Dear Anika Dreier , Well if we talk about cell culture, in general; Actual cell recovery is calculated after 24 hrs of culture after thawing. As you must be aware of the phenomena of cryopreservation, you never get 100% recovery of preserved cells.

What you are finding in your culture flask is % of cells are floating and idid not gain shape; i.e. they are possibly dead cells. In general people in their culture experiments, count the cells after 24 hours of thawing to get accurate cell numbers.

I think you should not be bothering about these cells. these will be removed in 1-2 passage of culture. So, if you need to treat your cells with some agent, it is recommended to treat only after 1-2 passage.

If you want to read further, there are numbers of articles available for cell recovery and people are working a lot to reach maximum recovery of cryopreserved cells.

I hope you question was answered.

Best wishes.

Jitendra Kumar Shandilya thank you so much for your input! So you think its kinda normal? The picture above was this moring, i attached a new one, which is actually after 24h. There seem to be even more of these floaters. Am i wrong that they dont look like dead cells? In my experience, dead cells are kind of dense and small under the mikroskope, these look so bloatet.

In the first try with these cells, they all died over some passages, so i am kind of worried. This time i changed the protocol from 5% FCS to 10% so attachement should be fine, right?

How do i count after 24h? Do i just take the Medium and count the cells which are not attached? Or do you mean, use trypsin and count all the cells in the flask? Wouldnt that be too much stress?

Thanks again for your help! I am really lost right now..

Dear Anika Dreier , Just take the non-adherent cells and check their viability or any other characterization marker for your cell type.

Jitendra Kumar Shandilya , ok i will do that, thank you! Do you think it is better to change the complete media after these 24h? In the protocol it says to change the media after 5 days. What would you recommend?

Hi Anika Dreier

I agree that they look a lot like floating dead cells that did not survive the thawing process. You shouldn't expect all cells to survive cryopreservation, but luckily there appear to be plenty of viable adherent cells. The dead cells will wash away with feeding and passaging.

5 days seems a bit long to change media, most protocols feed cells every 2-3 days (depending on the cell type and type of media). Does the protocol suggest changing to a different type of media after 5 days perhaps?

All the best,

Sam

Sam Siljee Thank you for your message! What could be the cause of the cells not surviving the thawing? Could it be the thawing process (which i did according to protocol) or is it something while freezing the cells?

No the there is no change to a different type of media, it stays the same. i think 5 to event 7 days, as suggested is a bit too long, so i change part of the media every 3 days and let a small amount of old media stay on the cells.

Thank you and i would be grateful for more input!

Some cells won't survive cryopreservation (could be that they die during freezing or thawing) due to the formation of ice crystals that disrupt the cell membrane during freezing. In order to reduce the formation of ice crystals, DMSO if added to most freezing medias, however, DMSO is also toxic to cells at high concentrations for extended periods of time, hence cells should be washed immediately on thawing before plating.

Remember that freezing is not a natural state for cells, so some death is expected and acceptable. This is also why most people do not do experiments on cells plated straight after thawing, but do a few passages first before doing any experimental work.

Hopefully this helps clarify some things!

Out of interest, how are the cells looking today? Are the surviving cells starting to expand?

Sam Siljee Thank you so much for your help! I really have a better feeling now! Today my cells look like this, which looks quite good i think... lets hope for the best.

I have some problems with evaluating the grade of concluency... i would say 30% based on the whole flask (in which the distribution luckily is very even). Would you confirm that based on the picture?

Thanks again!

Anika Dreier You're most welcome!

Your cells are looking good, you have nothing to worry about!

Even after a few years of cell culture, I still struggle estimating confluency myself. I would say based on the one field of view that it's significantly more confluent than 30%, I'd estimate around 80% (and passage today).

Best of luck!

Out of interest, what type of cells are you growing?

Sam Siljee Thanks again for your input! I just read your answer, the picture was from Friday last week.. Today they were even more dense and i splittet the cells 1:2

If you would say 80% in the picture, i hope today wasn´t too late. But the flask was not completly overgrown, so i hope it will be fine. When the cells are 100% konfluent, is it that bad? Do they die if this happens one time? Or is there a chance that after passaging they adjust? I know that it shouldnt happen and i think with my cells it wasnt 100% but just out of interest: i a one time 100% a death sentence for the culture?

And for your question: the cells are epithelial cells from fish gills

Also, if i have dead cells from the thawing process, how many changes of medium does it take to wash them out?

I splittet my cells and i still (or again?) have dead cells swimming around?

Anika Dreier Sorry I missed your reply. Growing to 100% confluence depends on your type of cell. Some cells don't mind being fully confluent for a little while (although it's not ideal), other cells quickly become senescent when they are over confluent.

The aim is to passage cells when they are in the exponential growth phase to keep them dividing. Ultimately if your cells are unhappy they will stop growing or start to differentiate (if they are stem cells).

Hopefully your cells are still growing!

Your cells should tolerate splitting better than cryopreservation, and there shouldn't be too much dead cells from splitting. What method are you using for splitting?

Dead cells and debris should wash out with a few feeds. If the debris gets worse over time, check under a high-power objective that there's no bacteria or other microbe contaminating your culture and killing the cells. It should be pretty obvious, the media will discolour and become turbid.

Hi Sam Siljee , thanks again for answering! i have epithelial like cells, i don´t know how good they tolerate being confluent. I have a feeling that now in the second passage ( i am on day 7 now) they again look not that good. Many vesikles and dead cells.. i really don´t know why they are not staying attached to the flask.

For splitting, i wash 2x with versene, then dissociate with trypsin. They medium is not cloudy and there are no bacteria etc visible. I get the feeling, that the cells might have not have been frozen idealy? could that be the cause?

Hi Anika Dreier They should have recovered from any freezing effects by now. I suspect that the likely cause is indeed the period of being over-confluent. I would passage sooner, and split them more than 1:2, especially if they are already over-confluent, splitting 1:2 will have them confluent again very quickly.

Another consideration is if these are primary cells or an immortalised cell line. A cell line will continue to expand almost indefinitely, where primary cells have a limited number of divisions before they become senescent. Check at which passage they were frozen down, if it's a high passage number they may just be at the end of their lifespan.