Isolation and primary culture of Octopus vulgaris cells: Assessment of their proliferative capacity

The octopus cell culture system represents a useful tool in various fields, such as biological studies and sustainable octopus production. In this paper, we describe the isolation procedure and culture protocols, metabolic activity, and the proliferative capacity assessment of the common octopus (Octopus vulgaris) cells, at three different life stages: embryo, paralarvae and adult. Isolation of cells was carried out with minimum essential medium (MEM) or Leibovitz L-15 medium, using 0.4 % type I collagenase for 2 (paralarvae and embryo) or 3 h (adult) at 25 {degrees}C. Isolated cells showed significant metabolic activity, demonstrating their viability for in vitro studies. The culture of isolated cells in MEM or L-15 supplemented with different sera (4 % fetal bovine serum (FBS), 10 % FBS and 5 % haemolymph (HEMO)) was viable for 168 h, although the survival of different cell types was variable. A detachment protocol using trypsin was also successfully developed. Finally, isolated muscle cells from both paralarval and adult specimens were marked with the mitotic indicator PHH3. Results from flow cytometry analysis showed that all developmental stages examined contained cells in mitotic phase, with embryos XII-XIII showing the highest proportion. This study provides novel guidelines for the in vitro culture of O. vulgaris cells, which are useful for the future development of cellular aquaculture, understood as the production of lab-grown meat for this species. Moreover, the results reported in this communication could contribute to reduce the use of animals for scientific purposes as well as providing a new option for human consumption of cephalopods.