Cryopreservation Enables Long-Term Studies of Octopus vulgaris Hemocyte Immune Functions

As with their nervous system and other physiological traits, the immune system of cephalopods, in general, and of the common octopus, Octopus vulgaris, in particular, could also present highly evolved characteristics compared to other classes of molluscs. However, to date, there is not much information about it, and studying the defense mechanisms is a key step in understanding their response system to external aggressions and, thus, having the tools to anticipate animal health problems and ensure their welfare. The lack of cell cultures in molluscs is a major problem when carrying out in vitro assays that help to deepen the knowledge of the main immune cells of this species. Cryopreservation becomes an alternative to maintain viable and functional cells after freezing/thawing processes, allowing a larger repertoire of studies to be performed with the same sample or to perform time courses. Having access to good quality cells for long periods will allow to cover a larger repertoire of studies with the same sample or to perform time courses, avoiding logistic bottlenecks that could arise due to the loss of viability and/or functionality of the cells on the lab bench, the lack of time to develop all the desired assays in the same day or the possible losses derived from transporting the samples between different laboratories. Additionally, high-quality suspensions of viable and functional cells are required for successful massive sequencing studies, such as single-cell analysis, where these aspects are the key to an optimal identification. We show here the first functional results from cryopreserved octopus hemocytes, where the cells were able to maintain viability above 80% after two months post cryopreservation and storage at -80{degrees}C and their functional ability to phagocytose bacteria similar to fresh cells. Our data revealed that the acclimation process after thawing was essential to recovering the functional activity of the cells. The results presented here will facilitate the study of the functions of the most important immune cells of this species and will provide tools for cell preservation in other molluscs species.