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Developing an Artificial Intestine for the sustainable farming of healthy fish

Periodic Reporting for period 1 - Fish-AI (Developing an Artificial Intestine for the sustainable farming of healthy fish)

Reporting period: 2019-04-01 to 2020-03-31

• 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 and key nutrients such as marine-derived omega-3 fatty acids, vitamin D, iodine and selenium. However, aquaculture’s sustainability has been criticized by consumer and environmental groups because farmed fish are fed with fishmeal and fish oil, produced largely from the processing of small oily species that are caught for non-food purposes by so-called “industrial fisheries”. Fishmeal or fish oil are considered unsustainable when derived from unsustainable fisheries and/or yield conversion ratios greater than one (i.e. more fish enters the system than comes out of it).
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.
Each partner began its activity with the recruiting and/or training of young researches in the techniques that they will need during the project. The consortium developed and standardised common procedures, acquired and validated specialized reagents and protocols to ensure the inter-laboratory validation of the platform that will constitute the next step of the project.
To coordinate project activities and to implement effective decision-making mechanisms and procedures we constituted the Management Committee (MC), established its procedures and meeting calendar and we met at our first annual meeting.
The Consortium implemented a project website and a Facebook page. Furthermore, the partners took part in dissemination events organised by their respective Institutions and in an event organised directly by the Consortium. All events were aimed at presenting the rationale and the aim of the project illustrating the benefits of developing an innovative 3D platform as an effective means for increasing the sustainability of aquaculture and, more in general, of healthy and humane food production.
The experimental work was devoted to the preliminary phase of the project:
i) RT intestine characterisation - These included the detailed characterization of the Rainbow trout intestine that lead to the accurate stereological quantification of the intestinal structures to be mimicked by the model. (Fig. 1). Furthermore, it showed some unexpected features of the intestinal mucosa that were described for the first time.
ii) RT intestinal cell lines derivation -The only available established RT intestinal cell line (RTgutGC) has been obtained through a collaboration activity from the ETH Zurich. The line has been propagated and tested. At the same time two more RT intestinal cell lines have been derived. The cells have been growing in vitro vigorously for over 6 months and represent a robust alternative to the RTgutGC cell line. Their characteristics are being tested and compared with the RTgutGC cells.
iii) 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 (Fig. 2).
iv) Predigestion - A preliminary feeding trial was conducted with rainbow trout for the purpose of extracting digestive enzymes and digesta for the development of an in vitro digestion (IVD) protocol that will be used to digest fish feed before its introduction to the artificial intestine. Procedures for the extraction of enzyme extracts were adapted and used as assays to determine pepsin and trypsin proteolytic activity in the extracts (Fig. 3).
v) Combination of cells and scaffolds - Preliminary attempts have been performed combining RTgutGC cells and new cell lines with the different materials developed so far and all showed biocompatibility with RT intestinal cells (Fig. 4).
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.
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.
Figure 1. Immunostaining of proliferating cells (red) localized at the intestinal folds base and his
Figure 4. Cell adhesion and growth of RTgutGC and primary cell lines on the different types of gelat
Figure 2. A shows a SEM image of an AUP moulded into pillar-like structures. B shows a perfusion cha
Figure 3. Extraction of enzyme extracts was performed at the NMBU facilities in Norway and samples w