Construction of donkey skin fibroblast immortalized cell line and comparative analysis with primary cells.

Ejiao, the traditional Chinese medicinal gelatin derived from donkey hide, faces sustainability challenges due to declining donkey populations and constraints on large-scale harvesting. We isolated primary dermal fibroblasts from donkey skin and generated an immortalized line by SV40 large T antigen transduction with puromycin selection. The immortalized cells preserved characteristic fibroblastic morphology while exhibiting enhanced proliferation, delayed senescence, and increased cell-cycle activity relative to primary fibroblasts. Transcriptomic profiling demonstrated enrichment of cell-cycle-related pathways after immortalization, whereas expression of collagen and other extracellular matrix-related genes remained largely consistent. Targeted LC-MS/MS analysis detected Ejiao-related collagen peptide fragments in culture supernatants from both primary and immortalized cells, supporting retention of relevant secretory functions. This study reports the first establishment of an immortalized donkey dermal fibroblast line that preserves fibroblastic features and provide a stable in vitro model for studying fibroblast biology, extracellular matrix metabolism, and skin-associated diseases. Detection of Ejiao-specific peptides further supports their potential as a proof-of-concept system for cell culture-based production of donkey-hide gelatin, addressing raw material scarcity and enabling sustainable applications in veterinary and pharmacological research.