Differentiating Otic Progenitor Cells Into Sensory Epithelial Cells Followed by Immunostaining

1. Maintaining Self-renewal in IMOP Cells

1. Prepare iMOP culture media: DMEM/F12, 1X B27 supplement, 25 µg/ml carbenicillin and 20 ng/ml basic fibroblast growth factor (bFGF). Make 50 ml of iMOP culture media using sterile reagents. Warm up 49 ml of DMEM/F12 in a 50 ml conical in a 37 °C water bath.
   1. Thaw 50X B27 supplement and filter-sterilized 100 mg/ml carbenicillin aliquots for 5 min in a 37 °C water bath. Thaw out 100 µg/ml bFGF aliquot at RT. Add 1 ml 50X B27, 10 µl of 100 µg/ml bFGF and 12.5 µl of 100 mg/ml carbenicillin into DMEM/F12.
2. Use 3 ml of media in a 60 mm tissue culture dish for culturing iMOP cells. Add fresh media to cultures every other day by doubling the volume of media. Ensure that the concentration of bFGF does not drop below 5 ng/ml.
   1. Culture iMOP cells at 37 °C with 5% CO_2. Passage cells in culture after 5 – 7 days as listed in the steps below. Transfer culture to a 15 ml conical using a 10 ml pipette tip.
3. Make 1 mM EDTA in HBSS by diluting 0.1 ml of 0.5 M EDTA pH 8.0 into 50 ml of HBSS. Pre-warm 1 mM EDTA made in HBSS in a 37 °C water bath.
4. Harvest cells by gravity sedimentation or centrifugation at 200 x g for 5 min at RT. For gravity sedimentation, place the 15 ml conical containing the cultures in the 37 °C incubator for 5 – 10 min. After that time, observe the otospheres collect at the bottom of the 15 ml conical.
   NOTE: Excessive centrifugal force can cause cell damage.
5. Carefully aspirate spent media using a 2 ml aspirating pipette without disturbing the cell pellet. Add 0.5 ml of pre-warmed 1 mM EDTA HBSS solution to the cell pellet. Using a P1000 pipette, gently pipet up and down 2 – 3 times. Place conical at 37 °C and incubate for <5 min to facilitate dissociation into single cells.
6. Gently swirl the cell solution to determine if otospheres are dissociated. If no otospheres sediment to the bottom of the conical, the cells are dissociated. The time of incubation may vary.
   NOTE: Prolonged incubation with EDTA will result in excessive cell death.
7. Remove cells from 37 °C, add 2 ml of media to neutralize and dilute the EDTA. Collect the cells by centrifugation at 200 x g for 5 min at RT. Aspirate out diluted EDTA using a 2 ml aspirating pipette and add 5 ml of 1X PBS to wash the cells.
8. Spin cells down at 200 x g for 5 min at RT and aspirate out 1X PBS using a 2 ml aspirating pipette. Resuspend the cells using a P1000 pipette in 0.5 ml of iMOP culture media by gently pipetting up and down 2 – 3 times.
9. Count cells using a microfluidic particle counter with the appropriate cassettes. A confluent plate of iMOP cells will contain ~6 X10⁶ cells. Dilute cells 1:100 in media and add 75 µl of cell solution into the cassette for counting. Plate 1 x 10⁶ cells in a 6 cm dish in iMOP culture media (~1:10 dilution). Passage iMOPs every 5 – 7 days.
   NOTE: Cells that form large otospheres or attach to the bottom of the tissue culture dish will differentiate. Cells will also die if the cultures are overconfluent.

2. Differentiating IMOP Cells into Sensory Epithelia

1. Prepare 50 ml of iMOP sensory epithelia differentiation media: DMEM/F12, 1X B27 supplement, 25 µg/ml carbenicillin. Make 50 ml of iMOP sensory epithelia differentiation media. Warm up 49 ml of DMEM/F12 in a 50 ml conical in a 37 °C water bath. Thaw 50X B27 supplement and 100 mg/ml carbenicillin aliquots for 5 min. in a 37 °C water bath. Add 1 ml 50X B27 and 12.5 µl of 100 mg/ml carbenicillin into DMEM/F12.
2. To harvest, dissociate, resuspend, and count cells, repeat steps 1.4-1.9
3. Plate 1 x 10⁶ cells in a 6 cm dish at Day -3 using iMOP culture media. On Day 0, transfer the cultures using a 10 ml pipette into a 15 ml conical. Collect the otospheres by gravity sedimentation, as stated in step 1.6. Aspirate out spent media using a 2 ml aspirating pipette and leave the otospheres on the bottom of the conical.
4. Gently add 2 ml of sensory epithelia differentiation media. Transfer otospheres to a 6 cm dish using a large bore 10 ml pipette.
   NOTE: Mechanical shearing from harsh pipetting can dissociate cells from the otospheres.
5. Add 2 ml of fresh sensory epithelial differentiation media to cultures every other day. If necessary, cells can be collected by gravity sedimentation and replaced media.
6. Collect otospheres at Day 10 by transferring to a 15 ml conical and allowing the otospheres to sediment as stated in step 1.6. Aspirate the spent media using a 2 ml aspirating pipette and leave the otospheres undisturbed.
7. Fix otospheres by incubating in 4% formaldehyde in 1X PBS for 15 min at RT. Remove the formaldehyde solution and wash otospheres with wash buffer (1X PBS containing 0.1% TritonX-100) before incubating them 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 and subject the otosphere to immunostaining. Transfer otospheres into 1X PBS and place otospheres into mounting media on a glass slide using a P1000 pipette. Put a coverslip over the sample and allow mounting media to dry at 4 °C.
8. Acquire epifluorescence images using an inverted microscope setup equipped with a 16 bit CCD camera and either a 20X 0.75 air or a 40X 1.3 NA oil immersion objective. Collect fluorescence from different color channels using the listed (excitation and emission) wavelengths: blue (377 ± 25 nm/447 ± 30 nm), green (475 ± 25 nm/540 ± 25 nm), red (562 ± 20nm/ and 625 ± 20nm) and infrared (628 ± 20nm/ and 692 ± 20 nm).