Establishment of cell lines from multipotent epithelial sheet in the budding tunicate, Polyandrocarpa misakiensis

We report here the in vitro culture of the atrial epithelium, which is the major formative tissue of the budding tunicate, Polyandrocarpa misakiensis. Preliminary studies suggested that both pH and osmotic pressure of the basic seawater medium should be lowered significantly (pH 6.8, 800-830 mOsm). In the growth medium consisting of modified millipore-filtered seawater, Dulbecco's modified Eagle medium and 3% fetal bovine serum, cells spread out from the epithelial explant and proliferated with a doubling time of about 13 hours. They could be cloned and cultured successively. They contained the Polyandrocarpa lectin gene, showing that they were indeed of tunicate origin. At a low cell density (< 3 x 10(2) cells/mm2), clonal cells took a spherical form and contained several granules in the cytoplasma. At a high cell density (> 3 x 10(4) cells/mm2), on the other hand, they gave rise to smaller cells without any specialized features and, finally, to dark flattened cells. Consistent with this observation, confluent cells lost the atrial epithelium-specific antigen, which reappeared on the cell surface when they were re-plated at a low density. In conclusion, we have established for the first time tunicate cell lines. They appeared to differentiate and dedifferentiate repeatedly in our culture system.