TABLE 3 |.

Troubleshooting table.

Steps	Problem	Possible reason	Solution
Box 1, step 6	Strong friction	Spin shaft is not smooth The teeth of gears are not perfectly aligned	Eliminate small protrusions by sanding Redo 3D printing Take off the gears and repeat steps 3–5
Box 1, step 16	Large noise when operating	Misalignment of assembly	Check assembly and dimensional precision of 3D-printed parts
	Motor dies	DC motor has a limited life span inside an incubator	This is normal after ~3 months of continuous operation. Monitor the motor daily for signs of failure, including slowing of rotational speed and jerky movements, and replace the motor periodically (every 1–2 months) to minimize the chance of motor failure
	Motor dies after less than a month	Humidity enters the motor	If motors other than the indicated one are used, make sure to use sealed motors and prevent liquid spills
		Strong friction	Check assembly and dimensional precision of 3D-printed parts
	Set screw rust	Liquid spilled onto the screw	Avoid spills. Use medical-quality stainless-steel screws instead
Box 1, step 17	Contamination inside the bioreactor	Long-term culture increases the risk of contamination 3D- printing defects increase the risk of contamination	We recommend having extra SpinQ units for routine sterilization Avoid using parts with crevices and protrusions from 3D printing
1	Colonies do not detach after 90 min of incubation	Collagenase was not fresh hiPSCs show signs of differentiation	Prepare collagenase solution right before use Check the morphologies and pluripotency of hiPSC colonies
8	Large variation in EB sizes	hiPSC colony size has large variation	Break iPSC colonies into similar-sized clumps during passaging. Let the larger clumps sink to the bottom of a microcentrifuge tube and remove smaller clumps from the top
10	EBs stick to each other	EBs are not distributed with enough spacing inside the plate	Gently shake the plate when placing it into the incubator. Make sure that the incubator shelf is horizontal and that the EBs do not sink to one side. When sticking starts to occur, pipetting the EBs with the medium twice up and down using a 5-ml pipette usually separates them with no damage to the EBs
11	Suboptimal EB morphology	EBs were damaged during handling hiPSC culture quality was suboptimal	Make sure to transfer the colony and perform medium changes gently to avoid disturbances Check the morphology and pluripotency of the hiPSC colonies. Start from earlier passages of hiPSCs
20	EBs fuse with neighbors during expansion in Matrigel	Insufficient spacing between EBs	Do not put too many EBs into one Matrigel cookie. Mix the EBs inside the microcentrifuge tube extensively. If necessary, use a sterile P200 pipette tip to gently push the EBs to fine-tune the spacing
21	No neuroepithelium formation	EB quality was suboptimal Lot batch-to-lot batch variations in Matrigel	Check EB quality and iPSC quality Try another lot batch of Matrigel
22	Organoids are still attached to Matrigel after extensive pipetting	Neural processes from differentiated cells cling onto Matrigel	Need to obtain pure neuroepithelium formation with minimal differentiation. Check EB and iPSC quality
		Incorrect dilution of Matrigel in Step 16	Make sure to dilute Matrigel with medium 3:2 as more-concentrated Matrigel is difficult to detach
		Lot batch-to-lot batch variations of Matrigel	Use another lot batch of Matrigel
	Neuroepithelium was damaged	Damage was caused during pipetting	Control the speed of pipetting and avoid air bubbles. Frequently check under the microscope to determine if sufficient pipetting has been performed
29	Organoids appear unhealthy and darkened after spinning	Spinning direction is opposite of intended spinning direction	Check the spinning direction. The opposite direction of spin pushes the organoid down and limits oxygen delivery. Refer to Box 1 and Supplementary Video 1
	Organoids form aggregates after spinning	Low spinning speed	At low spinning speeds, organoids may gather at the center of the well underneath the bottom of the spin shaft and form aggregates. Increase the speed until organoids are lifted in constant suspension
		Chunks of Matrigel remain in the medium	Perform the procedures in Step 24 to remove the remaining Matrigel
		DC motor stopped for a prolonged time	Keep an eye on the bioreactor and replace broken or worn-out motors
30	Medium color turns noticeably yellow	Organoids inside the well are overcrowded	Divide organoids into multiple wells