In Vitro Fish: New 3D models for nutritional and toxicological research

Future feeds for farmed fish are based on untraditional feed ingredients, which will change nutrient and contaminant profiles compared to traditional feeds based on marine ingredients. In fish nutrition and toxicology research, we aim to reduce the number of fish, refine the in vitro assays to better mimic the in vivo situation, and replace traditional fish feeding experiments. In vitro assays represent a more ethical way of conducting large-scale assessments of nutritional requirements and contaminant and mixture toxicity (cocktail effect). However, for in vitro models to reduce or replace the number of fish used in in vivo studies, it is critical to develop better and more advanced in vitro models resembling in vivo conditions to overcome the limitation of today's 2D models. To close the gap between the commonly used 2D mono cell culture assays and whole animal testing, the project In Vitro Fish intends to refine existing in vitro methods and develop novel 3D Atlantic salmon cultures; co-culture, triple culture and a fish "organs-on-a-plate" culture, using primary hepatocytes, adipocytes and head kidney and kidney epithelial cells isolated from the same fish. These new in vitro models permit the study of animal physiology in a more organ-specific, in vivo like context that will replace and refine existing methodology and reduce the number of fish used in nutritional and toxicological research. A triple culture, composed of primary Atlantic salmon hepatocytes, head kidney cells and visceral fat, has been established. Exposure to LPS (inflammation inducer) and resveratrol (may have antioxidant, anti-inflammatory action in salmon) have shown that signals from 3D visceral fat organ cultures affect other tissues in the triple culture. Primary kidney epithelial cells have been isolated to be used in a co-culture with hepatocytes. The kidney-hepatocyte co-culture have been exposed to the pesticides chlorpyrifos and glyphosate, the polycyclic aromatic hydrocarbon Benzo(a)pyrene (BaP) and the heavy metal cadmium (Cd). RT-qPCR transcriptomics and global metabolomic profiling were used to examine the toxicological effects in liver and kidney cells and to compare responses in 3D and 2D cultures. The glyphosate exposure affected lipid metabolism in hepatocytes and kidney cells. The 2D hepatocyte cell cultures were found more sensitive to BaP, chlorpyrifos and Cd exposure then 3D cultures. A fish "organs-on-a-plate" culture, using primary hepatocytes, adipocytes and head kidney and kidney epithelial cells isolated from the same fish, has been developed for toxicogenomics assessment of the pesticide diazinon. The cell viability assays LDH and MTT showed no cytotoxic response in exposed cells. Diazinon had also no significant effect on the cells choline esterase activity. Thus, the preliminary results suggests that diazinon has low toxicity in this salmon cell model. All cell models established in the project In Vitro Fish can replace and refine existing methods and reduce the large number of fish used in today?s research. These new cell models, with their whole fish approach, can become important to solve tomorrow?s nutritional and toxicological challenges for the salmon industry.

Prosjektet har klart å forbedre eksisterende cellemetoder og utvikle nye 3D laksekulturer; co-kulturer, trippel kulturer og en sirkulasjonskultur, ved å bruke primære celler fra lever, fettvev, hodenyre og nyre isolert fra samme fisk. Disse modellene vil bli benyttet i framtidige prosjekter på Havforskningsinstituttet. Prosjektet In Vitro Fisk førte videre til et samarbeid med Akvariet i Bergen som vi har hatt i hele prosjektperioden. Akvariet har holdt fisken vi har brukt i forsøkene våre i et utstillingsakvariet. Ved siden av utstillingsakvariet har det vært informasjon til publikum at fisken blir bukt til å utvikle nye cellemodeller og hvorfor dette er viktig. I prosjektet In Vitro Fisk ble det avdekket en kommersiell mangel på gode produkter til å dyrke fiskeceller 3D. Dette resulterte derfor i et samarbeid med SINTEF om et nytt NFR-prosjekt hvor vi skal utvikle et nytt 3D-dyrkningsprodukt for fiskeceller.

Future feeds for farmed fish are based on untraditional feed ingredients, which will change nutrient and contaminant profiles compared to traditional feeds based on marine ingredients. In fish nutrition and toxicology research, we aim to reduce the number of fish, refine the in vitro assays to better mimic the in vivo situation, and replace traditional fish feeding experiments. In vitro assays represent a more ethical way of conducting large-scale assessments of nutritional requirements and contaminant and mixture toxicity (cocktail effect). However, for in vitro models to reduce or replace the number of fish used in in vivo studies, it is critical to develop better and more advanced in vitro models resembling in vivo conditions to overcome the limitation of today's 2D models. To close the gap between the commonly used 2D mono cell culture assays and whole animal testing, the project In Vitro Fish proposes to refine existing in vitro methods and develop novel 3D Atlantic salmon cultures; co-culture, triple culture and a fish "organs-on-a-plate" culture, using primary hepatocytes, adipocytes and head kidney and kidney epithelial cells isolated from the same fish. These new in vitro models permit the study of animal physiology in a more organ-specific, in vivo like context that will replace and refine existing methodology and reduce the number of fish used in nutritional and toxicological research.