A systematic approach towards long-term, serum-free cultivation of fish cells with the RTgill-W1 cell line as example

Continuous cultures of vertebrate cells, referred to as cell lines, comprise invaluable biological material for a seemingly unlimited range of applications: from basic cell biology to toxicology to biotechnology, to name a few. Cell lines of fish, such as the cold-water fish rainbow trout (Oncorhynchus mykiss), are no exception but are still little tapped into compared to the widely applied mammalian cell lines. Yet, fish cell lines hold promises as alternatives to animal use in, e.g., toxicology and cell-based food production. However, while the number of fish cell lines is growing, a severe bottleneck for their defined and ethical use is lack of a fully described culture medium that supports long-term viability and proliferation, prerequisites to exploit them. More specifically, routine culture of these cells still depends on fetal bovine serum, an undefined, animal-derived component. Here, we report on the first systematically developed, fully defined medium for long-term culture of fish cells, using the rainbow trout gill cell line, RTgill-W1, as example. We first identified components that are apparently provided by serum. A purposefully designed short-term proliferation assay then allowed the screening of the serum replacement components regarding cell viability and proliferation support. Cells were then taken into long-term culture using the designed mix - yielding 35 passages in the fully defined medium thus far. They can be cryopreserved/thawed in this medium; and for the functions tested, they resemble their parent strain. We thus provide impetus to move fish cell line culture to fully defined media for basic research and application.