Table 2 -.

Troubleshooting

Steps	Problem	Possible Reason	Solution
1–10	ES cells do not attach to the plate	Insufficient gelatin coating	Re-plate the cells on a new plate with fresh gelatin coating
		Cells are not yet adapted to LIF-2i medium	Perform several passages
11–28	Epithelium does not form or thicken over time in control samples	Ectodermal medium was not made properly	Check reagent expiration dates; Make new Ectodermal medium
		Matrigel was not mixed into medium properly	Make sure that Matrigel is added to Ectoderm differentiation medium during preparation at 4°C.
29–31	Epithelium does not thicken and/or ruffle	One of the small molecules or proteins has expired or is not present at an optimal concentration	Test each treatment separately and replace small molecule or protein stocks
		Matrigel concentration is not optimal	Reduce/Increase Matrigel concentration
29–31	The aggregates themselves or the medium color in wells on the edge of the plate look different from the rest of the plate	Disproportionate evaporative loss in the edge wells	Check that incubator is properly humidified
			Only analyze aggregates from interior wells
32–40	Cellular rearrangement does not occur during days 8–12	Cell aggregates were transferred to floating culture too early/late	Transfer aggregates at a different time point between days 7–8.5
32–40	No Pax2, Pax8, Sox2, Ecad+ vesicles develop by day 12	Improper cellular rearrangement	Transfer aggregates at a different time point between days 7–8.5
		Carry-over of signaling molecules/proteins from day 0–8 culture	Wash aggregates with DMEM/F12 at least 3 times prior culture in maturation medium
			Check that proper morphology is observed during days 0–8
		Aggregates are damaged during transfer to maturation medium	Use wide-mouth or cut P1000 tips or a sterile transfer pipet to manipulate cell aggregates
29–40	Excessive development of beating myocytes, cartilaginous masses and adipose apparent between days 14–20	LDN-193189 concentration was suboptimal; insufficient suppression of mesenchyme development	Increase the concentration of LDN-193189 (1 μM should be sufficient)
32–40	Large translucent organoids do not develop by day 18–20	Too many aggregates were cultured together	Reduce the number of aggregates in each well on 24-well plates
		Matrigel concentration is too high during floating culture	Reduce Matrigel concentration to 0–0.5% at the start of floating culture
41–51	No hair cells observed in cyrosectioned organoids	Incomplete analysis	Ensure that the entire organoid is analyzed; cyrosections through nonsensory regions give the appearance of no hair cells