As long as you haven't had the problem before and it's all right, it is expected that the problem now came from the materials used in freezing.

Or that the cells get contaminated before freezing or after thawing and culturing them in the medium. It is not excluded that the contamination is from mycoplasma if it is not visible under the microscope.

Try using new materials from other sources and also use 0.22 micron filters when preparing them.

I wish you good luck

Dear Kiana,

It seems you are using a primary culture, isolated by yourself from the biopsies. The properties of primary cultures are much different from established cell lines. Primary cultures usually exhaust after several passages. They also may not withstand the harsh condition of freezing for several times. However, it is a undeniable fact that cells are hard to regrow after long-time freezings and you should be patient with these cells. some times they need 2-3 weeks to regrow and become refresh. My suggestion to you is being patient with the cells, even if you only a very low cell number in your culture vessel. I also suggest increasing the concentration of PBS in your medium to 15% and using glutamine supplement. This helps your cells to regrow faster.