Primary culture of Pacific oyster,Crassostrea gigas, heart cells

Summary

Pacific oyster,Crassostrea gigas, is the most economically important specie to the world shellfish breeding. It is important to note that infectious diseases, particularly viruses, may be hazardous for theC. gigas live-stocks. The study of these viral diseases and the development of diagnosis method need the establishment of in vitro methods for viral multiplication. As no oyster cell line is available actually, we have developed a procedure for primary culture of heart cells which could enable to study molluscan viruses in vitro, and could also provide a diagnosis method based on the search of eventual cytopathogen viral effects. Cells fromC. gigas ventricle of heart were dissociated by trypsin-EDTA treatment and the mechanical action of a Dounce type homogeneizer. The cells were inoculated in previously poly-D-lysin coated flasks. The optimised culture medium was L-15 (Leibovitz) prepared three fold concentrated, then diluted half with sea water, this mixture was supplemented with 10% FCS and 5%C. gigas hemolymph. Different cell types could be identified by transmission electron microscopy analysis, as mostly cardiomyocytes, fibroblast-like cells and pigmented cells, but also haemocytes were present in the cultures.