A Primary Cell Culture Platform for Studying Venom Gland and Brain Tissue in Octopus

Coleoid cephalopods, squids, cuttlefish, and octopuses, have emerged as powerful model organisms for studying neurobiology, development, and behavior, however, cellular tools for investigating their specialized tissues remain limited. In particular, their venom producing gland, the posterior salivary gland (PSG), has been extensively described anatomically and histologically, yet remains largely inaccessible to experimental investigation at the cellular level. Here, we report the first establishment of primary cell cultures derived from both the optical lobe and PSG tissues of Octopus bimaculoides. Building on recent advances in cephalopod brain cultures, we adapted and optimized dissociation and culture conditions to support short-term survival and attachment of cells in vitro. We show that passive cell release during tissue handling, rather than enzymatic treatment, yields viable cultures from both tissues, and poly-D-lysine markedly improves the adherence of PSG-derived cells. Morphological analyses and fluorescent staining confirm the presence and viability of distinct cell populations, while cell cycle analysis indicates that the majority of cells reside in G₀/G₁ phase. Notably, O. bimaculoides brain cultures exhibit features comparable to those previously described in squid, suggesting conserved aspects of coleoid cellular physiology. Together, our findings establish a foundational in vitro platform for studying octopus PSG and neural cell biology, providing a tractable system for probing venom biosynthesis, secretion, and neural regulation in coleoid cephalopods.