Name: Isolation and Culture of Primary Marginal Cells of the Stria Vascularis from Neonatal Mice (Video) | JoVE Journal
Uploaded: 2026-03-20T13:30:00.000+00:00
Duration: 8 min 27 s
Description: Learn to isolate and culture primary marginal cells from neonatal mouse stria vascularis using enzymatic digestion and differential plating. This method enables in vitro modeling of oxidative stress and cellular senescence for hearing loss research.

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5 minutes

We express our gratitude to the Department of Otolaryngology-Head and Neck Surgery, Wuhan University Renmin Hospital (Wuhan, China) and Research Institute of Otolaryngology-Head and Neck Surgery, Wuhan University Renmin Hospital (Wuhan, China) for their generous support and encouragement. This work was supported by the National Health Service Center Construction and Incubation Foundation of China (ZX0000000037), the National Natural Science Foundation of China (81700916, 82301294), the Natural Science Foundation of Hubei Province (2017CFB242) and the Health Commission Scientific Research Project of Hubei Province (WJ2017Q005).

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
<ol>
	<li>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 &#176;C oven for 4 h.</li>
	<li>Prepare 100 mL of epithelial cell medium-animal (EpiCM-a medium) according to the manufacturer&#39;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.</li>
	<li>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.</li>
</ol>
2. Dissection and temporal bone isolation for auditory epithelium collection
<ol>
	<li>According to the &#34;Guidelines for Euthanasia of Laboratory Animals GB/T 39760-2021&#34;, anesthetize mouse pups (p0&#8211;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.</li>
	<li>Immobilize the head, open the scalp along the sagittal suture using micro-dissection scissors, separate the scalp bilaterally, and excise it.</li>
	<li>Bisect the neonatal mouse&#39;s head along the midline using micro-dissection scissors. Remove brain tissue with forceps to expose the temporal bones (Figure 1A).</li>
	<li>Observe bilateral temporal bones at the cranial base, remove other unnecessary bones, and retain the temporal bone tissue containing the cochlea (Figure 1B).</li>
	<li>Transfer one temporal bone to a 35 mm sterile dish containing fresh Hank&#39;s Balanced Salt Solution (HBSS) on ice.</li>
	<li>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.</li>
	<li>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&#39;s Balanced Salt Solution (HBSS) (Figure 1E).</li>
	<li>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).</li>
	<li>Repeat the above steps to harvest the stria vascularis from the other side.</li>
</ol>
3. Enzymatic disaggregation for obtaining marginal cells of stria vascularis
<ol>
	<li>Incubate the microcentrifuge tube containing the harvested tissue in 1mg/mL collagenase type II solution at 37 &#176;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 KH&#8322;PO&#8324;, 137 mM NaCl, 10 mM Na2HPO4).</li>
	<li>Centrifuge the tube at 300 &#215; g for 8 min.</li>
	<li>Carefully remove the supernatant. Add 1 mL of culture medium to terminate digestion, gently triturate to resuspend the cells, and centrifuge again at 300 &#215; g for 8 min.&#160;</li>
	<li>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.</li>
	<li>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 &#176;C in a 5% CO2 incubator.</li>
	<li>Replace the medium daily and monitor cell status. Clustered growth of marginal cells can typically be observed 2 days after plating.</li>
</ol>
4. Purification of marginal cells
<ol>
	<li>On day 3 of culture, observe the appearance of numerous spindle-shaped fibroblasts surrounding the cobblestone-like clusters of marginal cells (Figure 2B).</li>
	<li>Exploiting differences in adhesion rates, gently pipette to partially remove the spindle-shaped fibroblasts. Using a 1 mL pipette, gently flush the D-Hanks&#39; 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.</li>
	<li>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.</li>
</ol>
5. Immunofluorescence staining
<ol>
	<li>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).</li>
	<li>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).</li>
	<li>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).</li>
	<li>Block cells with PBS containing 10% goat serum for 30 min at room temperature.</li>
	<li>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)&#160;overnight at 4 &#176;C.</li>
	<li>Wash cells three times with PBS (10 min per wash). Incubate with the corresponding&#160;secondary antibody (diluted 1:300 in PBS containing 3% goat serum) for 1 h at room temperature.</li>
	<li>Remove secondary antibody and wash cells three times with PBS (10 min per wash).</li>
	<li>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.</li>
</ol>
6. H2O2 treatment and ROS staining
<ol>
	<li>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.</li>
	<li>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&#39;s protocol.</li>
	<li>Remove the culture medium, then add diluted MitoTracker Red CMXRos (a mitochondria-selective dye) and DCFH-DA to the cells, and incubate at 37 &#176;C in a cell culture incubator for 30 min.</li>
	<li>Wash the cells three times with PBS (5 min each), mount the coverslips, and observe the samples using a confocal laser scanning microscope.</li>
</ol>
7. D-galactose treatment and SA-&#946;-gal staining
<ol>
	<li>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 &#176;C) to induce cellular senescence, while treating control groups with the same volume of Epi medium alone.</li>
	<li>Following 48 h of treatment, carefully remove the medium and wash cells with PBS. Assess cellular senescence in marginal cells using the SA-&#946;-gal staining kit according to the manufacturer&#39;s instructions.</li>
	<li>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).</li>
	<li>Incubate cells with SA-&#946;-gal staining working solution containing 5% X-Gal (1 mL per well of a 6-well plate) overnight at 37 &#176;C in a humidified incubator without CO2.</li>
	<li>Capture images of SA-&#946;-gal staining under bright-field illumination using a microscope.</li>
</ol>
8. Statistical analysis
<ol>
	<li>For comparisons between H&#8322;O&#8322;-treated and control groups regarding mean immunofluorescence intensity of ROS staining, and between D-galactose-treated and control groups regarding the proportion of SA-&#946;-gal-positive cells, apply t-tests following assessments for normality and log-normality.</li>
</ol>

<ol><li>Raphael, Y., Altschuler, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+and+innervation+of+the+cochlea%5BTitle%5D)+AND+%22Brain+Res+Bull%22%5BJournal%5D)">Structure and innervation of the cochlea.</a> Brain Res Bull. 60 (5-6), 397-422 (2003).</li>
<li>Bovee, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+stria+vascularis%3A+Renewed+attention+on+a+key+player+in+age-related+hearing+loss%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">The stria vascularis: Renewed attention on a key player in age-related hearing loss.</a> Int J Mol Sci. 25 (10), 5391(2024).</li>
<li>Schulte, B. A., Schmiedt, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lateral+wall+Na%2CK-ATPase+and+endocochlear+potentials+decline+with+age+in+quiet-reared+gerbils%5BTitle%5D)+AND+%22Hear+Res%22%5BJournal%5D)">Lateral wall Na,K-ATPase and endocochlear potentials decline with age in quiet-reared gerbils.</a> Hear Res. 61 (1-2), 35-46 (1992).</li>
<li>Hibino, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+is+the+highly+positive+endocochlear+potential+formed%3F+The+specific+architecture+of+the+stria+vascularis+and+the+roles+of+the+ion-transport+apparatus%5BTitle%5D)+AND+%22Pflugers+Arch%22%5BJournal%5D)">How is the highly positive endocochlear potential formed? The specific architecture of the stria vascularis and the roles of the ion-transport apparatus.</a> Pflugers Arch. 459 (4), 521-533 (2010).</li>
<li>Johns, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+the+stria+vascularis+in+neglected+otologic+disease%5BTitle%5D)+AND+%22Hear+Res%22%5BJournal%5D)">The role of the stria vascularis in neglected otologic disease.</a> Hear Res. 428, 108682(2023).</li>
<li>Yu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+the+stria+vascularis+in+the+pathogenesis+of+sensorineural+hearing+loss%3A+A+narrative+review%5BTitle%5D)+AND+%22Front+Neurosci%22%5BJournal%5D)">Role of the stria vascularis in the pathogenesis of sensorineural hearing loss: A narrative review.</a> Front Neurosci. 15, 774585(2021).</li>
<li>Ohlemiller, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+correlates+of+age-related+endocochlear+potential+reduction+in+a+mouse+model%5BTitle%5D)+AND+%22Hear+Res%22%5BJournal%5D)">Cellular correlates of age-related endocochlear potential reduction in a mouse model.</a> Hear Res. 220 (1-2), 10-26 (2006).</li>
<li>Mahendrasingam, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Relative+time+course+of+degeneration+of+different+cochlear+structures+in+the+CD%2F1+mouse+model+of+accelerated+aging%5BTitle%5D)+AND+%22J+Assoc+Res+Otolaryngol%22%5BJournal%5D)">Relative time course of degeneration of different cochlear structures in the CD/1 mouse model of accelerated aging.</a> J Assoc Res Otolaryngol. 12 (4), 437-453 (2011).</li>
<li>Lee, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+apoptosis+induced+by+cisplatin+in+marginal+cells+in+mouse+stria+vascularis%5BTitle%5D)+AND+%22ORL+J+Otorhinolaryngol+Relat+Spec%22%5BJournal%5D)">Mechanisms of apoptosis induced by cisplatin in marginal cells in mouse stria vascularis.</a> ORL J Otorhinolaryngol Relat Spec. 66 (3), 111-118 (2004).</li>
<li>Ohlemiller, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+endocochlear+potential+as+an+indicator+of+reticular+lamina+integrity+after+noise+exposure+in+mice%5BTitle%5D)+AND+%22Hear+Res%22%5BJournal%5D)">The endocochlear potential as an indicator of reticular lamina integrity after noise exposure in mice.</a> Hear Res. 361, 138-151 (2018).</li>
<li>Liberman, M. C., Kujawa, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cochlear+synaptopathy+in+acquired+sensorineural+hearing+loss%3A+Manifestations+and+mechanisms%5BTitle%5D)+AND+%22Hear+Res%22%5BJournal%5D)">Cochlear synaptopathy in acquired sensorineural hearing loss: Manifestations and mechanisms.</a> Hear Res. 349, 138-147 (2017).</li>
<li>Levano, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole+neonatal+cochlear+explants+as+an+in+vitro+model%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Whole neonatal cochlear explants as an in vitro model.</a> J Vis Exp. (197), e65160(2023).</li>
<li>Wu, K. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+culture+of+primary+cochlear+hair+cells+from+neonatal+mice%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Isolation and culture of primary cochlear hair cells from neonatal mice.</a> J Vis Exp. (199), e65687(2023).</li>
<li>Schmiedt, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+furosemide+applied+chronically+to+the+round+window%3A+A+model+of+metabolic+presbyacusis%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Effects of furosemide applied chronically to the round window: A model of metabolic presbyacusis.</a> J Neurosci. 22 (21), 9643-9650 (2002).</li>
<li>Kurata, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histopathologic+evaluation+of+vascular+findings+in+the+cochlea+in+patients+with+presbycusis%5BTitle%5D)+AND+%22JAMA+Otolaryngol+Head+Neck+Surg%22%5BJournal%5D)">Histopathologic evaluation of vascular findings in the cochlea in patients with presbycusis.</a> JAMA Otolaryngol Head Neck Surg. 142 (2), 173-178 (2016).</li>
<li>Wangemann, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Loss+of+KCNJ10+protein+expression+abolishes+endocochlear+potential+and+causes+deafness+in+Pendred+syndrome+mouse+model%5BTitle%5D)+AND+%22BMC+Med%22%5BJournal%5D)">Loss of KCNJ10 protein expression abolishes endocochlear potential and causes deafness in Pendred syndrome mouse model.</a> BMC Med. 2, 30(2004).</li>
<li>Ding, L., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((miR-106a+facilitates+the+sensorineural+hearing+loss+induced+by+oxidative+stress+by+targeting+connexin-43%5BTitle%5D)+AND+%22Bioengineered%22%5BJournal%5D)">miR-106a facilitates the sensorineural hearing loss induced by oxidative stress by targeting connexin-43.</a> Bioengineered. 13 (6), 14080-14093 (2022).</li>
<li>Wan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PARP1+inhibition+prevents+oxidative+stress+in+age-related+hearing+loss+via+PAR-Ca%282%2B%29-AIF+axis+in+cochlear+strial+marginal+cells%5BTitle%5D)+AND+%22Free+Radic+Biol+Med%22%5BJournal%5D)">PARP1 inhibition prevents oxidative stress in age-related hearing loss via PAR-Ca(2+)-AIF axis in cochlear strial marginal cells.</a> Free Radic Biol Med. 220, 222-235 (2024).</li>
<li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decreased+poly%28ADP-ribose%29+polymerase+1+expression+attenuates+glucose+oxidase-induced+damage+in+rat+cochlear+marginal+strial+cells%5BTitle%5D)+AND+%22Mol+Neurobiol%22%5BJournal%5D)">Decreased poly(ADP-ribose) polymerase 1 expression attenuates glucose oxidase-induced damage in rat cochlear marginal strial cells.</a> Mol Neurobiol. 53 (9), 5971-5984 (2016).</li>
<li>Fetoni, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathogenesis+of+presbycusis+in+animal+models%3A+A+review%5BTitle%5D)+AND+%22Exp+Gerontol%22%5BJournal%5D)">Pathogenesis of presbycusis in animal models: A review.</a> Exp Gerontol. 46 (6), 413-425 (2011).</li>
<li>Marie, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Senescence-accelerated+mouse+prone+8+%28SAMP8%29+as+a+model+of+age-related+hearing+loss%5BTitle%5D)+AND+%22Neurosci+Lett%22%5BJournal%5D)">Senescence-accelerated mouse prone 8 (SAMP8) as a model of age-related hearing loss.</a> Neurosci Lett. 656, 138-143 (2017).</li>
<li>Li, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rodent+models+in+sensorineural+hearing+loss+research%3A+A+comprehensive+review%5BTitle%5D)+AND+%22Life+Sci%22%5BJournal%5D)">Rodent models in sensorineural hearing loss research: A comprehensive review.</a> Life Sci. 358, 123156(2024).</li>
<li>Bommakanti, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cochlear+histopathology+in+human+genetic+hearing+loss%3A+State+of+the+science+and+future+prospects%5BTitle%5D)+AND+%22Hear+Res%22%5BJournal%5D)">Cochlear histopathology in human genetic hearing loss: State of the science and future prospects.</a> Hear Res. 382, 107785(2019).</li>
<li>Nicolson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Navigating+hereditary+hearing+loss%3A+Pathology+of+the+inner+ear%5BTitle%5D)+AND+%22Front+Cell+Neurosci%22%5BJournal%5D)">Navigating hereditary hearing loss: Pathology of the inner ear.</a> Front Cell Neurosci. 15, 660812(2021).</li>
<li>Liu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Applications+and+challenges+of+CRISPR-Cas+gene-editing+to+disease+treatment+in+clinics%5BTitle%5D)+AND+%22Precis+Clin+Med%22%5BJournal%5D)">Applications and challenges of CRISPR-Cas gene-editing to disease treatment in clinics.</a> Precis Clin Med. 4 (3), 179-191 (2021).</li>
<li>Guo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+methods+and+research+progress+of+livestock+and+poultry+immortalized+cell+lines%3A+A+review%5BTitle%5D)+AND+%22Front+Vet+Sci%22%5BJournal%5D)">Establishment methods and research progress of livestock and poultry immortalized cell lines: A review.</a> Front Vet Sci. 9, 956357(2022).</li>
</ol>

The authors declare that the research has no commercial or financial conflicts of interest.

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 drugs12,13. However, a growing body of resear...

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&#8314;/K&#8314;-ATPase and NKCC1, which are involved in regulating sodium and potassium ion transport in the stria vascularis. The function of Na&#8314;/K&#8314;-ATPase has been demonstrated to be directly associated with the EP2,3. KCNQ1/KCNE1 K&#8314; channels are located at the apical membrane of marginal cells and generate an additional K&#8314; 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 M&#233;ni&#232;re&#39;s disease (MD), although etiologically distinct, have also been shown by recent studies to involve the SV in their underlying pathogenic mechanisms5,6. 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&#160;throughout the murine lifespan, while the atrophy of the stria vascularis represents an early characteristic lesion in ARHL7,8. 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 applications5,9,10.
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.

<table><tbody><tr><td>15 mL Centrifuge Tube</td><td>Servicebio</td><td>EP-1501-J</td><td>using for culture</td></tr><tr><td>35 mm Cell Culture Dish</td><td>NEST</td><td>706001</td><td>using for dissection</td></tr><tr><td>4% Paraformaldehyde</td><td>Servicebio</td><td>G1101</td><td>immunofluorescent staining</td></tr><tr><td>6-well Plate</td><td>Servicebio</td><td>CCP-6H</td><td>using for culture</td></tr><tr><td>Alexa Fluor 488-Conjugated Goat Anti-Rabbit Antibody</td><td>Proteintech</td><td>SA00013-2</td><td>immunofluorescent staining</td></tr><tr><td>Antifade Mounting Medium with DAPI</td><td>Absin</td><td>abs9235</td><td>immunofluorescent staining</td></tr><tr><td>Cell Counting Kit-8</td><td>Beyotime</td><td>C0037</td><td>using for cell counting</td></tr><tr><td>Collagenase Type II</td><td>Biosharp</td><td>BS164</td><td>using for culture</td></tr><tr><td>Confocal Laser Scanning System</td><td>Olympus&amp;nbsp;</td><td>FV1200</td><td>immunofluorescent staining</td></tr><tr><td>DCFH-DA</td><td>Invitrogen</td><td>C2938</td><td>ROS staining</td></tr><tr><td>D-gal</td><td>Solarbio</td><td>D8310</td><td>using for cell modeling</td></tr><tr><td>EpiCM-a Medium</td><td>Sciencell</td><td>4131</td><td>using for culture</td></tr><tr><td>Fine forceps</td><td>RWD</td><td>F11020-11</td><td>using for dissection</td></tr><tr><td>Goat Serum</td><td>Beyotime</td><td>C0265</td><td>immunofluorescent staining</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Mkbio</td><td>MM0707</td><td>using for cell modeling</td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Biosharp</td><td>BL561A</td><td>using for culture</td></tr><tr><td>Isoflurane</td><td>RWD</td><td>R510-22-10</td><td>using for dissection</td></tr><tr><td>KCNQ1 Antibody (G-8)</td><td>Santa cruz</td><td>sc-365186</td><td>immunofluorescent staining</td></tr><tr><td>Microscope</td><td>Olympus&amp;nbsp;</td><td>SZX7-1063</td><td>using for dissection</td></tr><tr><td>Microscope</td><td>Olympus&amp;nbsp;</td><td>IX71</td><td>Used for observing the results of immunofluorescence staining</td></tr><tr><td>MitoTracker Red CMXRos</td><td>Invitrogen</td><td>M7512</td><td>ROS staining</td></tr><tr><td>Neonatal Mouse Pups (P0-P3)</td><td></td><td></td><td>provided by BIONT</td></tr><tr><td>PBS</td><td>Servicebio</td><td>G4202</td><td>immunofluorescent staining</td></tr><tr><td>Rabbit Anti-Mouse CK18 Monoclonal Antibody</td><td>Proteintech</td><td>10830-1-AP</td><td>immunofluorescent staining</td></tr><tr><td>Senescence &amp;beta;-Galactosidase Staining Kit</td><td>Beyotime</td><td>C0602</td><td>aging staining</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td>immunofluorescent staining</td></tr></tbody></table>

isolation and culture of primary marginal cells of the stria vascularis from neonatal mice

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 &#946;-galactosidase (SA-&#946;-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.

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...

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.

The stria vascularis is a key structure in the inner ear responsible for generating and maintaining the endocochlear potential (EP), and its dysfunction ...

We established a primary marginal cell model to investigate stria vascularis dysfunction in hearing loss. The protocol can be applied to investigate stria vascularis dysfunction in age-related, noise-induced, and hereditary hearing loss. To begin, prepare the dissection tools, including two pairs of micro-dissection forceps, one pair each of standard forceps, surgical scissors, and micro-dissection scissors.Sterilize all the tools using an autoclave and dry them at 60 degrees Celsius for four hours, then place the sterilized instruments in an alcohol beaker placed in the biological safety cabinet. Using 75%ethanol, disinfect the stereo microscope stage and housing thoroughly. Place the disinfected stereo microscope inside a biological safety cabinet and expose all items to ultraviolet light for 30 minutes to ensure complete sterility before initiating the dissection procedure.Carefully disinfect the euthanized mouse pup using 75%ethanol. Place the disinfected pup on a six-centimeter dish inside the biosafety cabinet for dissection. Immobilize the head firmly with straight forceps.Using microdissection scissors, open the scalp along the sagittal suture and separate the scalp bilaterally with scissors to excise it completely. Next, using micro-dissection scissors, bisect the neonatal mouse head along the midline. With forceps, remove the brain tissue carefully to expose the temporal bones.Observe the bilateral temporal bones at the cranial base and remove unnecessary surrounding bones while retaining the temporal bone tissue containing the cochlea. Transfer one temporal bone into a 35-millimeter sterile dish containing fresh Hanks'Balanced Salt Solution placed on ice. Transfer the other temporal bone to a sterile dish placed under the stereo microscope on ice.Using one pair of forceps, secure the temporal bone tissue firmly. Using another pair of forceps, perform blunt dissection to remove surface muscle tissue until the white semicircular ossicle ring becomes visible. Confirm the cochlear position visually and continue blunt dissection downward until translucent cochlear tissue is observed.Using microdissection forceps, carefully remove a small piece of the bone wall of the cochlea while keeping the tissue on ice. Gently insert one side of the forceps into the cochlea and carefully separate the bone wall from the membranous labyrinth. Remove the detached bone wall and extend the membranous labyrinth tissue into fresh Hanks'Balanced Salt Solution.Using one pair of forceps, secure the basilar membrane, then carefully separate the stria vascularis with another pair of forceps. Repeat the harvesting procedure on the contralateral side. Transfer the isolated stria vascularis into a 1.5-milliliter micro-centrifuge tube containing one milliliter of collagenase type II solution placed on ice.Place the sample in a 37 degrees Celsius incubator for 30 minutes. Next, using a centrifuge, spin the tube at 300G for eight minutes. Carefully remove the supernatant from the tube and add one milliliter of culture medium to the pellet.Gently titrate to terminate digestion and centrifuge the tube again at 300G for eight minutes. Next, aspirate and discard the supernatant. Add one milliliter of fresh culture medium to the pellet.Tritrate gently to obtain a single-cell suspension. Transfer the suspension into a 15-milliliter conical tube and add additional culture medium to adjust the total volume to four milliliters. Mix the cell suspension thoroughly by repeated pipetting.Transfer one milliliter of the suspension into each well of a six-well plate and add DMEM supplemented with 2%FBS, 1%EpiCGS-a, and 1%penicillin streptomycin. Place the plate in a 37 degrees Celsius incubator with 5%carbon dioxide to culture the cells. Replace the medium daily.To replace the media, aspirate the old medium from the well using a pipette. Add fresh Hanks'Balanced Salt Solution to wash and then aspirate it. Add fresh culture medium.Monitor the cell status regularly under a microscope. Observe the clustered growth of marginal cells approximately two days after plating. On day three, identify numerous spindle-shaped fibroblasts surrounding the cobblestone-like clusters of marginal cells.Using a one-milliliter pipette, gently pipette to partially remove the spindle-shaped fibroblasts based on their different adhesion rates. Gently flush D-Hanks'Balanced Salt Solution over large areas in each of the four quadrants of the culture dish three times to achieve uniform fibroblast distribution across the dish. After each round, examine the fibroblast density microscopically and apply localized gentle pipetting to densely populated fibroblast areas to achieve a sparse and uniform distribution across the culture dish.Using a pipette, remove the spent medium and add two milliliters of fresh culture medium to continue cultivation. Perform imaging following immunostaining marginal cells with anti-CK18 18 and KCNQ1 antibodies and treat the marginal cells with D-galactose to induce cellular senescence. On day two after cell purification, immunofluorescent staining revealed CK18 and KCNQ1 expression in the cultured cells.The half-maximal inhibitory concentration for hydrogen peroxide was determined from the dose response curve after assessing cell viability at different hydrogen peroxide concentrations using the CCK-8 assay. Compared to the untreated control group, treatment with one millimolar hydrogen peroxide markedly increased intracellular green fluorescence, indicating increased reactive oxygen species accumulation. Degalactose-treated marginal cells showed a significant increase in senescence-associated beta-galactosidase-positive staining, indicating cellular senescence compared to untreated controls.The key challenge while performing this protocol is preventing cell damage during dissection and optimizing purification, timing, and proportion. Researchers can perform ROS detection, senescence staining, electrophysiology, as well as Western blot and a qPCR analysis using this technique. Future studies can explore co-culture systems on growth factor, optimization, and the immortalization of marginal cells.

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Learn to isolate and culture primary marginal cells from neonatal mouse stria vascularis using enzymatic digestion and differential plating. This method enables in vitro modeling of oxidative stress and cellular senescence for hearing loss research.

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

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