Day 1. Sterilization and coating of glass coverslips.

1. Dry sterilize glass microscope coverslips in an autoclave.
2. Place the sterilized coverslips into two wells of a pre-sterilized four-well cell culture dish.
3. Coat the coverslips with 1:1 poly-L-ornithine and laminin supplemented with 20% Fetal Bovine Serum (FBS) overnight at 4 °C.
   1. 400 μL poly-L-ornithine (0.01% solution stored at 4 °C)
   2. 400 μL laminin (50 μg/mL stock solution stored in aliquots at -20 °C)
   3. 200 μL FBS (stored in aliquots at -20 °C)
4. Heat-sterilize the dissection tools overnight in a 150°C incubator.
5. Make culture medium containing 10% serum and 10 mg /mL ampicillin.
   1. 90 mL Dulbecco's Modified Eagle Medium
   2. 5 mL FBS (stored in aliquots at -20 °C)
   3. 5 mL horse serum (stored in aliquots at -20 °C)
   4. 10 μL ampicillin (10 mg/mL stock solution stored at 4 °C )

Day 2. Isolation of the organ of Corti.

1. Sterilize the positive flow dissection hood.
   1. turn on UV light for 20 min
   2. spray all surfaces with 70% ethanol and wait 5 min before use.
2. Decapitate mouse pup (P4) at the base of the foramen magnum using operating scissors.
3. Briefly rinse the head in 10 cm dish containing 70% ethanol.
4. Remove the epidermis using a scalpel blade.
5. Open the cranium along the sagittal suture using a scalpel blade and then bisect the forebrain. Retain the caudal forebrain for further dissection.
6. Remove the forebrain, cerebellum and brainstem using blunt dissection.
7. Remove the temporal bones (fig 2A), dip them briefly in 70% ethanol, and transfer them to a 3 mm dish containing sterile HBSS.
8. Using forceps, remove the bulla and surrounding tissue from the petrous portion of the temporal bone.
9. Locate the conch shaped cochlea (fig 2B) and separate it from the vestibular system using forceps.
10. At this stage of development, the bony labyrinth is not completely calcified and is easily dissected using forceps. Remove the bony labyrinth of the cochlea by careful separation starting at the basal end and moving apically using forceps.
11. The spiral ligament and attached organ of Corti is coiled along the spiral of the modiolus (fig 2C). Carefully remove the organ of Corti by securing the spiral ligament at the hook region of the base using forceps and unwinding it as you move apically.
12. Starting at the base, remove the spiral ligament from the organ of Corti using #55 fine forceps (fig 2D).
    Micro-isolation of the organ of Corti sensory epithelium (Optional).
13. Remove the organ of Corti hook region of the base using two ½ cc Insulin Syringes with permanently attached U-100 28G½ needles as forceps.
14. Starting at the apex, remove the spiral limbus from the row of inner hair cells and proceed basally (fig 2E-F).
    Plating the organ of Corti explant
15. Remove the poly-L-ornithine/laminin/FBS solution from the culture wells and add 130 μl of culture medium.
16. Transfer the dissected organ of Corti to the coated glass coverslip in the culture well and orient the explant so that the cilia of the hair cells are pointing up.
17. Remove the medium in the culture well using a 200 μL pipette. Make sure that the basilar membrane makes solid contact with the coated glass coverslip.
18. Carefully add 130 μL of culture medium to the organ of Corti using a 200 mL pipette. Apply two drops onto the surface of the organ of Corti and then slowly add the remaining volume to the side of the coverslip. Make sure that the organ of Corti does not float in the culture media, but remains affixed to the coverslip.
19. Incubate overnight at 37 °C in the presence of 5% CO₂.

Day 3. Electroporation of the reporter gene into cultured organ of Corti.

1. Remove the culture medium from the organ of Corti culture.
2. Add 130 μL H₂O for 1 min and then remove using a 200 μL pipette.
3. Add 30 μL of DsRed reporter plasmid (2mg /mL H₂O stored at -20 °C).
4. Advance the electrodes of the electroporator using a micromanipulator so that the anode and cathode are on either side of the culture.
5. Generate a pulse (27V, 30 ms duration, 10 pulse trains) to electroporate the reporter gene into the organ of Corti explant culture.
   1. Optional: reverse the polarity of the pulse to ensure electroporation of the transgene on both the modiolar and spiral ligament sides of the explant.
6. Wait 5 min.
7. Add 130 μL of the Fugene 6: DNA solution (3 parts Fugene to 2 parts DNA). This solution should be prepared prior to the start of the electroporation procedure (step 20) using a 3 mL round bottom polystyrene test tube in a laminar flow hood. To make this solution:
   1. Add 2.4 μL of Opti-MEM (stored at 4 °C) to the test tube.
   2. Add 0.6 μL of Fugene 6 reagent (stored at 4 °C) to the test tube. Make sure to add the Fugene directly to the Opti-MEM and avoid direct contact with the sides of the test tube.
   3. Vortex for 1 second.
   4. Incubate 5 min at room temperature.
   5. Add 2.0 μL DsRed reporter plasmid double stranded DNA (stored at -20 °C at 100 μg DNA/mL H₂O aliquots).
   6. Vortex for 1 second.
   7. Incubate 15 min at room temperature.
   8. Add 200 μL of culture medium.
   9. Vortex for 1 second.
8. Incubate overnight at 37 °C in 5% CO₂.
9. Add 2 mL culture medium to the culture well and incubate for 37 °C for up to 10 days.

Representative Results

We present a method for the isolation of the organ of Corti from a perinatal mouse. The procedure can be used for mice as young as embryonic day 16 up to about postnatal day 6, at which point the bony labyrinth becomes sufficiently calcified to render the dissection cumbersome. Once the organ of Corti is dissected, it may be plated and cultured either in its entirety (fig 3) or as micro-isolated sensory epithelium (fig 4). We have further presented a technique to express exogenous genes in the cultured organ of Corti. The organotypic culture is useful for many other types of study, such as the analysis of organ of Corti gene expression using RT-PCR or in situ hybridization, organ of Corti co-culture with spiral ganglion cells or exogenous stem cells using the micro-isolate³, or in vitro analysis of hair cell death and regeneration.

Figure 1. Cross section of the P4 murine organ of Corti. (A) A cross section from the basal turn of a cryosectioned cochlea obtained from a P4 mouse illustrates the general structures of the murine cochlea described in this protocol. The scala media is bordered by the spiral ligament and stria vascularis laterally, the Reissner s membrane superiorly, the spiral limbus medially, and the basilar membrane inferiorly. The box indicated the region expanded in B. (B) The organ of Corti is located on the superior side of the basilar membrane and includes one row of inner hair cells, three rows of outer hair cells, and their respective supporting cells. Dashed line 1 indicates the location along the basilar membrane that is removed during this organ of Corti dissection. Dashed line 2 indicates the location along the basilar membrane that is removed during the micro-isolation procedure. Green indicates immunohistochemical labeling of calbindin, which labels interdental cells of the spiral limbus, cochlear hair cells, spiral ganglion neurons, as well as cells of the spiral ligament and stria vascularis ⁷. DAPI labeling of nuclei is in blue.

Figure 2. Organ of Corti dissection. Images from the accompanying video of the organ of Corti dissection highlight A) the cochlea and vestibular system located within the isolated temporal bone (red), B) the bony labyrinth of the cochlea, C) the spiral ligament and attached organ of Corti after removal of the bony labyrinth, D) removal of the spiral ligament and stria vascularis (red) from the organ of Corti, E) micro-isolation of the sensory epithelium from the spiral limbus (red), and F) the isolated spiral limbus (left) and sensory epithelium (right).

Figure 3. Cultured organ of Corti explant. DIC image showing the organ of Corti of a P4 Atoh1-nGFP mouse that was isolated, plated, and cultured for five days as described. This mouse has been genetically engineered so that cells that express the pro-hair cell gene Atonal homolog 1 (Atoh1 aka Math1) exhibit a green fluorescent protein that is localized to the nucleus⁸. The organs of Corti from these mice exhibit a nuclear GFP label in all hair cell nuclei and therefore allow for easy visualization of the sensory epithelium using an epifluorescent microscope. The relatively large spiral limbus can be seen lateral to the sensory epithelium. Mesenchymal cells that have originated from the organ of Corti have migrated away from the explant. Blue nuclear label is DAPI.

Figure 4. Micro-isolated sensory epithelium. Epifluorescent image obtained from the sensory epithelium that has been isolated from a P4 murine organ of Corti as described and cultured overnight. The micro-isolate was then fixed in 4% paraformaldehyde and processed for immunolabeling of Myosin 7a which labels cochlear hair cells. Note the absence of the comparatively larger spiral limbus from the figure 3. Blue nuclear label is DAPI.

Figure 5. Electroporation of DsRed reporter gene into the cultured organ of Corti. Whole organs of Corti from P4 Atoh1-nGFP mouse pups were isolated, plated, and then electroporated with the DsRed reporter gene as described and then viewed under an epifluorescent microscope. Cells of the organ of Corti explant that expressed the transgenic DsRed reporter protein exhibit a red fluorescence and endogenous cells of the sensory epithelium exhibit a green nuclear fluorescence. Transgenic cells can be seen throughout the spiral limbus and sensory epithelium.