Abstract
This study aims to develop a comprehensive and standardized protocol for the primary cell culture of oyster hemocytes and their structural and functional analyses. Hemocytes are crucial for immune defense and overall bivalve health. Current primary culture systems for bivalve hemocytes are limited in duration, making long-term studies challenging. Here, we adopted the approach of utilizing hemolymph plasma as our complete and natural culture medium maintaining live, active, and immunocompetent oyster hemocytes throughout 21 days of culture period. Distinct types of hemocytes are described based on their morphology and function in primary culture. Granulocytes and hyalinocytes displaying various shapes and spreading patterns, in addition to blast-like cells, were cultured for up to 7 days. Between 14 and 21 days, blast-like cells and hyalinocytes decreased in number with signs of apoptosis, revealing a subpopulation of hemocytes with a higher resistance for in vitro maintenance. Live-cell imaging revealed that the remaining cells were kept alive and active throughout the 21 days of culture. Two primary cell motility behaviors were observed: pseudopod-dependent and lamellipodium-dependent membrane extensions, which are reminiscent of the mesenchymal and amoeboid migration modes, respectively. The proportion of about 40 % of the hemocytes displaying phagocytic activity within fresh hemolymph was maintained throughout the 21-day culture period. However, the phagocytic capacity was higher within phagocytes present after 14 and 21 days of culture. Metabolic assays revealed alterations in reactive oxygen species production and neutral lipid storage, suggesting physiological alterations in response to the culture environment. Our research established a robust protocol for long-term primary cell culture and assessment of bivalve hemocyte health, function, and metabolism. This protocol, potentially applicable to hemocytes of other marine bivalve species, paves the way to enhance our understanding of bivalve immunology and cellular physiology and ecophysiology. This valuable tool can be applicable for future research in marine biology, biotechnology, environmental science, ecotoxicology, and comparative physiology