Evaluation of different media for cell proliferation in mantle tissue culture of the green mussel, Perna viridis (Linnaeus, 1758)

Abstract

The aim of the present study was to establish a suitable culture system for tissue explants from the mantle of the green mussel, Perna viridis. The experiments were conducted using healthy, live green mussels in the size range of 75 to 110 g collected from Pulicat Lake, Tamil Nadu. Three different culture media namely M199, Leibovitz L-15 and sterile seawater were used to assess the most suitable medium for growth, proliferation and viability of mantle epithelial cells. The effect of the addition of two supplements viz., 10% foetal calf serum (FCS) and 0.1% yeast extract to the culture media was also evaluated. After carefully isolating the pallial layer from the mantle tissue, 1-2 mm2 size explants were successfully cultured in 12-well plates at 25°C for up to 14 days. Cultures were monitored under light and phase contrast objectives in an inverted microscope. Cell counts were made and cell size was measured for each treatment. Cells were observed to migrate from the periphery of the explant within 24 h after initiation of cultures. The liberated cells were mostly round and were either granulocytes or hyalinocytes. Fibroblast-like cells were also observed. Our results showed that proliferation of epithelial cells from mantle tissue was maximum in seawater medium (7.4x104 cells ml-1), followed by L-15 medium (2.55x104 cells ml-1). Average cell size in seawater medium was 10.72 μm and that in L-15 and M199 media was 8.56 and 6.39 μm, respectively. Adherent cells were also more prominent and higher in number in seawater medium. Supplementation of culture media with 10% FCS and 0.1% yeast extract improved both cell proliferation and cell size in all the three culture media. Four concentrations of 0.1% yeast extract (@ 50 μl, 75 μl, 100 μl, 150 μl ml-1 medium) were tested in the present study and best results were obtained with 100 μl ml-1, with respect to both cell counts and size.