Hi Sridharan. I guess the first thing to check is that you have TC treated flasks for adherent cells. For example Corning make a T25 flask that is for non-adherent cells (cat. 431463 this is the Corning catalogue and may be different if purchased from a different source).

If you are using TC-treated flasks the you should make sure you have FBS in the media (I'm sure you do). Cells do not stick directly even to TC treated flasks. The treatment allows ECM proteins such as Fibronectin in the FBS to stick to the plastic - the cells then stick to the ECM protein (which is why when using media without FBS people pre-coat their plates and dishes with e.g. Fibronectin, Laminin etc)..

If you are using TC treated and have FBS in the media then the most likely cause is that the cells were not frozen down well (ie had been left at -80 for 1-2 weeks before transferring to the LN2) and they are now mostly dead. Is their any indication that any cells are sticking down? (i.e. 1:100 stick down etc)?

Sorry I can't be more help.

Gary

Hi Sridharan, 

I agree with Gary. Check that you are using TC treated flasks, if your cells have been frozen down and brought back from LN correctly, this is the most likely cause. Ensure that they were frozen down correctly. We routinely use 10% DMSO in culture media or 90% FCS and cool them slowly at a rate of -1°C/minute by placing them in a Mr. Frosty at -80C before transferring to LN. We have seen cells recover from storage at -80C after longer periods than a few weeks (a few months) without any problems although I wouldn't recommend long term storage at -80C. Also, it is important to bring them out of LN correctly by rapidly defrosting the cells such as in a water bath at 37C. If they haven't been frozen or restored correctly they may be dead and therefore floating in the media.  

I hope this helps.

Pamela

An additional reason may be - but hopefully not -  a mycoplasma contamination. Usually contaminated cultures have problems to attach after thawing

I work in primary human skin cells that are adherent and use the Corning T75 flasks that are not pre-treated for cell culture. We store cell cultures in LN2 (better than storage at -80C) using freezing media that also contains 10% DMSO. If I take a cell line out of storage and the cells don't attach, it usually indicates that the cell line wasn't frozen down well and the cells were dead, so they will not attach to your flask.

Respected Professors :

 Thank you for your valuable comments. Am using TC treated plates, when am split the cells they are attached very well, but after freezing they are not able to attach. We don't have mr. frosty so am following this procedure for freezing my cells. ( Kept in 1 hr at -4 and 4 hrs at -20 and overnight at -80 then shifted into LN2 tank). 

Hello.. I would suggest you to use alcohol bath for slow freezing...You can put your cryovials in absolute ethanol at room temperature and keep it in -80 freezer for 1 day or longer.. and transfer it to liquid nitrogen... The temperature of alcohol goes down slowly.. It works very well....

Hi Sridharan, in that case I am rather at a loss as to what is wrong.

You might try to revive a different adherent cell line at the same time - this should (hopefully) tell you if the problem is in the materials/protocol you are following or in the HeLa cells themselves (i.e. if adherent line 2 stick and the HeLa does not then the problem is with the HeLa cells, if both fail to stick it is media/flask/freezing/storage).

In the case that it is the HeLa then it may be Norman above is correct (you have mycoplasma contamination). It may be worth checking for this anyway.

To save time in the long run I would be tempted to seek an alternative source of the HeLa cells - you may never find out what is wrong but at least you can continue your experiments.

Gary

Thank you so much  surchi and gray. I will follow your instructions !!!1

HI, I would agree with most suggestions above.

someone in our lab had a problem with HTERT fibroblasts which normally grow everywhere, they would attach one day and float the next!

If I were you to eliminate every possibility I would

1. Thaw 1 vial of the cells you are trying to thaw and they fail to attach and a vial that someone else has thawed in a different time point.

This will eliminate the flask adhesion question. Also this will tell you if your freezing technique is efficient and if the quality of your cells was good prior freezing.

Prior freezing cells make sure your flasks to be frozen to be of maximum 70% confluence and the cells have been fed the day before freezing.

2. Always make sure you cells are in -80C just for a day or 2 and always stored in LiqN2.

3. Always thaw your cells quickly at 37oC. Give them a couple of washed with fresh prewarmed medium and keep them in a small initially volume in the T25. This works nicely with both primary cells and cell lines I have been working for years.

As a freezing medium I use 70% TC medium, 20% FBS and 10%DMSO.

4. Have you checked if there is a problem with your culture medium? maybe one of the components is off. Or maybe try the medium with different cells if you are not already doing so.

5. I would agree with the mycoplasma suggestion as is quite a common problem in human TC.

I hope that helps.

Using 20% serum (or even more if you like) in your freezing media in addition to 10% DMSO, would definitely help cell survival after freezing. Honestly, if your cells do not attached after thawing, I believe it is a survival issue.

Best of luck...

All sound suggestions above.

In addition, you may like to consider a 90% serum/10% DMSO freezing medium, which I use for sensitive cells.

Freeze slowly, so once in freezing medium, cells can be placed at -80C directly (which approx. freezes at 1C per minute. Transfer within 24 h to liqN2.

Thaw quickly (remember, DMSO is highly toxic at room tempertature to mammalian cells), I suggest thawing within 2 min at 37C water bath and add to fresh 10% FCS/Medium of choice and centrifuge, before resuspending in fresh medium and plating.

To check cell viability on plating, cells should appear with a bright, glowing halo around the nuclear material when viewed using phase contrast. Small, predominantly darker cell bodies suggest poor viability/cell death.

Good luck.

In addition to all suggestions above, I would recommend to use 20% serum in cultivation medium after thawing. Furthermore, it is important not to centrifuge too harsh the thawn vials when washing. 150 - 200 x g are more than enough. That´s how you also get rid of dead cells and apoptotic vesicles, which presence in the HeLa culture would additionally affect viable cell by introducing pro-apoptotic signals. Good luck!

use for freezing medium

420 ul of cell pellets suspensions in media (contain atleast 15-20 lakhs cells) + 80 ul DMSO and 500 ul of FBS (serum)

and store in - 80 degree for short time and in liquid N2 for long time.

During reviving, centrifuge the old cell media and revive in fresh media. 

Did you test cell vitality after plating? Hela are quite strong cancer cells and normally do not give problem during the frozen and defrozen procedure. Therefore take care to the frozen procedure and don't use too much confluent cells and don't froze more than 2x106 cells vials

Rinse the cells in the medium, and check viability.

Cell supernatant, do not throw but leave a few days in the new Falcone tracking.

if everything is fine with sterility medium and serum?

It is common with Hela cells it used to be like that to me but I perhaps add more than one vial when starting. Please you may also try to freeze with 30% Serum. also avoid long freezing.

I suggest what Monica Bartucci, ·Radoslav Mladenov and the  90% serum/10% DMSO  suggested by dr. Vehid Salih, however Hela are reaaly strong cells

please change your flask type. HeLa cells are very strong and grow even in low concentration of serum and bad situation.

HeLa cells are strong enough to survive even in harsh conditions. If you use your complete medium with 7-10% DMSO your HeLas will be fine. If not, then make sure it is really HeLa cells.

You can consider the following:

With cell lines the rule is to freeze slow and thaw fast. 

After thawing cells must be washed in 5-10ml fresh medium before they are seeded. And this makes a difference.

Plastic some how affect the adherence of cells. You might need to try another plastic.

When you freeze it helps to use 20% FBS in the freezing medium.

Needless to say that FBS affects the well being of the cells.