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

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.