Primary culture of hematopoietic tissue cells from Procambarus clarkii and their susceptibility to WSSV and DIV1.

The absence of continuous cell lines from crustaceans highlights the urgent need for optimized primary cultures in aquatic pathology and disease research. This study describes the establishment of primary hematopoietic tissue (HPT) cell culture system from the crayfish Procambarus clarkii. Optimal cell isolation was achieved through digestion with a mixture of collagenase (types I and IV, each at 0.2%) at 30°C for 30 min, followed by maintenance in Leibovitz's L-15 medium supplemented with 2% fetal bovine serum (FBS) at 27°C. The cultures remained viable for up to 5 days, maintaining ∼70% viability. Crucially, these primary HPT cells were susceptible to infection by both white spot syndrome virus (WSSV) and decapod iridescent virus 1 (DIV1), as evidenced by viral gene expression and viral genome copies. This method provides an accessible and physiologically relevant in vitro model for investigating virus-host interactions in P. clarkii, thus facilitating the development of antiviral strategies.