Isolation and Culture of Primary Cochlear Hair Cells from Neonatal Mice

The inner ear is encased in the temporal bone, deep in the hardest bone of the body. Posing a challenging to investigations, the cultivation of primary hair cells is indispensable for investigating cochlear hair cells. This study aimed to discover a method for isolating and cultivating mouse hair cells.

We demonstrate the steps for isolating cochlear organs from neonatal mice and enzymatically detaching the abundant hair cells for individual studies. The nature of the cultured cells was confirmed using immunofluorescence staining. The auditory primary cultural method established by isolating living cells from cochlear organs and immediately culturing them pairs to be a valuable tool for extensive research on auditory system.

This method can enhance our understanding of the biological characteristics of in vivo cultured hair cells and demonstrate the efficiency of cochlear hair cell cultures establishing a solid methodological foundation for further auditory research. To begin securely hold the head of the euthanized newborn mice in place. Using micro operating scissors, open the scalp along the sagittal suture.

Then separate and immobilize the scalp bilaterally with the fingers. Use scissors to sever the cranium and flip it forward to access the skull. Employ a small periosteal elevator to extract the brain and then use the tip of the scissors to gently scrape the brain revealing the base of the skull.

Now examine the bilateral temporal bones at the skull's base. Employ scissors to incise the base of the skull along the midline. Then scrape off the skin and eliminate any unnecessary bone.

Preserve and transfer the temporal bones to 35 millimeters sterile Petri dishes filled with fresh HBSS. Next, use a pair of five number pointed forceps to eliminate the bola and surrounding tissue from the petrus portion of the temporal bone. Hold the forceps with one hand to stabilize the semi-circular part of the temporal bone.

Use the other hand to insert the lower tip of the forceps into the round window niche separating the lateral bone of the cochlea from the scale of vestibule. Carefully remove the petrus portion of the temporal bone without contacting the organ of corti epithelium. Then meticulously detach and extract the bony labyrinth of the cochlea starting from the basal to the apical end.

Using five number pointed forceps, carefully micro-isolate the sensory epithelium of the organ of Corti from the modiolus. Next, employ micro operating forceps to hold the spiral ligament and gently separate it from the Stria vascularis. Transfer the clean auditory epithelium using a 200 microliter pipette to a three millimeter sterile culture dish containing HBSS.

Place the isolated auditory epithelium in a 100 millimeter sterile Petri dish filled with HBSS for the subsequent preparation steps. To begin, isolate the auditory epithelium from the temporal bone of newborn mice and transfer it to 10 milliliters of fresh DMEM containing 0.25%Trypsin. Incubate the mixture at 37 degrees Celsius for 12 minutes.

Using a 200 microliter pipette tip, gently separate the hair cells from the basal lamina and other cells using an operating microscope. Then add another 10 milliliters of culture medium to inhibit the disaggregation. Filter the suspended cells through a 70 micrometer filter.

Collect the filtrate in a clean 50 milliliter tube and centrifuge it at 300 G for five minutes. Using a 1000 microliter pipette tip, resuspend the hair cells in five milliliters of culture medium by gentle pipetting. Now place a cover slip at the bottom of a six well plate in advance.

Count the cells and culture them at 10 to the sixth cells per milliliter density in the six well plate. Grow the adherent cells in two milliliters of DMEM at 37 degrees Celsius and 5%carbon dioxide. After one day of culturing the hair cells adhered firmly to the bottom of the dish.

By the third day, the number of cells had doubled.