Developing cultivated mollusks through establishing primary cell culture methods of Eastern Oyster, Crassostrea virginica, as a model bivalve

Cultivated seafood is a potential alternative protein source that can address the rising global food demand with exponentially rising human population growth. Cultivated seafood is made by growing animal cells in vitro using stem cells for edible food, eliminating the need to raise the entire animal. A crucial first step in developing cultivated seafood is creating a well-characterized cell line that can continuously grow and differentiate into desired cell types. Due to difficulties in determining optimal primary cell culture conditions, no continuous cell lines of food-relevant mollusks have been established so far. This study used the adult Eastern Oyster, Crassostrea virginica, as a model bivalve to study the decontamination, cell dissociation, and culture conditions suited for mollusk adductor muscle cells. Oyster adductor (OAD) cells were obtained via tissue explant, mechanical and enzymatic digestion. The cells were routinely monitored using an inverted microscope for phase-contrast and fluorescence imaging. Culture vessels were coated with surface proteins such as fibronectin, laminin, matrigel, and poly-d-lysine to promote cell attachment. The tissue decontamination with Penicillin-Streptomycin (100 µg/mL), Amphotericin B (0.25 µg/ml), and algaecide solution (0.03%) was effective in controlling microbial growth. OAD cells grew best at lower nutrient levels in a one-to-one ratio of Lebovitz L-15 media and artificial seawater. Lower fetal bovine serum levels, 1-5%, provided a high number of cell attachments and consistent growth in combination with 1% adult oyster whole-body or larvae extract. The tissue explant method resulted in the optimal cell dissociation from the three methods, and proceeding cultures had attached cells surviving for up to 10 days. All the plate coatings promoted cell attachment, but fibronectin provided optimal cell attachment of OAD cells. Fibroblast-like, neuron-like, epithelial-like, and rounded cells were observed. Fluorescence cell staining confirmed the presence of cytoskeleton and nuclei in the OAD cell cultures. These advances in primary cell culture methods of OAD cells may be beneficial for establishing mollusk cell lines for cultivated seafood production.