Cetacean cell cultures: towards standardized protocols for cell bio-banking

The Mediterranean Marine Mammal Tissue Bank, established in 2002 at the University of Padova, stores more than 3400 tissue samples from more than 280 specimens, belonging to 15 species. Its main mission is to provide samples for free to all institutions interested in marine mammals research, under motivated request. We recently began to deal with tissue cultures as a new tentative division of the tissue bank, to extend our efforts in the conservation and protection of marine mammals. Primary cell cultures are of special interest in cetacean research as they are comparatively less exploited than other mammalian species and they allow a wide range of reliable in vitro experiments with controlled settings. They are however more susceptible to stressors and senescence, so standardized protocols of immortalization are necessary to allow long-term storage of viable cells. We have obtained a cell line from the skin of a bottlenose dolphin (Tursiops truncatus), which was immortalized and characterized with immunocytochemistry. We also tried to set up a suitable protocol to obtain cultures derived from the muscle longissimus dorsi of a Risso’s dolphin (Grampus griseus), in particular of satellite cells, which comprise the myogenic stem precursors in postnatal and adult muscles. Cells were isolated by trypsin and collagenase solution and were maintained in rich growth medium. The morphology of myogenic cells was monitored with a phase contrast microscope. During early days of culture we observed round satellite cells. After 5 days the myoblasts in culture appeared with typical elongated shape and after 15 days they began to migrate and align. The fusion of myoblasts yielded multi-nucleated myotubes, clearly identifi able in vitro, whose contractility however could not be observed. Future efforts are aimed at improving the protocols for the isolation of satellite cells to gain higher yields and viability.