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

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 transporters, Na⁺/K⁺-ATPase and NKCC1, which are involved in regulating sodium and potassium ion transport in the stria vascularis. The function of Na⁺/K⁺-ATPase has been demonstrated to be directly associated with the EP^2,3. KCNQ1/KCNE1 K⁺ channels are located at the apical membrane of marginal cells and generate an additional K⁺ diffusion potential that contributes to the formation of the EP⁴. Marginal cells are enriched in multiple potassium-related proteins and serve as the core cellular unit driving potassium recycling in the cochlear endolymph. Thus, marginal cells are the key cells responsible for generating and maintaining the EP.

The stria vascularis (SV) has been demonstrated to play a critical role in the pathogenesis of multiple disorders associated with sensorineural hearing loss (SNHL), including age-related hearing loss (ARHL), noise-induced hearing loss (NIHL), hereditary hearing loss (HHL), and drug-induced hearing loss (DIHL). In addition, several other hearing disorders, such as autoimmune inner ear disease (AIED), autoinflammatory inner ear disease (AID), and Ménière's disease (MD), although etiologically distinct, have also been shown by recent studies to involve the SV in their underlying pathogenic mechanisms^5,6. Therefore, establishing reliable cellular models for investigating the pathogenesis of hearing loss is essential. Studies have demonstrated that reduced marginal cell density serves as a strong predictor of the EP throughout the murine lifespan, while the atrophy of the stria vascularis represents an early characteristic lesion in ARHL^7,8. In vitro culture of marginal cells can provide a reliable model for studying the pathogenesis of ARHL and exploring potential therapeutic targets. Beyond ARHL, recent studies have revealed a high correlation and possible underlying relationship between the stria vascularis and other auditory disorders, suggesting that in vitro marginal cell models may have broader applications^5,9,10.

This study describes a protocol for the dissection and culture of the stria vascularis from neonatal mouse cochleae, followed by the purification of marginal cells using differential plating, thereby providing a reliable cellular model for research on stria vascularis-related pathologies.

Protocol

The neonatal mouse pups (P0-3) were provided by Biont. All experimental procedures were in compliance with the Guidelines for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication, revised 2011) and approved by the Animal Ethics Committee of Renmin Hospital of Wuhan University (WDRM202400019).

1. Sterilization and material preparation

1. Prepare the dissection tools, including two pairs of micro-dissection forceps, one pair of standard forceps, one pair of surgical scissors, and one pair of micro-dissection scissors. Sterilize all tools using an autoclave and dry them in a 60 °C oven for 4 h.
2. Prepare 100 mL of epithelial cell medium-animal (EpiCM-a medium) according to the manufacturer's instructions. Add fetal bovine serum (FBS), epithelial cell growth supplement (EpiCGS-a), and penicillin/streptomycin solution to the basal medium to achieve final concentrations of 2% FBS, 1% EpiCGS-a, and 1% penicillin/streptomycin.
3. Disinfect the stereomicroscope stage and housing with 75% ethanol. Then, place the autoclaved dissection tools and the disinfected microscope inside a biological safety cabinet. Expose all items to UV light for 30 min to ensure complete sterility before initiating the dissection procedure.

2. Dissection and temporal bone isolation for auditory epithelium collection

1. According to the "Guidelines for Euthanasia of Laboratory Animals GB/T 39760-2021", anesthetize mouse pups (p0–3) with 4% isoflurane, then use a hemostat to pinch the hind toe. After observing the disappearance of the toe-pinch reflex to confirm that the mouse is in a deep anesthetic state, perform euthanasia. Afterwards, disinfect with 75% ethanol and transfer to a biosafety cabinet. If necessary, use other ethically approved methods.
2. Immobilize the head, open the scalp along the sagittal suture using micro-dissection scissors, separate the scalp bilaterally, and excise it.
3. Bisect the neonatal mouse's head along the midline using micro-dissection scissors. Remove brain tissue with forceps to expose the temporal bones (Figure 1A).
4. Observe bilateral temporal bones at the cranial base, remove other unnecessary bones, and retain the temporal bone tissue containing the cochlea (Figure 1B).
5. Transfer one temporal bone to a 35 mm sterile dish containing fresh Hank's Balanced Salt Solution (HBSS) on ice.
6. Transfer the other temporal bone under the microscope, secure the temporal bone tissue with one pair of forceps, and use another pair to perform blunt dissection of the surface muscle tissue until the white semicircular ossicle ring is visible (Figure 1C). After confirming cochlear positioning, continue blunt dissection downward until translucent cochlear tissue is observed (Figure 1D).
   NOTE: Steps 2.6-2.7 should be performed on ice.
7. Using micro-dissection forceps, carefully remove a small piece of the bone wall at the apical turn of the cochlea, gently insert one side of the forceps into the cochlea, and separate the bone wall from the membranous labyrinth. Remove the bone wall and extend the membranous labyrinth tissue in fresh Hank's Balanced Salt Solution (HBSS) (Figure 1E).
8. Secure the basilar membrane with one pair of forceps, and carefully separate the stria vascularis with another pair of forceps. Transfer it to a 1.5 mL microcentrifuge tube containing 1 mL of 1mg/mL collagenase type II solution placed on ice (Figure 1F).
9. Repeat the above steps to harvest the stria vascularis from the other side.

3. Enzymatic disaggregation for obtaining marginal cells of stria vascularis

1. Incubate the microcentrifuge tube containing the harvested tissue in 1mg/mL collagenase type II solution at 37 °C for 30 min.
   NOTE: The collagenase type II solution was prepared by diluting collagenase type II in phosphate-buffered saline (PBS; 2.7 mM KCl, 2.0 mM KH₂PO₄, 137 mM NaCl, 10 mM Na₂HPO₄).
2. Centrifuge the tube at 300 × g for 8 min.
3. Carefully remove the supernatant. Add 1 mL of culture medium to terminate digestion, gently triturate to resuspend the cells, and centrifuge again at 300 × g for 8 min. 
4. Aspirate the supernatant and add 1 mL of fresh culture medium. Triturate to obtain a single-cell suspension, transfer the suspension to a 15 mL conical tube, and add additional medium to adjust the total volume to 10 mL.
5. Mix the cell suspension thoroughly by repeated pipetting, then transfer 1 mL of the suspension to each well of a 6-well plate. Culture the cells in 2 mL of DMEM supplemented with 2% FBS, 1% EpiCGS-a, and 1% penicillin/streptomycin solution at 37 °C in a 5% CO₂ incubator.
6. Replace the medium daily and monitor cell status. Clustered growth of marginal cells can typically be observed 2 days after plating.

4. Purification of marginal cells

1. On day 3 of culture, observe the appearance of numerous spindle-shaped fibroblasts surrounding the cobblestone-like clusters of marginal cells (Figure 2B).
2. Exploiting differences in adhesion rates, gently pipette to partially remove the spindle-shaped fibroblasts. Using a 1 mL pipette, gently flush the D-Hanks' balanced salt solution over large areas in each of the four quadrants. Monitor the cell purification under a microscope, and adjust the force and direction of pipetting accordingly. Generally, large-area flushing is performed twice. After each round, examine the fibroblast density microscopically; based on these observations, apply localized gentle pipetting to densely populated fibroblast areas to achieve a sparse and uniform distribution across the culture dish.
   NOTE: An appropriate density of fibroblasts was retained, as complete removal or excessive purification may restrict the growth of primary cells. We recommend maintaining an approximate 2:1 ratio of marginal cells to fibroblasts.
3. Cease purification when fibroblasts appear sparsely distributed among marginal cell clusters under microscopic examination. Remove the supernatant and add 2 mL of fresh culture medium to continue cultivation.

5. Immunofluorescence staining

1. On day 2 post-purification, examine cell morphology under a microscope. When observing adequate cell cluster formation and appropriate density, aspirate culture medium and wash cells three times with phosphate-buffered saline (PBS, pH 7.4).
2. Fix cells with 4% paraformaldehyde for 15 min at room temperature. After removing fixative solution, wash cells three times with PBS (5 min per wash).
3. Permeabilize cells with 0.5% Triton X-100 for 15 min. Remove the solution and wash cells three times with PBS (5 min per wash).
4. Block cells with PBS containing 10% goat serum for 30 min at room temperature.
5. Incubate cells with rabbit anti-mouse CK18 monoclonal antibody (diluted 1:300 in PBS containing 3% goat serum) or KCNQ1 Antibody (G-8) (diluted 1:100 in PBS containing 3% goat serum) overnight at 4 °C.
6. Wash cells three times with PBS (10 min per wash). Incubate with the corresponding secondary antibody (diluted 1:300 in PBS containing 3% goat serum) for 1 h at room temperature.
7. Remove secondary antibody and wash cells three times with PBS (10 min per wash).
8. Apply 1-2 drops of antifade mounting medium containing DAPI onto slides, mount coverslips, and examine specimens using a confocal laser scanning system.
   NOTE: Except for washing steps, all incubation solutions, including antibodies, were added in volumes sufficient to completely cover the cells.

6. H₂O₂ treatment and ROS staining

1. After 7 days of culture, when marginal cells have formed a confluent monolayer, induce oxidative stress by treatment with 1 mM H₂O₂. Prepare the 1 mM working solution by diluting the 30% H₂O₂ stock solution 1:10000 in epithelial cell medium. Incubate the experimental groups with this working solution, while treating control groups with epithelial cell medium alone.
2. Following 16 h of treatment, carefully remove the medium and wash the cells with PBS. Evaluate intracellular ROS levels in marginal cells using a Reactive Oxygen Species Assay Kit according to the manufacturer's protocol.
3. Remove the culture medium, then add diluted MitoTracker Red CMXRos (a mitochondria-selective dye) and DCFH-DA to the cells, and incubate at 37 °C in a cell culture incubator for 30 min.
4. Wash the cells three times with PBS (5 min each), mount the coverslips, and observe the samples using a confocal laser scanning microscope.

7. D-galactose treatment and SA-β-gal staining

1. After 7 days of culture, when marginal cells form a confluent monolayer, D-galactose powder was dissolved in EpiCM-a medium to prepare a 40 mg/mL D-gal working solution. Incubate experimental groups with this solution (2 mL per well of a 6-well plate, 48 h, at 37 °C) to induce cellular senescence, while treating control groups with the same volume of Epi medium alone.
2. Following 48 h of treatment, carefully remove the medium and wash cells with PBS. Assess cellular senescence in marginal cells using the SA-β-gal staining kit according to the manufacturer's instructions.
3. Fix cells with fixative solution for 15 min at room temperature. After removing the fixative, wash cells three times with PBS (5 min per wash).
4. Incubate cells with SA-β-gal staining working solution containing 5% X-Gal (1 mL per well of a 6-well plate) overnight at 37 °C in a humidified incubator without CO₂.
5. Capture images of SA-β-gal staining under bright-field illumination using a microscope.

8. Statistical analysis

1. For comparisons between H₂O₂-treated and control groups regarding mean immunofluorescence intensity of ROS staining, and between D-galactose-treated and control groups regarding the proportion of SA-β-gal-positive cells, apply t-tests following assessments for normality and log-normality.

Results

Growth and morphological characteristics of cultured cells
Using this protocol, we successfully established primary cultures of marginal cells isolated from the murine cochlea. On day 2 of culture, light microscopy revealed that the cells had adhered to the substrate and begun to form clustered colonies (Figure 2Aa). On day 3, we purified the cells (Figure 2B). Subsequent observations over the following days revealed that the primary margina...

Discussion

For decades, research on hearing loss has primarily focused on the role of sensory hair cells and their synaptic connections with spiral ganglion neurons¹¹. Numerous published studies have been devoted to establishing and optimizing in vitro culture models of hair cells, which have played an indispensable role in investigating hair cell damage, regeneration, and the effects of ototoxic drugs^12,13. However, a growing body of resear...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 mL Centrifuge Tube	Servicebio	EP-1501-J	using for culture
35 mm Cell Culture Dish	NEST	706001	using for dissection
4% Paraformaldehyde	Servicebio	G1101	immunofluorescent staining
6-well Plate	Servicebio	CCP-6H	using for culture
Alexa Fluor 488-Conjugated Goat Anti-Rabbit Antibody	Proteintech	SA00013-2	immunofluorescent staining
Antifade Mounting Medium with DAPI	Absin	abs9235	immunofluorescent staining
Cell Counting Kit-8	Beyotime	C0037	using for cell counting
Collagenase Type II	Biosharp	BS164	using for culture
Confocal Laser Scanning System	Olympus	FV1200	immunofluorescent staining
DCFH-DA	Invitrogen	C2938	ROS staining
D-gal	Solarbio	D8310	using for cell modeling
EpiCM-a Medium	Sciencell	4131	using for culture
Fine forceps	RWD	F11020-11	using for dissection
Goat Serum	Beyotime	C0265	immunofluorescent staining
H2O2	Mkbio	MM0707	using for cell modeling
Hank's Balanced Salt Solution (HBSS)	Biosharp	BL561A	using for culture
Isoflurane	RWD	R510-22-10	using for dissection
KCNQ1 Antibody (G-8)	Santa cruz	sc-365186	immunofluorescent staining
Microscope	Olympus	SZX7-1063	using for dissection
Microscope	Olympus	IX71	Used for observing the results of immunofluorescence staining
MitoTracker Red CMXRos	Invitrogen	M7512	ROS staining
Neonatal Mouse Pups (P0-P3)			provided by BIONT
PBS	Servicebio	G4202	immunofluorescent staining
Rabbit Anti-Mouse CK18 Monoclonal Antibody	Proteintech	10830-1-AP	immunofluorescent staining
Senescence β-Galactosidase Staining Kit	Beyotime	C0602	aging staining
Triton X-100	Sigma-Aldrich	X100	immunofluorescent staining