Development of three-dimensional (3D) spheroid culture system from rainbow trout kidney cell line (RTK) for in vitro production of fish viral pathogen

The present study aimed to generate 3D spheroid cultures from three rainbow trout (RTE, RTK and RTS) cell lines by simple and reproducible methods and evaluated them for the propagation of fish viral pathogen for the first time. The 3D spheroids were generated in the present study by scaffold-free method using 3D-orbital shaker. The growth of spheroids was documented on each day until 15 days and their viability was assessed by using AO/EB and Hoechst 33258 fluorescent staining under bright field microscope. The successfully generated RTK spheroids (RTKs) from the RTK cells were further used for the propagation of SJNNV virus in direct comparison of viral growth with conventional 2D monolayer culture of RTK cell line. Immunofluorescent staining confirms the expression of SJNNV major capsid protein in infected RTK spheroid earlier than the RTK monolayer. The samples collected from both RTK spheroid and RTK monolayer at different days of post infection was confirmed by RT-PCR and their viral copy numbers found high in RTK spheroid while quantified by qRT-PCR. Further, SJNNV major capsid protein was confirmed by western blot and ELISA. The viral load obtained using RTK spheroid was found to be high when compared to the monolayer and this study suggests that the spheroid generated from RTK cells is more suitable in vitro tool that can be used for propagation of fish viral pathogens in large-scale for production of whole virus vaccine.