Sustained viability and profileration of hemocytes from the cultured pharynx of Styela clava

In order to acquire more in-depth information on the site(s) of hemopoiesis in marine animals, we successfully cultured pharyngeal explants of the tunicate Styela clava over a period of 82 d. Transmission electron microscopy of resident hemocytes within explants revealed changes in hemocyte composition. Hemoblast-like cells increased shortly after commencement of the cultures (8.1% at Day 0 increasing to a maximum of 28.7% at Day 7). Autoradiography using ³H-thymidine incorporation confirmed that hemocyte proliferation in pharyngeal explants still continued after 37 d culture. During culture, the migration of many free cells into the medium resulted in sparse, residential hemocytes in the pharyngeal explants. Hemocyte migration increased by up to 4.3x10⁵ cells/explant (max.) at 17 to 24 d, but finally decreased to 4.9x10⁴ cells/explant at 75 to 82 d. Vital neutral-red staining revealed that many emerging cells were not hemocytes such as those found in the normal hemolymph. The continued development of in vitro approaches will strengthen analyses of immune-defense responses in tunicates which, as protochordates, are the immediate invertebrate ancestors of vertebrates.