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

Yutong Bai; Qingyan Zhou; Qingquan Hua; Yuanyuan Zhang

doi:10.3791/69916

A subscription to JoVE is required to view this content. Sign in or start your free trial.

Method Article

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

321 views

⸱

DOI:

10.3791/69916

⸱

March 20th, 2026

Yutong Bai^*¹^,²^,³ , Qingyan Zhou^*¹^,²^,³ , Qingquan Hua¹^,² , Yuanyuan Zhang¹^,²

¹Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University, ²Research Institute of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University, ³The First Clinical School of Wuhan University

^* These authors contributed equally

Summary

Here, we present a protocol for the isolation, purification, and culture of primary marginal cells from the mouse stria vascularis, and for establishing in vitro models of oxidative stress and cellular senescence to study cochlear lateral wall disorders.

Abstract

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.

Introduction

In the auditory system, various tissues within the cochlea work in concert to convert complex sound waves into electrical signals¹. The stria vascularis regulates the ionic composition of the endolymph and generates the Endo-cochlear Potential (EP), thereby maintaining cochlear homeostasis and ensuring the normal function of inner and outer hair cells. It is composed of three distinct cell types: marginal cells, intermediate cells, and basal cells. Marginal cells form a polarized epithelial monolayer that directly faces the endolymph and is sealed by tight junctions. The basolateral membrane of marginal cells is enriched with two key potassium ....