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. Author manuscript; available in PMC: 2022 Sep 9.
Published in final edited form as: Nat Protoc. 2021 Mar 1;16(4):1802–1829. doi: 10.1038/s41596-020-00476-z

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.