithelial–mesenchymal interface as found in vivo. We therefore initiated and characterized the first fibroblast cell line from the intestine of rainbow trout, which has been termed RTgutF. Co-culture studies of RTgutGC and RTgutF were performed on commercially available electric cell substrate for impedance sensing (ECIS) and on newly developed ultrathin, highly porous alumina membranes to imitate the cellular interaction with the basement membrane. Cellular events were examined with non-invasive impedance spectroscopy to distinguish between barrier tightness and cell density in the ECIS system and to determine transepithelial electrical resistance for cells cultured on the alumina membranes. We highlight the relevance of the piscine intestinal fibroblasts for an advanced intestinal barrier model, particularly on ultrathin alumina membranes. These membranes enable rapid crosstalk of cells cultured on opposite sides, which led to increased barrier tightening in the fish cell line-based epithelial–mesenchymal model.

Highlights

Rainbow trout (Oncorhynchus mykiss) cell lines have been established from a variety of different tissues and organs and are indispensable for mechanistic investigations in fish based research (Bols and Lee 1991; Castano et al 2003; Bols et al 2005, 2017)
Focusing on the recreation of basic intestinal architecture, the aim of this study was to combine absorptive epithelial cells, which face the intestinal lumen in vivo, and supportive fibroblasts, which are the main cell type in the underlying connective tissue
A novel fibroblast cell line from rainbow trout, RTgutF, was initiated and characterized. This cell line was used alone or in combination with the intestinal epithelial cell line RTgutGC on solid, non-porous supports to evaluate their electrical properties

Summary

Introduction

Rainbow trout (Oncorhynchus mykiss) cell lines have been established from a variety of different tissues and organs and are indispensable for mechanistic investigations in fish based research (Bols and Lee 1991; Castano et al 2003; Bols et al 2005, 2017). The attractiveness of rainbow trout cell lines as economic and easy to handle source to obtain reliable and reproducible results is further increasing with the development of functional tissue analogues (Malhao et al 2013; Geppert et al 2016; Minghetti et al 2017), technical innovations in label free bio-sensing (Brennan et al 2012; Curtis et al 2013; Widder et al 2015; Tan and Schirmer 2017) and computational modelling (Stadnicka et al 2012; Stadnicka-Michalak et al 2014, 2015). The current in vitro model of the fish intestine is based on the epithelial-like cell line RTgutGC (Kawano et al 2011). Optimal mimicry of basic intestinal architecture calls for further refinements by e.g. having additional intestinal cell types that help to improve the intestinal barrier function and allow for a broader application of the model

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Conclusion