Development of two haemocyte culture systems (in attachment and in suspension) for shrimp immunity studies

Abstract

For studying shrimp immunity, in vitro haemocyte cultures are essential. Despite various reported attempts, well-described and reproducible culture techniques are lacking. The current work aimed to establish two in vitro haemocyte culture systems for Penaeus (Litopenaeus) vannamei. Haemocyte suspensions were either seeded in conventional Nunc® Nunclon™Δ Surface 24-well cell culture plates with glass cover slips (haemocytes in attachment) or in Nunc® Hydrocell Surface 24-well cell culture plates (haemocytes in suspension). The culture medium was based on L-15 (Leibovitz), and was further supplemented with L-glutathione and protease inhibitors in an attempt to improve haemocyte survival. Parameters such as number of living adherent and non-adherent single cells, number and average diameter of clusters and survival of cells inside clusters were evaluated. Additionally, live-cell imaging videos were recorded. It was found that haemocytes cultured for 1h on glass coverslips in Nunc® Nunclon™Δ Surface plates could be separated in two cell fractions: adherent or non-adherent. Shrimp haemocytes cultured in Nunc® Hydrocell Surface plates remained in suspension and over time formed cell clusters which melanised. L-glutathione supplementation clearly improved haemocyte survival up to 48h and delayed clustering and melanisation; addition of protease inhibitors did not. To validate the system, the phagocytic and antibacterial activities of adherent haemocytes towards Vibrio campbellii were evaluated. After 1h of co-culture, 11.5±0.14% of haemocytes showed phagocytosis with an average of 2.4±0.1 bacteria internalised per haemocyte. Furthermore, haemocytes clearly demonstrated an antibacterial activity. It was concluded that these systems were reproducible and could keep haemocytes functionally active during the time required for the study of innate immune processes. Consequently, these techniques represent powerful tools for studying a variety of cell-mediated and humoral immune responses of shrimp in vitro.