Employing marine invertebrate cell culture media for isolation and cultivation of thraustochytrids

Thraustochytrids, a common group of marine eukaryotic protists, have drawn considerable scientific and industrial interest due to their ability to synthesize high levels of bioactive compounds, including polyunsaturated fatty acids, docosahexaenoic acid, squalene and carotenoids, and their new applications for biofuels. The pharmaceutical and industrial potential of thraustochytrids necessitate effective isolation of new strains and establishment of axenic cultures. To date, existing isolation protocols have used baiting and direct plating methods to generate axenic cultures with varied media compositions that contain peptone and yeast extracts as nitrogen sources, glucose as carbon source, seawater and antibiotics. Here we reveal a new approach for the isolation of thraustochytrids from tissues of marine invertebrates using (a) primary cell cultures in a liquid medium containing basal medium, 50% artificial seawater, vitamins, proteins and antibiotics, and (b) cultivation in the same cell culture medium. Using the colonial tunicate Botryllus schlosseri as a model system, thraustochytrid cells thrived in the medium from the day of extraction, grew and proliferated for the next five weeks (five-passages, up to 1.9 x 10(6) cells ml(-1) in passage 5; 1.45-fold multiplication week(-1)). This new approach for isolation and cultivation of axenic thraustochytrid cultures enables the isolation of new species with promising bioactive compounds.