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 estab lishment of in vitro metamorphosis from aseptic tadpole larvae are des cribed in the cosmopolitan, shallow water colonial tunicate, Botryllus schlosseri. A total of more than 1,600 embryos, collected from differ ent colonies at various stages of blastogenic cycles, were used in ten experiments (up to 350 embryos/experiment). Embryos were fully dissoc iated by mechanical or chemical treatments. In three experiments we su cceeded in initiating continuous cell lines from embryonic tissue (des ignated NIO-BSE-1 to -3). Using different substrates and supplements, we found that embryonic cells cultured in botryllid cell culture mediu m (Rinkevich and Rabinowitz, 1993) supplemented with either chick embr yo extract or heat inactivated Botrylloides hemolymph and on plastic o r gelatin coated substrates may acquire cell-line characteristics. Tun ic cells of maternal origin were found in all wells of primary culture s but subsequently disappeared. These cultures grew slowly for the fir st several weeks, but after transfer to 25 ml culture flasks, prolifer ation accelerated. When co-cultured with freshly collected blood cells from allogeneic colonies, the embryo-derived cells exhibited antibact erial properties. Two of the cell lines were frozen for future experim ents. By using 24-well culture plates and 1-8 embryos/well under asept ic 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 an d improve the state of oozooids' health. The potential use of the embr yo-derived cell cultures and the in vitro culture of metamorphosed lar vae for studying developmental biology and organogenesis of colonial p rotochordates is discussed.