It could be because the cells are not healthy. Maybe try to revive the cells to healthy state first. and here are some general factors affecting cell adhesion: https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/poor-adherent-cell-attachment-troubleshooting

https://lab.plygenind.com/factors-that-affect-cell-adhesion-in-mammalian-cell-culture

Hi Mohamed Lahmadi

Some cell lines, especially the non-cancerous, take a very long time to adhere and divide. If you work with such non-cancerous cell lines, please keep them undisturbed for 36-42 hours. Also, you may try increasing the concentration of FBS to 12-14%.

Hello Mohamed Lahmadi

A. Could long storage of cells cause this problem?

No, cells may be stored in liquid nitrogen for years (> 10 years) without any decrease in viability unless you have been late to refill the liquid nitrogen tank from time to time. If you notice low viability after thawing the cells stored in liquid nitrogen, then there may be other factors which you would need to investigate.

1. Have you cryopreserved the cells in an appropriate freezing medium and using the gradual freezing technique (i.e., -1 degree C/min) with the help of Mr. Frosty container? This container is designed to achieve a rate of cooling very close to -1°C/minute, the optimal rate for cell preservation. This will help to maintain the viability.

2. Have you thawed the frozen cells rapidly (< 1 minute) in a 37°C water bath? This too will preserve the viability of cells.

3. If yes, then you may have to consider environmental stress as a possibility, and what could cause environmental stress?

Look out for contamination, temperature fluctuation, improper gas mixture in the environment, inappropriate substratum for cell attachment (are you using tissue culture treated flask?) and inadequate or altered nutrients in the culture media.

B. Are there factors that promote cell adhesion?

Yes, serum contains adhesion-promoting factors, You may increase the amount of serum from the normal (10%) to 15% in the culture media as mentioned by V H Krishna Prasad .

C. It is possible to use 3D cell culture in this case?

I would suggest you first get the 2D cell culture in place.

How to save cells that are still alive?

Centrifuge the cell suspension at low speed (800 rpm for 5 mins). Resuspend the cell pellet in a small volume of culture media containing 15% FBS. Seed the cells in a smaller vessel (say in a well of a 6-well plate or in a 35mm petri dish).

Hope it works!

Best.

Thank you all

Hi Malcolm Nobre

Thank you for the detailed answer

These lines are cancerous and I've already used 15% serum with an appropriate incubation ...

- Apparently, the cells are not healthy but even though they proliferate in suspension "high density" ;

- The use of dishes coated with substrates "fibronectin or collagen" could save the cells in culture?

Hello Mohamed Lahmadi , I had thought of coated dishes, try it out! But that may not work because you already have a low cell viability.