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.

Lipoprotein-induced cell growth and hemocyanin biosynthesis in rhogocytes

“…On the contrary, cells cultured in Leibovitz's L-15 demonstrated a lower cell growth. The current finding is in contrast to previous studies which suggested L-15 as a suitable media for molluscan cell culture of various tissues (Van Der Merwe et al 2010;Dessai 2012;Daugavet and Blinova 2015;Suja et al 2017;Barrick et al 2018). An earlier study by Suja et al (2007) on Haliotis varia mantle tissue demonstrated that L-15 promotes cell proliferation while M199 enhances cell adherence.…”

“…The effectiveness of lipid supplementation was previously demonstrated on oyster's heart cell (Cecil 1969;Domart-Coulon et al 1994;Cornet 1995). On the other hand, fetal bovine serum were shown to enhance mollusc cell culture (Dessai 2012;Suja et al 2017;Barrick et al 2018). High concentration of FBS, however, may cause a detrimental effect to some cells (Kusumoto et al 1997), whilst, batch to batch variations may also have an impact on cell growth.…”

“…The difficulty is due to the lack of vital information regarding the cell requirements and their physiology (Rinkevich 2005(Rinkevich , 2011. Several studies had attempted to establish a marine invertebrate primary culture with limited success (Lebel et al 1996;Giard et al 1998;Sera et al 2000;Suja et al 2017). The lack of knowledge on rhogocyte cell physiology has been attributed to the difficulty of isolating and sorting out rhogocyte cells and the poor understanding of optimal culture conditions.…”

In vitroproliferation ofMytilus edulismale germ cell progenitors

“…The addition of BCS was essential to sustain proliferation, and yeast extract provided stability to the culture media, reducing the variability between cultures. Yeast extract was previously used in culture media as a source of vitamins and other supplements [30]. It is thus reasonable to presume that its addition complements the L-15 formulation and provides unknown useful components for mussel cell maintenance [31].…”

Cell Culture and Gene Expression Studies in Relation to Biomineralization in the Black-lip Pearl Oyster, Pinctada margaritifera

“…However, in this study a relatively simple and cost effective culture medium composed of sterilized seawater supplemented with 0.1% yeast, developed by Jayasankar et al (2018) was used successfully for culture of P. margaritifera mantle explants. Sea water, with or without added factors has been used in earlier studies too as a culture medium for invertebrate cells (Cecil, 1969;Qiao et al, 2003;Suja et al, 2017;van der Merwe et al 2010).The close resemblance of these media to natural seawater, the habitat of marine molluscs, in terms of pH and osmolarity is what makes it an attractive _________________________________________________________________________________________________ Journal of Coastal Research, Special Issue No. 86, 2019 Figure 5.…”