do you have the name of the cell lines? you might need to check the nature of growing of those cell lines, whether they grow as adherent culture or floating suspension if they are well-established cell lines that have information somewhere online. It is perfectly normal to have cell culture that grows in suspension such as many of the established small cell lung cancer cell lines. For suspension cell lines like these, you know that they are alive if they get more and bigger in time. They tend to form necrotic core if they grow in spheroid form. Hence, you will need to disaggregate them either by pipetting vigorously or by digesting them with trypsin. I personally prefer to disaggregate them just by vigorous pipetting. How do you exclude dead cells you may ask? Routine collection by centrifugation at low speed should be able to remove some cell debris and routine subculturing/splitting should dilute them further, so the dead cells and cell debris will likely not accumulate that much.

Hello Mohamed Lahmadi

The cells are not looking great! I feel they are stressed.

When you cryopreserve cells, the following should be included in the freezing protocol.

1. Cell Cultures should be healthy with a viability of >90% and no signs of microbial contamination.

2. Cultures should be in log phase of growth (this can be achieved by using pre-confluent cultures that are below their maximum cell density and by changing the culture medium 24 hours before freezing).

3. A high concentration of serum/protein (>20%) should be used. In many cases serum is used at 90%.

4. The use of cryoprotectant such as DMSO help to protect the cells from rupture by the formation of ice crystals. The most used cryoprotectant is DMSO at a final concentration of 10%.

5. Slow freezing by decreasing the temperature approximately 1°C per minute and by using Mr. Frosty freezing container may aid in successful cell cryopreservation.

6. Keep a log of the level of liquid nitrogen present each time the liquid nitrogen tank is checked.

Did you check the viability of cells after thawing? You may try to revive these cells by centrifuging the cell suspension at 700-800rpm for 5 mins and plating these cells in a culture vessel of a smaller growth area such as 6-well plate or 35mm petri dish. The closer these cells are to each other, there are chances that they may revive.

Best.

Thank you for your responses ...

- Cells are probably stressed due to freezing procedures.

- Even cells look abnormal, but they continue to proliferate!

- I've tried to culture them on small surfaces, even for adherent cell it's hard to form a colony (confluence) and look abnormal under microscope.