Cell cultures of the Manila clam and their possible use in biomonitoring and species preservation

A huge progress has been achieved in mammalian in vitro technique. Instead, of the many trials to develop marine invertebrate cell cultures, only a few have obtained them and only from few tissues. Since in vitro cell culture of invertebrates could be very useful for many aspects of basic and applied science, in this work we investigate and describe the development of a technique for the establishment of cell cultures from gill, mantle and gonadic tissue of the Manila clam (Ruditapes philippinarum). We maintained viable cultures for up to 25 days. Culture viability and proliferation were tested with 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) and with trypan blue, while an antibody against the ATP-dependent RNA helicase VASA, a protein expressed in the germline, and in multipotent stem cells of some animals, was used to verify the presence of these cell types. Following the described protocol: 1) explant resulted the better source to obtain cell cultures, when compared to enzymatic dissociation; 2) cultures of suspended cells were viable for longer period than adherent cells; 3) cell cultures obtained from tissues sampled in September-October performed better compared to other periods of the year, regarding maintenance and growth; 4) the tissue from which we obtained longer-lived cell cultures was gonadic tissue, especially form samples that show more undifferentiated germ cells and more VASA-stained cells. This study describes the challenges concerning the development of in vitro culture techniques for aquatic invertebrates.