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 300G for five minutes. Using a 1000 microliter pipette tip, re suspend 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 adherence 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.
