The overall goal of this procedure is the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants to be used as an in vitro screening tool in hearing research. This method can help answer key questions in the hearing research field, such as enabling the simultaneous study of cochlear hair cells, synapses, neurites, and neurons in an ex vivo model and providing insight into mechanisms of action of molecules new to the cochlea, including factors found in human secretions. The main advantage of this technique is that the screening process of candidate therapeutic compounds can be accelerated prior to their testing in vivo.
Begin with a freshly collected neonate head contained in 70%ethanol in the lid of a Petri dish. Use a scalpel blade to make a superficial cut in the skin from the anterior to the posterior end of the skull. Then cut both external auditory canals and fold the skin anteriorly to expose the whole cranium.
Next, open the cranium along the sagittal suture by gently moving the scalpel blade up and down from anterior to posterior to avoid compression of the cochlea. Remove and discard the snout by making a vertical cut directly posterior to the orbits. The posterior part of the skull should still contain the brain and cochlea.
Then use 4 forceps to bluntly dissect and dispose of the forebrain, cerebellum, and brain stem. Place the two halves of the cranium into a 60 millimeter plastic Petri dish filled with cold HBSS. Then use 4 forceps to completely expose the cochlea which is adjacent to the surrounding stapedial artery and bluntly separate the organ from the temporal bone.
Transfer the cochlea into a 50 millimeter clear walled glass bottom Petri dish containing HBSS and position the dish under the microscope. Place the 60 millimeter plastic Petri dish with the other half of the cranium on ice. Next, use 4 forceps to carefully dissect the cochlear otic capsule.
Now, use a micro knife or two 55 forceps to carefully separate the spiral ligament that is adhered to the organ of Corti from the rest of the cochlea and the medialis. This can be done by grasping the spiral ligament by the most basal aspect and gently unwinding it while moving apically. After the spiral ligament is removed, position the specimen to obtain a longitudinal sagittal view of the cochlea.
Then cut the cochlea into two or three sections using the micro knife, classifying these sections as the apical and basal turns. Remove the tissue superior for the more apical piece and inferior for the more basal piece, down to the actual layer of hair cells and neurites to allow the cochlear explant to lay horizontally on the Petri dish. Next, use forceps to gently grasp and peel away the tectorial membrane, a very thin translucent layer immediately superior to the organ of Corti.
The most technically challenging step is likely the removal of the thin translucent tectorial membrane as one can easily damage the hair cells underneath. By starting from the edges of the tissue, hair cells along the length of the explant are preserved, except at the very edge where peeling of the tectorial membrane began. Lastly, remove Reissner's membrane immediately superior to the spiral ganglion neurons by touching it with forceps on both sides and peeling it away.
The cochlear specimen is now ready for culturing after removing any damaged areas on the edges. Begin the culture procedure by wetting the tip of a one milliliter pipette with approximately 120 microliters of DMEM to prevent the explant from adhering to the inside of the tip. Then use the pre-wetted tip to pipette a maximum of 70 microliters from the glass bottom Petri dish and place the specimen along with the liquid on one of four inlays in the four-well Petri dish.
Check under the microscope that the explant is in the correct orientation. Ensure that the area of hair cells from which the tectorial membrane was removed faces upwards and that the basilar membrane together with the trimmed basal part of the medialis faces downwards and adheres to the coverslip inlay. If the specimen is orientated upside down upon inspection, as shown here, add HBSS from the pipette tip and reposition the piece until it is correctly oriented.
Once the explant is correctly oriented, aspirate excess HBSS then wait for 15 seconds. Now, pipette 60 microliters of warm culture medium directly unto the specimen, taking care not to touch the specimen with the pipette. Replace the inner and outer Petri dish covers and incubate overnight at 37 degrees Celsius.
The next day, transfer the cochlear explant containing Petri dishes from the incubator to the laminar flow hood. Aspirate the old culture medium which does not contain FBS from the inlay. Add 60 microliters per well of the prepared warm treatment medium.
Place the Petri dish back into the incubator until staining of the cultures after a maximum of seven days. As seen in this confocal image, this technique preserves the anatomy of the entire cochlear turn including the spiral ganglion neurons or SGNs, allowing analysis of treatment effects on neurites and somata of SGNs in addition to the organ of Corti. As seen here, these explants can be used to simultaneously compare the effect of treatment on apical and basal turns after immunofluorescent staining.
Hair cells are shown in green and neuronal structures appear red. Intact synapses can be assessed by labeling pre-synaptic proteins shown in red and post-synaptic proteins shown in green together with the staining of neuronal structures in blue. This figure shows representatives images of apical and basal cochlear explants after seven days in culture with either 3%FBS on the left or 1%FBS on the right.
Cells incubated in 3%FBS start to migrate between days four and five as shown by the light gray arrows for hair cells and the red arrows for neurites. Explants in 1%FBS maintain organizational integrity until day seven. While attempting this procedure, it's important to remember to visually ensure the structural integrity and orientation of the cochlea tissue before adding the final culture media and substances of interest.
Following this procedure, 3D reconstruction can be performed to confirm cell type specific localization of GFP expressing cells within the medialis.
