299 CryoLetters 22, 299-310 (2001)  CryoLetters, c/o Royal Veterinary College, London NW1 OTU, UK CRYOPRESERVATION OF PRIMARY CELL CULTURES OF MARINE INVERTEBRATES Nelly Odintsova*, Konstantin Kiselev**, Nina Sanina** and Edward Kostetsky** *Institute of Marine Biology, Far East Branch of Russian Academy of Sciences, Vladivostok, Russia 690041. E-mail: inmarbio@mail.primorye.ru. **Far Eastern State University, Vladivostok, Russia 690090. Summary Primary cell cultures obtained from somatic and larval tissues of bivalve molluscs and from embryos of sea urchins were frozen to –196ºC by two-step freezing using 10% dimethyl sulfoxide (DMSO) or/and trehalose (3-30mg/ml) as cryoprotectants. We estimated both cell viability and the RNA synthetic activity after freeze-thaw. Total lipid extracts from the tissues of echinoderms examined as possible cryoprotective agents demonstrated a weak cryoprotective capacity. Mussel lipid extract was found to possess a considerable cryoprotective activity. Cryoprotective capacity of tested lipids correlated with their thermotropic behaviour. DMSO + trehalose combination was shown to be a favourable cryoprotectant and sea urchin blastula cells the most freezing-tolerant cells. Keywords: Cryopreservation, primary cell culture, mollusc, sea urchin, trehalose, total lipids, phase transition INTRODUCTION It has been well established that the cells of a wide variety of organisms may be successfully cryopreserved. Nevertheless, there have been very few studies on freezing tolerances in the cells of invertebrates. Single examples of cryopreservation of primary cell cultures of invertebrates, insects (16, 19) and molluscs (22), have been reported. Development in methods of cryopreservation permits the removal of season and geographic limitations of investigations on sea animals, helps to establish cryobanks of rare and disappearing species and would be a useful tool in cell technology of marine bioactive materials. Sperm cells of marine invertebrates and fishes are successfully preserved in liquid nitrogen (2, 4, 20, 38). However, cryopreservation methods described for embryos and larvae of these animals are still inadequate in maintaining the structure and activity of embryos after freeze - thawing (2, 4, 12, 21, 34, 38). Post-thaw development in cryopreserved embryos often showed abnormalities in structure and colour, which were difficulty to analyse quantitatively (11). Cultured cells of marine invertebrates could be used as a model system in studies on cryoprotection and help to choose an optimal cryoprotectant quickly and estimate quantitatively the chilling injury. The main site of damage during freeze-thaw cycles is the bilayer structure of membranes (15). During freezing and thawing, cell damage is due to the destructive action of the