Differentiating Immortalized Multipotent Otic Progenitors into Spiral Ganglion Neurons and Evaluating the Differentiation

1. Differentiating iMOP-Derived Neurons

1. Make 50 ml of neuronal differentiation media: Neurobasal media, 1X B27 supplement, 2 mM L-Glutamine. Thaw bottle of 50X B27 and 200 mM L-Glutamine in 37 °C water bath for 5 min. Add 1ml 50X B27 and 0.5 ml 200 mM L-Glutamine to 48.5 ml of Neurobasal media.
2. Coat coverglass by placing 12 mm round 1.5 glass coverslips in a sterile 10 cm plate and add 70% ethanol (EtOH) to the plate to sterilized and clean the coverslips.
3. Gently agitate the coverslips to ensure they are covered in ethanol. Leave the plate for 10 min at RT. Rinse the coverslips 3 times with sterile 1X phosphate-buffered saline (PBS) to wash out the remaining ethanol. Rinse the coverslip once with sterile H₂O to wash out the remaining 1X PBS.
4. Aspirate the H₂O using a 2 ml aspirating pipette and let the coverslips dry. Expose the coverslips to UV light in the tissue culture hood for 15 min. Store coverslips in a sterile environment if not immediately used.
5. Place one 12 mm round coverslip in each well of a 24 well dish. Shake plate gently to ensure that the coverslips lie flat on the bottom of the well.
6. Thaw 2 mg/ml poly-D-lysine stock solution at room temperature (RT) and dilute to a 10 µg/ml poly-D-lysine concentration in 1X PBS. Add 0.25 ml of 10 ug/ml poly-D-lysine to the wells. Leave the plate in the 37 °C incubator for 1 hr.
7. Wash the wells 3 times with sterile 1X PBS. Thaw 10 mg/ml laminin stock solution at RT and dilute to 10 µg/ml in 1X PBS. Add 0.25 ml of 10 µg/ml laminin working solution into a single well of a 24 multi-well dish. Incubate the plate at 37 °C incubator. Aspirate out the laminin solution using a 2 ml aspirating pipette.
8. Wash the coverslip 3 times with 1X PBS by adding in 1 ml of 1X PBS with a P1000 pipette and removing the 1X PBS by aspirating with a 2 ml aspirating pipette. Leave 1X PBS from last wash in the well until cells are ready to be plated. Initiate neuronal differentiation by harvesting, dissociating and counting cells. Plate 1 x 10⁶ cells in a 6 cm dish at Day -3.
9. On Day 0, harvest, dissociate and count cells. Seed 1 x 10⁵ – 1.5 x 10⁵ iMOP cells into 0.5 ml pre-warmed neuronal differentiation media per well in a 24 multi-well dish. Aspirate and add pre-warmed neuronal differentiation media to cultures every other day.
10. Fix iMOP-derived neurons in 4% formaldehyde for 15 min. at RT on Day 7. Remove formaldehyde solution, wash iMOP-derived neurons with wash buffer (1X PBS containing 0.1% TritonX-100) before incubating in blocking buffer (1X PBS containing 10% normal goat serum and 0.1% Triton X-100) for 1 hr.
    1. Replace buffer and incubate samples in blocking buffer with diluted antibody. Incubate at 4 °C. Wash samples with 1X PBS containing 0.1% Triton X-100 subject cells to immunostaining. Wash the coverglass with 1X PBS before placing onto mounting media. Allow the mounting media to dry at 4 °C before acquiring epifluorescence images.