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 |