Isolation and Culture of Primary Marginal Cells of the Stria Vascularis from Neonatal Mice

The stria vascularis is a key structure in the inner ear responsible for generating and maintaining the endocochlear potential (EP), and its dysfunction is closely associated with various forms of hearing loss. The stria vascularis is composed of marginal cells, intermediate cells, and basal cells, among which marginal cells play a central role in the generation and maintenance of the EP. Therefore, establishing an in vitro primary culture model of marginal cells is essential for investigating the biological characteristics of the stria vascularis and its involvement in hearing loss-related disorders. In this study, we successfully isolated and cultured primary marginal cells from the stria vascularis of neonatal mice. Strial tissues were obtained by microdissection, digested with type II collagenase, and purified by differential plating to remove most fibroblasts, with cultured cells reaching over 80% confluence by day 7. Immunofluorescence staining demonstrated stable expression of cytokeratin 18 (CK18) and potassium voltage-gated channel subfamily Q member 1 (KCNQ1), confirming successful isolation, high purity, and preserved physiological function of marginal cells. Based on this model, in vitro oxidative stress and cellular senescence models were established using hydrogen peroxide (H2O2) and D-galactose, respectively. Treated cells exhibited significantly increased intracellular reactive oxygen species (ROS) levels and senescence-associated β-galactosidase (SA-β-gal) positivity, indicating high sensitivity of primary marginal cells to oxidative stress and senescence-inducing stimuli. Collectively, this study establishes a stable and reproducible system for the culture and modeling of mouse primary marginal cells, providing a reliable in vitro platform for investigating stria vascularis dysfunction and related mechanisms.