This protocol incorporates a straightforward approach to streamline culture steps, obtaining high-quality explants and will contribute to exploring and unveiling molecular mechanisms of cochlear cells. The main advantage of this technique is minimal direct organ handling by coordinating the dissection steps, appropriate polymer surface coating, optimized medium volume, and attachment time of the explants. This technique represents a popular in-vitro approach to study sensorineural hearing loss.
This can be used to establish ototoxic models, perform structural analysis, evaluate signaling pathways, and conduct drug screening studies. In principle, this method can apply to different tissues explants. It can also be employed in inner ear research to gain insight into embryonic developmental stages.
To begin, place the decapitated rat's head on the sterile pad. Use scissors and forceps to remove the mandible, then lift the skin and peel it back from the skull. Place the forceps in the orbital cavities to hold the skull.
Using a sharp scalpel blade, carefully cut the skull along the sagittal suture and then the coronal suture area without damaging the cochlea. Now, remove the brain from the two skull halves. Under the microscope, localize the cochlea in the temporal bone, and loosen the surrounding tissue between the cochlea and the temporal bone.
Then place the forceps in the superior semicircular canal. Gently pull the temporal bone away from the cochlea and hold the cochlea attached to the vestibule with the temporal bone. Insert the forceps tips into the apex region which is visible as a white line, and remove the cartilaginous cochlear capsule piece by piece to expose the cochlear duct.
Place the forceps under the cochlea, and detach them from the vestibular organ and temporal bone. Transfer the cochlea into a new 60-millimeter dish containing ice-cold PBS. To isolate Corti explant organs, hold the organ at the base and the cochlear duct at the basal hook region, and gently unwind cochlear duct from the modiolus without tearing it.
Then hold the cochlear duct at the base and pull away the spiral ligament and stria vascularis. For isolating cochlear explants, use forceps to detach the spiral ganglion from the osseous spiral lamina, and unwind the modiolus during the detachment. With forceps, grasp the hook region and remove the spiral ligament with the stria vascularis.
Further, pull off Reissner's membrane piece by piece. To transfer the cochlear explants, dip a laboratory spatula into the dish containing the explant. Lift the explant with the hair cells facing up along with a few microliters of PBS by waving a spatula.
Place the explant into an eight-well chamber slide by gently waving the spatula in the medium. Allow the explant to slide into the chamber. Using a 100-microliter pipette, remove 80 microliters of medium and discard it.
Check the visibility of hair cells and spiral ganglion neuron cells under the microscope. If necessary, use forceps to push apart overlapping tissue. Remove the remaining medium from the well.
The explant will now lie flatter on the surface of the chamber. After 10 seconds, pipette back one or two drops of the medium just next to the explant, and the remaining medium some distance away to avoid explant detachment. Ensure the explants do not move while adding the medium.
Incubate the chamber for two hours to attach the organs firmly to the chamber's bottom. After incubation, aspirate the medium. And using a 100 or 200-microliter pipette, carefully add 300 microliters of fresh, pre-warmed complete medium.
The spinning disc confocal microscopic images showed that the rat Organ of Corti explants maintained their structure and total length under both normal and stressed conditions. In addition, a scanning confocal microscope demonstrated surviving hair cells of explants from the mice organs along with hair cells undergoing apoptosis. The protocol enabled the monitoring of biological processors in living cochlear cells using appropriate cell permeable probes and a spinning disc confocal microscope.
The culture of these cochlear explants enhances the analysis of the interaction between hair cells and spiral ganglion cells. The hair cells of cochlear implants were detected with the hair cell marker, MYO7A. Similarly, healthy and damaged spiral ganglion cell bodies and neurites were identified using the neuronal marker, Tuj 1.
In the magnified overview, the cell bodies of the hair cells labeled with MYO7A antibody were visible. Outer hair cell stereocilia, and deconvoluted images of individual inner hair cell stereocilia labeled with phalloidin were identified. Care must be taken during the procedures under the microscope, especially when separating the spiral ganglion from the boney spiral lamina.
The intact explants should be transferred and aligned with care. This protocol optimized in-vitro studies on inner-ear explants. The outcomes of these studies, such as signaling pathways and drug toxicity can guide the design of farther in-vivo studies.
