Introduction
The Rainbow trout (Oncorhynchus mykiss) is one of the widely farmed fish species worldwide. This makes it to a key model organism in aquaculture research. Therefore, in vitro models are developed in eco-physiological and eco-toxicological research for analysis at the cellular level. In this way, a wide variety of scientific questions can be precisely measured under controlled conditions.
In present study, we are introducing you to our new cell model OMYlar5, derived from larval rainbow trout. With this model, we are able to examine for example, the impact of increasing temperatures or hypoxic conditions on physiological parameters as well as genetic and metabolic alterations. In accordance with the 3Rs principle, cell-based studies can replace or reduce the number of animal-based studies used in ecology and physiology research.
Material and Methods
The cell line OMYlar5 was isolated from one rainbow trout larva and is currently in passage 56. Proliferation of OMYlar5 cells was analysed and compared at 18°C, 20°C, and 22°C to evaluate the optimal growth condition and possible temperature-related effects. All cells were cultivated in Leibovitz-15 Medium (L-15) supplemented with 10% fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin (P/S). Growth and vitality were measured by trypan blue staining and image-based automated cell counting (EVE��� Plus).
Morphological cell changes were visualized by phase-contrast (AE2000 microscope with a Moticam 5 Plus camera) and actin staining with Phalloidin-iFluor 488 Reagent (DM400B fluorescent microscope). Additionally, mRNA abundance of selected temperature- and stress-related genes was measured by quantitative reverse transcription PCR (RT-qPCR). Furthermore, immunofluorescence staining with heat shock markers Hsp70 and Hsp90 (Cell Signaling, cat. #4872 and #4874), expressed during stress conditions, was conducted. The impact of temperature on OMYlar5 mitochondrial function was analyzed by measuring oxygen consumption rates using Seahorse technology (Agilent). Statistical significance was calculated using two-way ANOVA followed by Tukey's post- hoc modification. A p value < 0.05 was considered significant.
Results
Morphological observation and cytoskeletal analysis revealed that confluent cells growth at 18 °C and 20 °C displayed a round, epithelial-like morphology. In contrast, at 22 °C, fibroblast-like cells were present with a mosaic-like arrangement. Cells also tended to form multi-layered structures more frequently. Growth analysis revealed that OMYlar5 cells incubated at 22���°C exhibited the highest proliferation rate, which corresponded with the greatest cell density and the shortest doubling time (not significant). Cell size and viability were not affected by temperature.
Gene expression analysis of stem cell-markers, temperature-related genes, and stress-related genes characterized the larval rainbow trout cell line at the molecular level. Furthermore, we detected immunofluorescence signals of Hsp70 appearing in the nucleus and vesicle-like structures of cells, and Hsp90 showing a uniform distribution within the cytosol in all three temperatures.
In regard to metabolic analysis, we determined mitochondrial oxidative respiration by measuring basal and FCCP-mediated oxygen consumption rates at 18, 20 °C and 22 °C. Comparison of key parameters like basal respiration, mitochondrial ATP production, and maximal respiration allows conclusions regarding temperature-induced metabolic changes in OMYlar5 cells.
Discussion
Aquaculture research, as a rapidly growing sector, requires simple, consistent diagnostic tools to support efficient research. Therefore, in vitro models derived from fish offer a cost-effective and controllable approach for studying environmental variables such as temperature, oxygen saturation or ecotoxic substances.
In regard to global warming, increasing water temperatures and reduced oxygen levels pose major problems for fish populations as well as aquaculture facilities. There are numerous interesting and important in vivo studies on rainbow trout that provide valuable knowledge. These studies could be supplemented or replaced by research using cell-based models. Using the larval cell line OMYlar5, our study demonstrates that increasing temperatures induce measurable changes in cell growth, morphology, gene expression, and metabolism. These results underscore the value of this in vitro system for eco-physiological and eco-toxicological research, as well as to develop a deeper understanding of the cell physiology of O. mykiss.
Using the OMYlar5 cell line in experimental approaches, aligns with the 3Rs principles by reducing and replacing the use of animals and refining experimental methods in aquaculture research and therefore providing a practical alternative to experiments using live fish.