An engineered epididymal cell line provides a sustainable source of functional extracellular vesicles for in vitro sperm preservation: A multi‐omics and functional study

ABSTRACT This study investigated the function of epididymal extracellular vesicles (EVs) in overcoming sperm post‐testicular modification deficiencies. Although EVs derived from the epididymis play a crucial role in intercellular communication and molecular exchange, there is limited research addressing how EVs released by in vitro cultured epididymal epithelial cells affect sperm function. Pig epididymal caput epithelial cells (pEECs) were successfully established and verified through immunofluorescence, Western blotting, semiquantitative analyses, and RNA sequencing. EVs from pEECs were isolated by low‐temperature ultracentrifugation and characterized using NanoSight and scanning electron microscopy. Proteomic sequencing showed that these EVs are enriched in cargoes beneficial to sperm, including antioxidants, motility modulators, sperm‒egg recognition molecules, and proteins associated with antigen processing and presentation. To validate their function, sperm were incubated with EVs at 17°C, demonstrating that EVs could attach to sperm, enhance antioxidant capacity and motility, and reduce plasma membrane oxidation, mitochondrial impairment, and DNA damage. These findings indicate that pEECs can sustainably secrete multifunctional EVs in vitro and that these EVs confer protective effects on sperm in a time‐dependent manner.