Development and characterization of primary cell culture from the spinal cord of Asian seabass, Lates calcarifer.

Abstract

Asian seabass, Lates calcarifer, is one of the most important fish species in aquaculture. An attempt was made to develop a primary cell culture from the spinal cord of Lates calcarifer by the enzymatic and mechanical dissociation method. The primary cell culture was sub-cultured for 20 times in Leibovitz's L-15 medium with 20% fetal bovine serum (FBS) and 0.5nM of human neurotrophin-3 at 28°C. The primary cell culture was cryopreserved at different passage levels and recovery of cells after long-term storage was estimated about 75-85%. The authenticity of origin of primary cell culture from L. calcarifer was confirmed by polymerase chain reaction assay using species-specific mitochondrial 12S rRNA primer. The primary cell culture was designated as seabass spinal cord cells (SBSC). The cells morphologically resembled the neurons due to their neural-like prolongations and star-like structure. Immunophenotypic analysis of the SBSC revealed that they are of neuronal origin. The SBSC were found to be highly susceptible to striped jack nervous necrosis virus (SJNNV) and infection in the cells was confirmed by RT-PCR. In conclusion, this is the first innovative euryhaline fish neuronal primary cell culture of L. calcarifer now available for neurophysiological and neurotoxicological studies.