3D spheroid culturing ofAstyanax mexicanusliver-derived cell lines recapitulates distinct transcriptomic and metabolic states ofin vivotissue environment

Abstract

AbstractIn vitroassays are crucial tools for gaining detailed insights into various biological processes, including metabolism. Cave morphs of the river-dwelling fish species,Astyanax mexicanus, have adapted their metabolism allowing them to thrive in the biodiversity-deprived and nutrient-limited environment of caves. Liver-derived cells from the cave and river morphs ofAstyanax mexicanushave proven to be excellentin vitroresources to better understand the unique metabolism of these fish. However, the current 2D cultures have not fully captured the complex metabolic profile of theAstyanaxliver. It is known that 3D culturing can modulate the transcriptomic state of cells when compared to its 2D monolayer culture. Therefore, in order to broaden the possibilities of thein vitrosystem by modeling a wider gamut of metabolic pathways, we cultured the liver-derivedAstyanaxcells of both surface and cavefish into 3D spheroids. We successfully established 3D cultures at various cell seeding densities for several weeks and characterized the resultant transcriptomic and metabolic variations. We found that the 3D culturedAstyanaxcells represent a wider range of metabolic pathways, including cell cycle changes and antioxidant activities, associated with liver functioning as compared to its monolayer culture. Additionally, the spheroids also exhibited surface and cave-specific metabolic signatures, making it a suitable system for evolutionary studies associated with cave adaptation. Taken together, the liver-derived spheroids prove to be a promisingin vitromodel for widening our understanding of metabolism inAstyanax mexicanusand of vertebrates in general.