The four-finger threadfin (Eleutheronema tetradactylum) is a marine fish species of high economic value. Although artificial breeding and selection have been achieved in China, challenges such as high stress susceptibility and diseases persist in aquaculture. This study aimed to establish a cell line from this species to provide a research platform for non-model economic fish, paving a new path for in-depth gene function analysis and genetic improvement. Heart tissue from E. tetradactylum was used for primary culture via protease digestion. The established cell line was then systematically characterized. Optimal growth conditions were determined by testing different culture parameters. Cells were cryopreserved to assess recovery rate, and karyotype and species origin analyses were performed. The cell line was transfected using Lipofectamine 3000. Sensitivity tests were conducted using media containing three heavy metals at varying concentrations, as well as against two bacterial species.Results A stable cell line, designated EtH, has been subcultured to passage 70. It exhibits a normal diploid karyotype (2N=26) and a post-thaw viability exceeding 81.3%. The optimal growth conditions were identified as Leibovitz's L-15 medium supplemented with 10% fetal bovine serum at 28°C. The transfection efficiency for a pEGFP-N1 plasmid was approximately 16%. The EtH cells showed dose-dependent toxic sensitivity to manganese, chromium, and copper, and were susceptible to both Streptococcus agalactiae and Nocardia seriolae.

The EtH cell line demonstrates stable genetic characteristics and high post-thaw viability, making it a reliable and reusable research tool. Its specific sensitivity to various heavy metals and bacteria indicates strong application potential in environmental pollutant monitoring, toxicology, and fish disease research for E. tetradactylum and related species. Successful transfection confirms its utility as an in vitro tool for exogenous gene expression studies. Addressing the current research bias towards model species, this study fills the gap of a "dedicated" cell line for E. tetradactylum, enriches the research system for non-model economic fish cell models, and provides a practical pathway for future gene function analysis and germplasm improvement.