Table 2.

Troubleshooting

Problem	Possible reason	Solution	Step
Cells detach from the culture plate prior to Collagen I transfer	High density of the culture (culture detaches as a monolayer)	If detachment occurs before day 15, reduce the DE plating density (step 19) or replate the cells a few days earlier (steps 23 and 37)	30 and 39
		If detachment happens after day 15, increase the splitting ratio at step 37
	Loss or dysfunction of attachment substrate (cells detach as single cells or small cell clumps)	Replate the cells immediately into newly prepared 1% (vol/vol) growth factor reduced Matrigel-coated plates
		If Matrigel solution has been thawed and kept at 4°C for more than 1 week, thaw a new vial or aliquot to coat plates or increase the concentration to 1.5–2% (vol/vol)
Low cell viability after collagen I embedding	Collagen I gel mix pH suboptimal (although rare, we have observed lot-to-lot pH variations in commercially available rat collagen I solutions)	When using a new lot, we recommend determining the pH of the prepared mix using a pH test strip prior to embedding the cells. If the pH is below or above 7, optimize the amount of NaOH to add to the collagen I mix	55–56
	Size of the cell clumps transferred too small (bigger sized cell clumps exhibit better survival after embedding in collagen I as opposed to single cells or smaller cell clumps)	Transfer bigger sized cell clumps
		Be gentler with the mechanical disruption of the monolayer at step 49
		Avoid over trypsinization at step 47, shorten the trypsinization time
	Cell density in the collagen I too low	Increase cell replating density at day 25 (step 44)
Low cell viability later in the maturation stage	Insufficient feeding	Feed more often or increase the volume of media	55–56
	Collagen I gels have contracted and cells are in direct contact with media	Repair the gel layer (see CRITICAL STEP in step 55)
Cells retain expression of NKX2.1 and express poor or incomplete set of lineage specific markers	Cell density in the collagen too high at the time of embedding	Plate at lower density in step 44	55–56
		Increase the duration of the maturation stage, delay the time point of analysis
Collagen I gels detach from the walls of the well, contract or thin out	This might happen due to the contracting forces of the cells growing inside the gel, matrix remodeling by the cells and/or long periods of culture	Repair the gel layer (see CRITICAL STEP in step 55)	55–56
		Add a new layer of collagen I on top of the old one
Overgrowth of contaminating neuronal or non-endodermal lineages in the collagen I	Differentiation started from DE with low purity	Improve DE yield (see Huang et al. 201519)	55–56
	Suboptimal trypsinization at day 15	Increase the trypsinization period, wash the wells with 1x PBS prior to trypsinization, discard bottles of trypsin-EDTA that have been thawed for more than 1–2 months.
	Too many non-endodermal cells were carried over when replating	Do not centrifuge at step 35. If necessary, wash the cells a few times with PBS until the supernatant gets clear
	Overgrowth of non-endodermal cells from day 6 to day 15 of the differentiation protocol	Replate the cells twice into 1% (vol/vol) growth factor reduced Matrigel-coated plates. The first replate can be done a few days before day 15 and the second replate 1 to 2 days after the cells reach confluence.