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Developing an Artificial Intestine for the sustainable farming of healthy fish

Periodic Reporting for period 2 - Fish-AI (Developing an Artificial Intestine for the sustainable farming of healthy fish)

Reporting period: 2020-04-01 to 2022-09-30

• What is the problem/issue being addressed?
A healthy, balanced diet has a fundamental role in preventing a wide range of chronic diseases. Aquaculture plays a substantial role in this perspective because fish is an important source of well-balanced proteins, however, aquaculture’s sustainability has been criticized.Indeed the industry is trying to address these concerns by actively implementing the substitution of marine-derived protein and lipid sources by using plant-based feeds from sustainable agriculture. At the same time insects and microorganisms are being explored. However, alternative feedstuffs often have characteristics that make them less suitable for use in aquafeeds compared to fishmeal, for example by not supporting optimal fish health. The search for a viable and sustainable alternative to fishmeal requires a continuous and extensive raw material evaluation program.

• Why is it important for society?
This project promotes a substantial leap forward of the current knowledge on fish nutrition by
combining state of the art bioengineering and biomaterial science with the latest concepts in intestinal
stem cell biology. Such science-based knowledge will be used to develop an innovative 3D in vitro platform that will enable the feed industry to predict the nutritional and health value of alternative feed sources accurately and efficiently. More importantly, such a platform will surpass substantially current technological paradigms because the correlation between an in vitro model of a complex organ and the organ itself will be directly determined, and the 3D in vitro platform will replace the use of a large number of experimental animals without losing valuable physiological data.

• What are the overall objectives?
The overall objective is the development of the first 3D in vitro platform able to screen efficiently and
reliably the health and nutritional value of sustainable feeds that the industry is currently developing.
This will be achieved through these intermediate objectives:
i) The development of a functional fish artificial intestine (Fish-AI) able to mimic the complex
functions of the intestinal mucosa, ii) the assessment and development of the Fish-AI prototype
capability to predict the nutritional and health value of feeds and bioactive components, and iii) The
validation of Fish-AI through the analysis of previously untested fish feeds.
All the partner continued their work mostly as planned but a few changes were implemented in order to adapt to the results obtained and to the disruption brought by the delays caused by the pandemic situation.
The Management Committee (MC) met several times mostly on line to coordinate project activities and to implement effective changes along the project to keep the pace of the experimental work

The experimental work was devoted to the development and the implementation of the in vitro platform through different aspects.

i) Materials and process for the 3D-platform development - Different kinds of gelatin derivatives and acrylate-endcapped urethane-based precursors (AUPs) were synthesized to investigate the effect of this structure on the processability, mechanical properties, and diffusion properties. A polydimethylsiloxane (PDMS) mould fabricated via laser ablation was used to process gelatin derivatives and AUPs with promising results to create cone-shaped materials. Different resins have been tested using our high-resolution 3D printing systems to ensure a complete control over the dimensional properties of 3D printed objects. New designs of perfusion chambers have been produced and the one considered best will be processed for further testing.

ii) RT intestinal cell lines derivation – The consortium obtained two new stable RT intestinal cell lines one of which originates from the proximal intestine and now complement the only other available established RT intestinal cell line (RTgutGC) that belongs to the ETH Zurich and originated from the distal part. These cell lines are the only one available for any fish species. Using the data obtained during the initial part of the project it was determined that: a) the cell lines include different cell types; b) it is possible to modulate the cell composition through that change of the culture environment; c) this generates differentiated cells that reproduce the key properties of the intestinal mucosa.

iii) Combination of cells and scaffolds - Extensive attempts have been performed combining
the new cell lines with the different materials described above. While they all showed a good biocompatibility with RT intestinal cells, they revealed some critical shortcomings that prevented their use for the assembly of a functional prototype. The consortium implemented a contingency plan that lead to the identification of two alternative scaffolds. Their properties were suitable for the development of a functional prototype (Fig. 1).

iv) Predigestion - An in vitro digestion (IVD) protocol based on the use of rainbow trout digestive enzymes has been developed. The procedure enables the extraction of the bio-accessible fraction from feed pellets and to transform it in a format compatible with its exposure the cell lines (Fig. 2).

v) Preliminary test of the platform prototype - Several trials have been performed to determine the conditions necessary to expose the cells to the in vitro digested pellets. Thanks to such detailed protocol the procedure are now available for determining the presence of a functional epithelial barrier and establishing the amino acid profile absorbed by the epithelial cells.

vi) Identification of diverging diets –To validate the predictive power of the Fish-AI prototype two or more diets with diverging nutritional properties are required. This has been achieved through an in vivo trial of seven diets (Fig. 3). These diets are now being tested in vitro.
Fish-AI will be used to predict the nutritional and health value of raw materials whose effects on fish intestine have not been characterized before. Besides its general significance, the implementation, characterization and validation of the Fish-AI will tell if it is possible to reliably predict the nutritional and health value of novel feedstuff in vitro not only in aquaculture but in any other species. This will open unforeseen possibilities towards carefully engineering animal feed to enable a widespread improvement in livestock sustainability and animal health. Industry productivity will increase thanks to a better feed-conversion rate and to a substantial reduction of expensive in vivo trials. Consequently, it would also ensure that European Agriculture remains a global leader.
Furthermore, the model will enable to also test the effect on the heath of the intestinal mucosa of the so-called functional feeds, or to study the effect of environmental contaminants and their possible absorption, like nano-plastics, or the efficiency of oral vaccines. All this reducing, at the same time, the number of animals required for the experiments, thereby supporting and effective 3R policy.
A fully functional Fish-AI would pave the way for a wide array of innovative products based on 3D models of complex organs creating new markets opportunities that strengthen the competitiveness and growth of active and ambitious high-tech SME or academic spin-offs.
Seven contrasting diets have been tested in vivo. The same are being tested in vitro
The use of different scaffolds leads to increasing resemblance of the in vitro epithelium with its i
Cell can be exposed to up to 50% in vitro digested feed with no alteration of their functions