Acquiring embryo-derived cell cultures and aseptic metamorphosis of larvae from the colonial protochordate botryllus schlosseri

The initiation of the first embryo-derived cell cultures and the establishment of in vitro metamorphosis from aseptic tadpole larvae are described in the cosmopolitan, shallow water colonial tunicate, Botryllus schlosseri. A total of more than 1,600 embryos, collected from different colonies at various stages of blastogenic cycles, were used in ten experiments (up to 350 embryos/experiment). Embryos were fully dissociated by mechanical or chemical treatments. In three experiments we succeeded in initiating continuous cell lines from embryonic tissue (designated NIO-BSE-1 to -3). Using different substrates and supplements, we found that embryonic cells cultured in botryllid cell culture medium (Rinkevich and Rabinowitz, 1993) supplemented with either chick embryo extract or heat inactivated Botrylloides hemolymph and on plastic or gelatin coated substrates may acquire cell-line characteristics. Tunic cells of maternal origin were found in all wells of primary cultures but subsequently disappeared. These cultures grew slowly for the first several weeks, but after transfer to 25 ml culture flasks, proliferation accelerated. When co-cultured with freshly collected blood cells from allogeneic colonies, the embryo-derived cells exhibited antibacterial properties. Two of the cell lines were frozen for fiiture experiments. By using 24-well culture plates and 1-8 embryos/well under aseptic conditions (produced by a cocktail of antibiotics), almost 100% of 399 embryos at all developmental stages successfully metamorphosed and produced oozooids under those in vitro conditions. The oozooids could be obtained in vitro without feeding up to 40 days. Retinoic acid and different illumination regimens shorten the time for metamorphosis and improve the state of oozooids’ health. The potential use of the embryo-derived cell cultures and the in vitro culture of metamorphosed larvae for studying developmental biology and organogenesis of colonial protochordates is discussed.