ABSTRACT
Marine molluscan cell lines, required for virus screening and cultivation, form essential tools for developing health management strategies for these animals in the blue economy. Moreover, they are also crucial to develop cultivated seafood. As there is no valid marine molluscan cell line, primary cell cultures are relied upon for all investigations. A sound protocol for generating primary cell cultures from molluscs is entailed, but existing protocols often involve heavy antibiotic usage and depuration that invariably affect gene expression and cell health. This work presents an easy-to-adopt, time-saving protocol using non-depurated mollusc Crassostrea madrasensis, which requires only initial antibiotic treatment and minimal exposure or no use of antibiotics in the cell culture medium. The important experimental considerations for arriving at this protocol have been elucidated. Accordingly, sodium hypochlorite and neomycin sulfate were chosen for disinfecting tissues. The study is the first to use shrimp cell culture medium (SCCM) as a cell culture medium for molluscan cell culture. Despite being osmoconformers, the oysters exhibited stable intracellular osmotic conditions and pH, which, when provided in vitro, promoted effective cardiomyocyte formation. The cell viability could be enhanced using 10% fetal bovine serum (FBS), but healthy cell culture could also be obtained using SCCM without FBS. The optimized culture conditions allowed for regular beating cardiomyocyte clusters that could be retained for a month. Limited cell proliferation, as shown by the BrdU assay, demands further interventions, such as possibly producing induced pluripotent stem cells. The optimized protocol and culture conditions also align with some requirements for producing cultivated meat from marine molluscs.
ABSTRACT
Effluent produced during the electroplating process can contain high concentrations of heavy metals that can enter the environment and induce toxicity to aquatic organisms. Relatively high concentrations of zinc (Zn) and mercury (Hg) have been detected in treated electroplating industrial effluent (TEPIE), though the cytotoxic potential of these compounds has not been well assessed in fish gills. A novel cell line, Danio rerio gill (DrG), were exposed to TEPIE and concentrations of Zn, Hg, and Zn + Hg previously measured in treated effluent to evaluate the use of the DrG cell line following exposure to environmental pollutants. Several cytotoxic assays were employed to assess the effect of TEPIE, Zn, and Hg on this cell line. The percent cell viability was significantly reduced in a concentration-dependent manner following exposure to TEPIE, Zn, Hg, and Zn + Hg (p < 0.05) for 24 h, with additional morphological changes observed in exposure treatments relative to controls. Additionally, there was a significant induction of DNA damage detected in all exposure treatments determined through comet assay tail length. An increase in intracellular ROS generation was also observed in cells exposed to TEPIE, Zn, Hg, and Zn + Hg, corresponding to dose-dependent increases in apoptosis. Our study confirmed that TEPIE and the metals present in it induced cytotoxicity in the DrG cell line, demonstrating its usefulness as a model to explore relationships between pollutants and fish gills.
