When using Biolaminin 521 to culture human stem cells, I have an issue with uneven cell distribution. The cells don’t attach to some areas of the well, whereas they attach and grow normally in other areas. What can I do to solve this issue?
The problem could be caused by the following:
- The coating concentration used is too low for your specific cell line. Try to increase it to 10 μg/ml. Once the cells are adapted to the Biolaminin 521 matrix, you can try to reduce the coating concentration to 5 μg/mL.
- The coating volume used is not enough. This is often a problem seen with smaller well sizes due to surface tension. For 24-well plates, 500μL of coating solution per well should be enough for even coverage. For a 6-well plate, at least 1ml should be used.
- The coating solution has dried out before cell seeding. If the coating matrix goes dry, this will inactivate to coating and it will not support cell growth. This could happen if the coating volume is not sufficient or if the time during the exchange of the coating solution to the culture medium was prolonged. Make sure to seal the plate during overnight coating in the fridge. If a quick coating at 37 degrees is used, do not leave the plate for more than 3 hours to prevent it from drying out.
- The cells were not seeded evenly in the seeding step. Make sure that you rock the plate side-to-side after seeding to get an even cell spread.
- The problem might also be in the cultureware plastic. At least Falcon, Sarstedt and Corning tissue culture plates have been tested to work fine for Biolaminin coating.