Table 4 |.

Troubleshooting table

Step	Problem	Possible reason	Solution
19	The tissue is not noticeably digested after 2 h	Digestion was not suffcient	Centrifuge the 15-ml Falcon tube at 450g for 5 min at 8 °C and repeat Steps 16–19
40	Expansion medium appears very yellow (and dead cells are present in the culture)	Plating density is too high	Passage organoids at lower density
		Insufficient expansion medium	Refresh expansion medium more often and/or use 1 ml of expansion medium per well
	BME drops are floating	The drops were not properly attached to the plate	Skip Step 51 and/or incubate for 30 min in Step 50
45, 86	The organoid pellet after centrifugation is fuzzy and contains undissolved BME	The TrypLE treatment was insufficient to dissolve the BME, or the amount of adDMEM/F12+++ was insufficient to wash away the BME, or the adDMEM/F12+++ was not cold enough to aid dissolving the BME	(1) Resuspend the pellet in 1 ml of TrypLE, incubate for 5 min at RT and wash with adDMEM/F12+++; or (2) wash once more in >10 ml of adDMEM/F12+++ following Steps 44–46; or (3) place the tube on ice for 10 min, resuspend the well using a 10-ml pipette and centrifuge at 300g for 5 min at 4 °C
	The organoid pellet after centrifugation is smaller than expected	Single organoid cells or fragments are stuck to the side of the tube	Add 2% (vol/vol) FBS to the tube, resuspend the well using a 10-ml pipette and centrifuge at 300g for 5 min 4 °C
53	The culture is growing slowly or reduces growth rate	Organoids are growing at too low density	Increase plating density by passaging with low split ratio (sometimes lower than 1:1, thus plating in a smaller total volume of BME compared to the previous passage)
	The organoid culture contains a lot of dead cells/cell debris	Passaging conditions are too harsh	Passage with milder conditions by reducing TrypLE concentration, incubation time or times of resuspension
	Culture is changing in growth speed or morphology	The culture is cross-contaminated with a different culture	Avoid cross-contamination by following the critical steps at 41–43 and 49 Perform a SNP analysis (see ‘Human material’ section) to check for culture purity
			Discard the culture and continue with an early passage
65A(x)	Lipofectamine 2000 transfection efficiency is low	The specific organoid culture is difficult to transfect	Try electroporation or select a different organoid culture
			Try electroporation
		The plasmid is difficult to transfect (e.g., too large)
65B(v), (ix)	Organoid survival after transfection by electroporation is low	The settings are too harsh for the cells	Use lower voltage and pulse length settings
		The specific organoid culture is sensitive to electroporation	Use a different organoid culture or try Lipofectamine 2000-based transfection
	Transfection efficiency after transfection by electroporation is low	The settings are too mild	Use higher voltage and pulse length settings
65C(iv)	Organoid survival after lentiviral transduction is low	MOI is too high	Lower the MOI by adding less virus or by increasing the number of organoid cells (up to 300,000)
		The specific organoid culture is sensitive to lentiviral transduction	Use a different organoid culture
	Transduction efficiency is low	MOI or incubation time is too low	Increase the MOI (up to 10) by adding more virus or by decreasing the number of organoid cells (>50,000) or increasing incubation time
77	Low efficiency of clone outgrowth	Passaging conditions were too harsh	Reduce TrypLE incubation time or times of resuspending
		Organoids were small while picking	Grow larger organoids (e.g., >300 μm in diameter) before picking