Table 6:
Troubleshooting table
| Step | Problem | Possible reason | Solution |
|---|---|---|---|
| 17 | Organoid morphology is not spherical or cyst-like | The organoids are too differentiated, i.e., the shape is not cyst-like, but has thick and folded edges (Figure 3). | Break down the fragments more when passaging the organoids. Reduce cell density by increasing the split ratio |
| 17 | Organoid yield low | Organoids were seeded too sparsely or are not healthy (Figure 4). We observed that this occurs occasionally after thawing a new vial of organoids | Decrease the splitting ratio, (e.g., to 1:2 split ratio) Check the quality of the WRN-Conditioned medium by assessing Wnt activity.57 |
| 17 | Organoids stop growing after a few passages | Unknown – suspect genetics are deficient for growth, we observed that this is donor specific and organoids derived from diseased donors are more prone to stopping growing. We encountered organoids from one donor that stopped growing a few passages following thawing. Mycoplasma contamination |
Thaw a new vial of organoids, or switch to using organoids from another donor. Check the WRN-Conditioned medium has high Wnt activity.57 Check for mycoplasma contamination regularly. |
| 56 | Low cell viability after single cell dissociation (empirical threshold: 75%) | Dissociation too harsh, organoids not healthy. | Avoid incubating for too long in CRS or trypsin. Avoid too much pipetting during organoid dissociation. |
| 80 | Monolayer detaching from the edge of the Transwell membrane (Figure 5a) | High passage number in continuous organoid culture or monolayers are not mature enough at the start of differentiation. | Avoid culturing organoids for more than 15 consecutive passages after thawing. Let the monolayer grow in seeding medium for 1–2 more days before starting the differentiation. |
| 83, 88 | Bacteria do not form a colony when culture is started or during CFU plating assays | High oxygen levels, agar too dry | Exchange the instrument catalyzer in the anaerobic chamber regularly (at least once every two weeks), use freshly prepared and pre-reduced agar and preserve the agar in sealed bags. |
| 94 | Cells in monolayer die after introducing bacterial media | Bacterial media is toxic (Figure 5b) | Evaluate the toxicity of the bacterial media. |