You may check your cell line for Mycoplasma contamination.

Alternatively, use a preventive concentration of Mycoplasma removal agent from MP Biomedical.

Use the recommended media constituents:

low glucose Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 2 mM glutamine, 0.01 mg/ml insulin, and 1% penicillin/streptomycin mix.

Ensure you make a single-cell suspension after trypsinization and use same seeding density every time. (may be around 0.1-0.2 million cells per ml).

Maintain the split ratio very precisely.

Hope this may solve your issue.

Hello Shahad Alsharif

There could be various reasons.

1. If the cell suspension is not mixed adequately before addition to the culture vessel, it could result in uneven distribution, attachment, and growth of cells. You may add the media with cells and gently tilt the vessel in an appropriate manner which would result in an effective way of mixing medium thoroughly without creating bubbles or foam.

2. Uneven growth can occur because of the shear forces generated by medium sweeping across the cell monolayer during medium changes or while moving cultures between the laminar flow hood and the incubator.

3. Unusual patterns of growth in the culture vessel could also occur when the vessel in the incubator is not at a leveled base. If you use shelves in the incubator that have not been leveled prior to use, it may often cause this effect. The shelves in the incubator should be checked with a spirit level and adjustments should be made following the incubator manufacturer’s recommendations. You should periodically check and make sure that the shelves are leveled, especially when they are removed for cleaning purposes.

4. Vibrations can cause unusual cell growth. Vibrations can come from a loose fan motor in the incubator or from any other source such as the motorized appliances. Keep incubators on sturdy surfaces that don't have any other pieces of equipment that vibrate.

5. Temperature differences within the incubator may create such a problem even when the differences are only a few tenths of a degree. For instance, cells prefer the warmer areas which are directly over the metal portion of the shelf. These conditions usually occur when incubators are frequently opened, especially during the first few hours after freshly inoculated cultures are placed inside the incubator.

On the other hand, if the temperature in the incubator is a bit too high for the cells, then the cells would prefer the holes in the shelf for cell growth and attachment. This clearly shows that cells are sensitive to very small temperature changes.

Maybe one of the above could be the cause of the problem. Please check!

Best.