Cetacean cells in tissue culture can provide excellent model systems for elucidation of the many biochemical and physiological specializations peculiar to the Cetacea, and expands into the general fields of health and medicine, genetics, and environmental toxicology. Cells in culture derived from longer lived species tend to undergo a greater number of population doublings before undergoing "crisis". Cetacean cells therefore have the potential to be robust in vitro tools if the proper culture parameters can be maximized.

Epidermal keratinocytes from eight species of cetaceans in four families were cultured with varying degrees of success. These include beluga whale, long-finned pilot whale, killer whale, pygmy sperm whale, sperm whale, humpback whale and spotted dolphin. The primary species of interest is the bottlenose dolphin, Tursiops truncatus, the most common cetacean inhabiting the U.S. coast, and for which the most comprehensive biomedical data are available. Epidermal keratinocytes were chosen for several reasons. First and possibly most important, skin biopsies can be taken relatively easily from live animals without causing any harm. Second, the structure of the skin allows the isolation of many keratinocytes from a single biopsy. Third, keratinocytes retain significant capacity for in vitro cytochrome P450 induction and the metabolism of xenobiotic compounds by the mixed function oxidase system in culture. The third is important for projects in our laboratory which include the development of methods for the in vitro analysis of environmental contaminants in dolphin cells and tissues.

Skin samples were collected from live wild and captive animals utilizing dart and punch biopsies respectively, as well as from stranded animals which died during rehabilitation. Methodologies established for the in vitro culture and isolation of human epidermal keratinocytes were used as guidelines for the development of the culture system for cetacean epidermal keratinocytes. Several modifications were made based on the differences between cetacean and human skin, cetacean and human serum osmolarity, and the relative viability of cells in the tissue sample when culture procedures were initiated. Cells generally attached in 8-15 days and small colonies became apparent shortly thereafter.

Epithelial cell type was confirmed by immunofluorescence microscopy using a rabbit anti-cytokeratin IgG which reacts with both basic and acidic keratins and has demonstrated reactivity with keratins from several species. Data concerning growth and colony formation were collected under a variety of culture conditions to optimize the system. The culture environment was manipulated by varying media osmolarity, temperature, attachment factors and growth factor supplementation.

It has been difficult to establish a cetacean epidermal keratinocyte cell line that remains mitotically active for many cell generations. This is most likely a reflection of the lack of high quality skin samples. Cetacean epidermal keratinocytes can be successfully cultured but in order for the maximum utility of the system to be realized, a temporally consistent source of high quality skin samples must become available.