The search for alternative sustainable raw materials to improve current fish diets would be supported by reliable and predictive in vitro models. 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 used two different bicameral culture platforms to evaluate the effects of prolonged exposure to fish feed on intestinal health. We compared a simple system consisting only of an epithelial monolayer and a complex organotypic system that combined a stroma-like scaffold populated by rainbow trout (RT) fibroblasts with a functional epithelium.
Material and Methods
To generate the in vitro platforms, RT proximal and distal intestine epithelial cells (RTpiMI, RTdiMI) were seeded onto ThinCert (TC) inserts (Greiner BioOne, 0.4 µm pore size), a permeable PET membrane or onto the synthetic scaffolding Alvetex™ (Reprocell) (AV) previously populated with RTskin01, a fibroblast cell line derived from the trout dermis. Both were cultured at 20°C under ambient atmosphere. We used a diet rich in fish meal with crude protein and lipid level of 46 and 23% respectively. Feed pellet were digested in vitro with enzymes extracted from the RT gastric and intestinal segments of the digestive tract.
Barrier integrity and functionality were assessed through the measurement of transepithelial electrical resistance (TEER), apparent permeability (Papp) to 100 µg/mL 4 KDa FITC-Dextran (FD4) and in situ detection of the alanine aminopeptidase (ALP), a brush border enzyme. Once the models established an effective epithelial barrier, they were exposed for 21 days to increasing concentrations (6, 12, 25, 50%) of in vitro digested feed (IVD) diluted in Leibovitz L-15 medium supplemented with 10% FCS. Controls were run incubating cells with L-15 medium without IVD. Samples were fixed in 4% paraformaldehyde in PBS, processed for histological analysis and stained with hematoxylin and eosin (HE).
During the 21 days of exposure to IVD, barrier integrity was conserved along the course of the experiment as indicated by the limited paracellular flux of FD4 compared to the respective inserts without cells. TEER values significantly increased from the baseline values in both cell lines cultured in both systems. The morphological analysis revealed that in all the combinations of IVD concentration, cell lines and culture platform, epithelial cells lost their monolayer arrangement and formed a multilayered barrier, with the only exception of RTpiMI exposed to 6% IVD cultured in AV, in which the cubic and polarized monolayer observed in the control was preserved. In AV, ALP activity significantly decreased in both cell lines exposed to 25 and 50% IVD affecting cellular functionality. This was not evident in the TC system, in which high IVD concentration did not reduce the enzymatic activity but rather increased the proteolytic activity in the RTdi-MI cell line.
High concentrations of IVD generated a mildly stressful environment in both platforms since long-term exposure led to increased proliferative activity at the expense of cellular differentiation. The fact that irritation occurred even with high levels of marine ingredients may be due to the lack of mucus-secreting cells that would provide the physiological protection for the epithelium. Interestingly, this reaction reminds our recent data, demonstrating that nutritional stress induces de-differentiation and proliferation of mature enterocytes in the RT proximal intestinal mucosa1. The fact that we observed a similar response in vitro, suggests that the data generated by the artificial intestinal platforms can be representative of in vivo trials. Since the cells grown on the Alvetex™ scaffold reacted with higher sensitivity to the exposure to the digesta this looks as the more reliable platform.
This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 828835.
 Verdile, N.; Cardinaletti, G.; Faccenda, F.; Brevini, T.A.L.; Gandolfi, F.; Tibaldi, E. Ectopic stem cell niches sustain rainbow trout (Oncorhynchus mykiss) intestine absorptive capacity when challenged with a plant protein-rich diet. Aquaculture 2022, 564, 739031, doi:10.1016/j.aquaculture.2022.739031.