| Step | Problem | Possible reason | Solution |
| Throughout | Cultures were contaminated | Inadequate sterile technique | Improve sterile technique. Wear
laboratory coat. Use 70% ethanol to spray your hands and any equipment or
reagents that go into the safety cell culture cabinet. Autoclave all
non-sterile equipment. Change HEPA filters in ncubators regularly. Autoclean
incubators regularly. Identify sources of contamination in the incubator or
cell culture room. Clean the water bath in the incubator and in the cell
culture room on a weekly basis. |
| 29 | Individual spheroids do not form | Overfilled individual wells of
the AggreWell plate | Count cell number carefully and
add the required amount of cells to each well. Centrifuge the plate with optimal speed |
| 31 | Spheroids do not form | hPS cells are differentiating | Always check hPS cell quality
before use |
| | | hPS cell colonies are too small | Allow hPS cell colonies to grow
larger. Do not use until the optimal day for splitting (80–85% confluency) |
| | | Over-crowding of hPS cell
Colonies | Start differentiation by using
hPS cell colonies with the optimal confluency (80–85%) |
| | | Mycoplasma or other contamination | Perform mycoplasma testing
regularly (ideally, once per month) and discard the cells and use new hPS
cell stock if mycoplasma positive |
| 38 | Difficulty in inducing forebrain fate (low FOXG1 expression) | hPS cells are challenging to differentiate into forebrain
cells | Consider adding XAV-939 (1.25
µM) from days 1–6 of differentiation |
| | Spheroids stick to one another | Too many spheroids cultured per plate | Transfer some spheroids to a new or larger low-attachment plate. If
already attached, separate them by gently pipetting up and down by using a
10-or 25-ml pipette (for spheroids up to day 25) or two P20 pipette tips at
later stages |
| | Spheroids are too small | hPS cells are differentiating | See solutions for Step 31 |
| | | hPS cell colonies are too small | See solutions for Step 31 |
| | | Mycoplasma or other
contamination | See solutions for Step 31 |
| | Spheroids are disintegrated | Excessive pipetting | Be gentle when handling
spheroids at all times |
| 38 and 39 | Growth of spheroids is fast | Abnormal genome integrity in the
hPS cell line | Check the genome integrity of
the hPS cell line by using SNP array, and confirm that there is no de novo
large duplication or deletion |
| 39 and 40 | Medium turns acidic overnight | Too many spheroids cultured per
plate | In the initial stages of differentiation, 12 ml of medium per 10-cm
plate is sufficient. As spheroids grow, use 12–15 ml of medium per plate.
Maintaining <30 spheroids per 10-cm plate is recommended |
| 40A | Sparse or no viral labeling | The viral titer was too low | Make sure that viral titers are
sufficiently high |
| | | Insufficient time between
infection and fluorescent protein expression | Wait ≥1–2 weeks for viral gene
expression |
| | | Incorrect serotype | Use optimal AAV serotypes |
| 40B | Unable to assemble hCS and
hStrS | hCS or hStrS at late stages of
differentiation | Try to fuse smaller or
early-stage spheroids (days 47–80). After day 80, the success rate is lower |
| 40F | Unable to find GCaMP6+ cells inassembloids
under a confocal microscope | The signal of GCaMP6 is weak | Check expression of GCaMP6 by using an epifluorescence microscope (e.g.,
EVOS or REVOLVE). Increase laser intensity |
| | | Imaging only a few z-slices | Increase thickness of slices by
changing the pinhole size and/or image more slices |
| 40G | Cells are hard to find under
bright field | Cell bodies are not always easy
to identify by using bright field | Identify cells by using
fluorescence imaging |
| | Cells are blown away by pressure | The pressure is too high | Apply minimal positive pressure |
| | | Pipette tips are too large | Use a pipette with smaller tips |
| | It is dif ficult to form a tight
seal. Cells cannot form a fully tight seal even though cells are partially
sucked into the pipette | Pipette tips are too large | Use a pipette with smaller tips |
| | Photobleaching of fluorescence when checking cells | The LED power is too high | Lower the LED power and increase
gain and/or exposure time in the camera software setting |
| | Cells are hard to break in | For some cells, it is hard to
break in with just suction | Try ‘zap’ in the 700B panel for
multiple trials; start from short durations such as 50 μs and then increase
durations step by step; try strong suctions immediately after zaps. Use
pipettes with slightly bigger tips |
| | Cells get significantly resealed after break-in | Pipette tips are too small | Use pipettes with slightly bigger tips |
| | | Osmolarity of the external
solution and/or the internal solution is not appropriate | Check osmolarity of both the
external solution and the internal solution |
| | | The relative position of the
pipette tip and the cell changes in whole-cell mode | Slightly adjust the position of pipette tips |