Troubleshooting.

Step	Problem	Possible reason	Solution
7	Fibers are not straight and are curly Fibers are clumped	Fibers have been shaved too much Fibers have not been separated well enough	Try a more upright angle of the scalpel blade in order to cut individual fibers off without shaving them or dragging the blade along the fibre Use a narrower angle in order to separate individual fibers while cutting Press down on the suture to splay the individual fibers before cutting
10	Cells are not coating the fibers and are clumped in the middle Microscaffold EBs are very round with fibers inside the sphere	Fibers are too long Fibers are too short	Make sure to cut fibers about 1mm in length Same as above
12	EBs are very dark	Lack of neuroectoderm EBs are too large	Check the quality of starting PSCs. PSCs grown in StemFlex or E8 tend to be more efficient at neural induction. Try the commercially optimized kit. Small molecules (dual SMAD inhibitors) may be needed. Check the accuracy of the cell counting
31	A lot of outgrowth of non-neural identities	Failed neural induction Too much neural crest	Same as above Try again, it may be an issue with the batch Try the commercially optimized kit
39	Buds remain small and underdeveloped Lack of formation of a cortical plate	Shaker speed is not optimal Tissue culture vessel or volume is not optimal Shaker speed or vessel is not optimal Matrigel is not fully dissolved	Be sure to adjust shaker speed depending on orbit diameter Use a 60mm dish with the shaker speed at 57rpm. A smaller or larger dish will result in non-optimal centrifugal forces Same as above Be sure to add Matrigel to cold media just before feeding to avoid Matrigel precipitation
51	The organoid tissue detaches from the agarose matrix during sectioning	Leftover Matrigel or media around the organoid can cause the tissue to detach	Remove all matrigel from the surface of the organoid prior to embedding Rinse the organoid in HBSS and incubate in warm agarose for a few minutes before placing in embedding mold and cooling
51	The organoid is damaged during sectioning	Sectioning speed and/or vibration frequency may be too high Necrosis has already developed in the core The organoid is too large	Decrease the sectioning speed and the frequency of vibration Section at earlier stages Section at earlier stages and do not let the organoids fuse
52	Difficulties handling sections and maintaining tissue integrity	Too much or too little agarose left around the organoid The bristles of the brush come into contact with the tissue	Cut the agarose around the organoid leaving ~2-mm space all around the organoid Orient the block on the specimen plate so as to minimize the travel distance of the blade Only manipulate the agarose surrounding the organoid section Use two scalpels to manipulate the sections
58	Poor ALI-CO health after sectioning Contamination has spread across multiple inserts	Too slow organoid transfer to agarose Too long incubation on ice The antibiotic-antimycotic solution used became spoiled The same tip was used to remove spent medium from multiple wells The repeated pipette combitip came into contact with a contaminated surface	Embed fewer organoids at one time and work quickly Embed fewer organoids at one time After thawing an aliquot do not refreeze and discard Thaw the antibiotic solution at 4 °C overnight For each well, use a new 1 ml tip to remove the medium Do not touch the side of the wells containing the ALI-COs or any other potentially non-sterile surface with the combitip
Box 3,step 13	Problems loading or expelling the micropipette	Bubbles in the micropipette that interfere with the vacuum	Use a 10 ml syringe to push the air bubbles out of the micropipette. Wash with PBS and ensure there are no further bubbles.