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) | ||
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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) | ||
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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 | |||
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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. |