In vitro dynamics of oyster hemocytes in plasma-based primary cultures

Hemocytes are specialized cells found in the circulatory system of invertebrates, such as marine bivalves, and play crucial roles in physiology and health. However, in bivalves, in-depth studies on their in vitro biology beyond one or two days remain scarce, particularly in Crassostrea (Magallana) gigas. In this study, we characterized the hemocytes isolated from triploid C. gigas oysters, maintained in vitro in their natural medium, hemolymph plasma, to assess their morphological, behavioral, and metabolic status over 21 days. A diverse population of hemocyte morphotypes, including granulocytes, hyalinocytes, and blast-like cells, was preserved for up to seven days, displaying various shapes and spreading patterns. By days 14 and 21, blast-like cells and hyalinocytes were less represented, showcasing granulocytes as the most resilient subpopulation in vitro. Despite this decline in subpopulation diversity, we confirmed through cell viability and metabolic assays, combined with live-cell imaging, that the remaining cells remained alive and active throughout the culture period. Notably, key indicators of cellular homeostasis such as membrane integrity, ATP levels, mitochondrial content, and mitochondrial reactive oxygen species (ROS) remained stable in these surviving cells. Phagocytic activity persisted over the full 21 days, with a marked increase after two weeks in culture. Certain time-dependent changes were observed, including a decrease in total basal ROS production from day 7 and a significant increase in neutral lipid storage by day 21. Live-cell imaging revealed two modes of cell motility: pseudopod-dependent and lamellipodium-dependent membrane extensions. Fibroblast-like hyalinocytes remained non-motile. Interestingly, not all motile granulocytes display phagocytic behavior and non-motile fibroblast-like hyalinocytes lack phagocytic activity entirely, highlighting the functional diversity within the different morphotypes identified in our cultures. By presenting an innovative method for sustaining primary marine invertebrate cell cultures using hemolymph plasma and leveraging different cell-based assays and live-cell imaging, this study provides a platform for investigating morphology, behavior, metabolism, and immune function of these cells under controlled in vitro conditions. Additionally, by highlighting the selective decline of certain hemocyte subpopulations, this study offers an in vitro model well-suited for future research into hemocyte lifespan and cell death mechanisms. Importantly, our framework not only advances research on bivalve hemocyte biology but also contributes to the broader field of experimental and comparative cell biology, with potential with applications in immunology, ecotoxicology, ecophysiology, molecular screening, and health innovation.