Aquaculture Europe 2023

September 18 - 21, 2023


Add To Calendar 21/09/2023 15:45:0021/09/2023 16:00:00Europe/ViennaAquaculture Europe 2023DEVELOPMENT AND CHARACTERIZATION OF NOVEL RAINBOW TROUT Oncorhynchus mykiss CELL-BASED ORGANOTYPIC INTESTINAL PROTOTYPESStolz 2The European Aquaculture Societywebmaster@aquaeas.orgfalseDD/MM/YYYYaaVZHLXMfzTRLzDrHmAi181982


N. Verdile*1, F. Camin1, M. Stuknyte4 R. Pasquariello1, R. Pavlovic5, I. De Noni3, T. A.L. Brevini2 and F. Gandolfi1


1Department of Agricultural and Environmental Sciences, 2Department of Veterinary Medicine and Animal Sciences, 3Department of Food, Environmental and Nutritional Sciences, 4Unitech COSPECT – University Technological Platforms Office, University of Milan, Italy, 5Proteomics and Metabolomics Facility, IRCCS San Raffaele Scientific Institute, Milan, Italy.

*E-mail address:



Cell-based models provide valuable support in the search for alternative feed formulations in the aquaculture sector. A powerful in vitro tool should consist of epithelial and fibroblast cells. Indeed, as we recently demonstrated1, subepithelial fibroblasts modulate epithelial cell proliferation and differentiation in the rainbow trout (RT) intestinal mucosa. Moreover, growing data2,3 showed that increasingly elaborated in vitro models that closely recapitulate the morphological and functional features of the native organ, could generate feasible data for the in vitro versus in vivo correlations studies. The H2020-FETOPEN project Fish-AI, aims to develop such an in vitro screening platform based on rainbow trout digestive enzymes and intestinal cells to evaluate nutritional and health values of novel aquafeeds. As part of this project, we developed, characterized, and compared four gradually increasingly complex RT intestinal in vitro platforms from a morphological and functional point of view.

Material and methods

Rainbow trout proximal intestine epithelial cells (RTpi-MI) have been seeded on 1) the culture inserts ThinCert™ (TC) (Greiner BioOne, 0.4 m pore size, cat. No. 665640); 2) the same inserts but coated with the solubilized basement membrane matrix Matrigel® (MM); 3) the same but with the rainbow trout fibroblast cell line RTskin01 previously embedded within the Matrigel® matrix (MMfb); 4) the highly porous synthetic scaffolding Alvetex™ (Reprocell) (AV) previously populated with the same fibroblast cell line (AV). The generation of effective epithelial barriers in vitro has been investigated measuring the transepithelial electrical resistance (TEER) and the apparent permeability (Papp) to 4kDa FITC-dextran (FD4). Moreover, epithelial cell functionality has been assessed by investigating the enzymatic activity of alanine aminopeptidase (ALP), leucine aminopeptidase (LAP), and alkaline phosphatase (AP), 3 well-known enterocytes’ brush border enzymes. Thereafter, the 4 platforms have been characterized for their morphological properties using histological, and immunohistochemical techniques.


All models successfully established an efficient barrier preventing the paracellular flux of FD4 and showing a significant increase of the trans-epithelial electrical resistance (TEER) compared to baseline values of their respective controls (inserts without cells). The value at plateau became higher going from the simple through the more complex models (TC-MM-MMfb-AV). Moreover, the activity of the brush border enzymes alanine aminopeptidase (ALP), leucine aminopeptidase (LAP), and alkaline phosphatase (AP), followed the same gradual pattern, suggesting that the presence of an epithelial and mesenchymal interface significantly boosted epithelial cells differentiation. Morphological analysis showed that RTpi-MI had a flat shape when cultured alone onto TC and MM systems. Conversely, they assumed a more in vivo-like phenotype when fibroblasts were present, acquiring a cubic shape on MMfb and a cylindrical shape on AV with brush border enzymes neatly located on the apical membrane. In addition, fibroblast organization was also different depending on the culture support. While in MMfb they were densely packed with only limited extracellular space from one cell to the other, cells assumed a loose arrangement in AV more like what occurs in the connective tissue. Moreover, fibroblasts that populated the AV scaffolding synthesized and remodelled their own extracellular matrix, indicating a higher degree of cell differentiation.


The presence of fibroblasts boosted epithelial cell differentiation and polarization providing a more physiological environment. Therefore, our results indicate that reconstructing the epithelial-mesenchymal interface in vitro is important for the development of intestinal artificial platforms able to generate reliable, predictive data suitable for in vitro/ in vivo correlations studies. This observation suggests that, as we recently observed in vivo, also in vitro fibroblasts may represent an essential source of growth and signaling factors actively involved in the modulation of epithelial cell functions1. Overall, the platform based on the Alvetex scaffold better recreated the morphological and functional complexity of an artificial the intestinal mucosa therefore it is being tested for its ability to reliably predict the nutritional value of feed formulations.


This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 828835.


  1. Verdile N., Pasquariello R., Cardinaletti G., Tibaldi E., Brevini TAL, Gandolfi F., Telocytes: active players in the rainbow Trout (Oncorhynchus mykiss) Intestinal Stem-Cell Niche Animlas 2022, 12, 74.
  2. Fournier, E.; Etienne-Mesmin, L.; Grootaert, C.; Jelsbak, L.; Syberg, K.; Blanquet-Diot, S.; Mercier-Bonin, M. Microplastics in the human digestive environment: A focus on the potential and challenges facing in vitro gut model development. J. Hazard. Mater. 2021, 415, doi:10.1016/j.jhazmat.2021.125632
  3. Delon, L.; Gibson, R.J.; Prestidge, C.A.; Thierry, B. Mechanisms of uptake and transport of particulate formulations in the small intestine. J. Control. Release 2022, 343, 584–599, doi:10.1016/j.jconrel.2022.02.006