Hello Mahsa Daneshmand

The CHO cell culture may be contaminated, especially with mycoplasma contamination. Check for mycoplasma because mycoplasma contamination always goes undetected for many passages. They can proliferate within the cell, tolerate antibiotics and their growth always does not have any obvious microbial evidence like turbidity and pH changes or cytopathic effect. Mycoplasma contamination affects function, growth, metabolism, morphology, attachment, and many other properties of the cells.

The best way to check for mycoplasma contamination is by PCR, though the DNA staining method is a simple method that relies on staining of bacterial nuclei with Hoechst or DAPI which is visible by fluorescence microscopy. There are several PCR-based mycoplasma detection kits available which may help.

If you detect mycoplasma contamination in your culture, you may either eliminate the contamination from culture or start over again if you have extra frozen vials. The most reliable and efficient treatment of mycoplasma contamination is the addition of suitable antibiotics, such as quinolones, tetracyclines and macrolides. Alternatively, mycoplasma elimination kits are available which are a convenient and efficient choice for ridding cell cultures of contamination.

Best.

Hello Malcolm Nobre

Thank you very much for your complete answer.

Honestly I guess so and I was really worry about it.

Does it affect on adhere of cells after thawing?

Mahsa Daneshmand, yes, mycoplasma contamination does affect the adherence of cells to the substratum after thawing.