Establishment of grass carp (Ctenopharyngodon idella) fibroblast cell line and its application in elucidating pathogenic mechanisms of pathogen infection.

We successfully established and characterized a fibroblast cell line derived from the epidermal tissue of grass carp (Ctenopharyngodon idella), designated as Ctenopharyngodon idella epidermal fibroblast (CIEF). The cell line was maintained in M199 medium supplemented with 20% fetal bovine serum under standard culture conditions (28 °C, 5% CO2), exhibiting characteristic fibroblast-like morphology. CIEF cells have demonstrated stable proliferation through 38 successive passages and retained viability after cryopreservation. Species confirmation was achieved through 18 S ribosomal RNA sequencing, verifying the cell line’s origin from grass carp. Cytogenetic analysis revealed a diploid chromosome count of 48, consistent with the species’ karyotype. Dual immunofluorescence demonstrated co-expression of fibroblast-specific markers α-smooth muscle actin (α-SMA) and fibroblast-specific protein 1 (FSP1), confirming cellular identity. The cell line exhibited sensitivity to major piscine pathogens, showing pronounced cytopathic effects (CPE) following challenge with Aeromonas hydrophila, Aeromonas veronii, and grass carp reovirus (GCRV). Ultrastructural analysis via transmission electron microscopy revealed pathogen-induced cellular alterations, including increased apoptotic bodies and phagocytic vesicles, accompanied by significant organelle damage particularly affecting endoplasmic reticulum and mitochondrial structures. Quantitative real-time PCR analysis demonstrated activation of antimicrobial immune responses post-infection, coupled with observed mitochondrial membrane potential depolarization. This established in vitro model provides a valuable platform for investigating host-pathogen interactions, pathogenic mechanisms, and cellular responses to aquatic pathogens in cyprinid species.