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 optic lobe (OL) 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 OL cultures share several features with the recently described squid brain cultures, including passive cell release, and an adherent fibroblast-like cell population, but additionally contain a predominant population of spherical cells, suggesting both conserved and species-specific coleoid cell behavior in vitro. Together, our findings establish a foundational in vitro platform for studying octopus venom and neural cell biology, providing a tractable system for probing venom biosynthesis, secretion, and neural regulation in coleoid cephalopods. We thank the Marine Biological Laboratory’s Cephalopod Program team including Bret Grasse, Taylor Sakmar, and Miranda Vogt, for their extensive assistance with animal husbandry, euthanasia protocols, and the provision of octopus for the experiments.
Yoonji Kim for meeting with our laboratory, sharing the protocol she developed in squid primary cultures to develop our work with octopus samples, and providing training in cephalopod OL dissection. We would also like to thank the Life Science Editors Foundation’s JEDI award initiative and Dr. Stephen Matheson for assistance in manuscript development..
This research and the open accessibility were funded by the National Institutes of Health – Pioneer Award, grant number 5DP1AT012812 to M.H. and an EE Just Marine Biological Laboratory (MBL)Whitman Fellowship to M.H. Manuscript contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
James Vincent Parziale and Saurabh Attarde have contributed equally to this work. Department of Chemistry, Hunter College, CUNY, New York City, NY, USA James Vincent Parziale, Fatima Khalid & Pollena Devi Sangana Programs in Biochemistry and Biology at CUNY Graduate Center, New York City, NY, USA Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, USA Malacology, Harvard Museum of Comparative Zoology, Cambridge, MA, USA Invertebrate Zoology, American Museum of Natural History, New York City, NY, USA The authors declare no competing interests. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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