RESUMO
A dissociation protocol for Pecten maximus heart has been established that makes it possible to obtain functional primary cultures routinely (9, 10); freezing assays of isolated cells were performed with the aim of making it possible to cultivate these cells in vitro after thawing to provide a constant standardized source of cells for applied research. Various parameters such as the nature and the concentration of the cryoprotectant, the cooling rate, and the incubation time of cells with the cryoprotective agent were evaluated. Best results were obtained by freezing cells in 12% dimethyl sulfoxide (Me2SO) in Leibovitz L15 medium at a cooling rate of approximately 2-3 degreesC/min. Thawed cells in culture attached to the substrate and survived for at least 3 weeks. They exhibited similar morphology and synthesised proteins, DNA, and lipids in vitro at levels close to those observed in fresh cells. To our knowledge, it is the first time that cultures have been obtained from cryopreserved marine invertebrate cells. Copyright 1998 Academic Press.
