Dear Ruchi,

As said by colleagues above, a lot of possibilities. Simply deduced of yours observations : it appears after changing the medium, all the medium, for fresh medium. So, something is "toxic" in the new one, or the new conditions. When changing the medium, it necessitates to have the medium not too cold before use; also, some cells necessitate to have a part of the "old" medium diluted with the new one , because some cells secretate their own growth factor, but an equilibrium has to be find because they also excretate tsome metabolic products that could be toxic: ie you can mixed 1/4 old + 3/4 new medium.

Regards

Didier

That seems an interesting problem. It may be a some kind of contamination (e.g., mycoplasma). You may try another lot of medium. We prepare our medium by ourselves and never used commercial medium. Changing medium, sterilizing incubator, checking CO2 incubator may help solve your problem. But it is obvious that you are having a problem with the medium.

It could be many things, your fresh medium could be too old, wrong pH, too cold because what you describe looks like cells are dying. As in the comment above, I would definitely avoid changing medium just prior transfection, if you do not have a particular reason for that I would not do it. Most transfection protocol require that a certain number of cells be plated the day before transfection (in some case without antibiotic to avoid a rush of them inside the cells with the transfection), in that case unlike there is something wrong with your medium it should not need to be changed so soon, quite the contrary because cells need the factors they secrete into the medium. If your transfection protocol require medium without serum it is different but from your description it does not appear to be the case..

One other thing is you might be plating too many cells and instead of being in exponential growth, they are already stationary and starting to die, changing the medium just makes it visible, in that case you should plate fewer cells, ideally they should not reach confluency before you are ready to do whatever experiments you are planning with them.

Same thing happened to me before. I was working with HEK293T cells. Somebody told me it could be a contamination in my stock of cells because the medium and everything else was working perfectly for other cell lines. I borrowed some fresh cells from a neighboring lab and that solved the problem.

Epithelial cells are hard to work with. They don't stick to the plate very well. So, you need to be very careful while changing the media.

Dear Ruchi,

As said by colleagues above, a lot of possibilities. Simply deduced of yours observations : it appears after changing the medium, all the medium, for fresh medium. So, something is "toxic" in the new one, or the new conditions. When changing the medium, it necessitates to have the medium not too cold before use; also, some cells necessitate to have a part of the "old" medium diluted with the new one , because some cells secretate their own growth factor, but an equilibrium has to be find because they also excretate tsome metabolic products that could be toxic: ie you can mixed 1/4 old + 3/4 new medium.

Regards

Didier

Dear Ruchi,

I agree with all our friends answered. In particular I agree with didier Jambou, when he writes about keeping some old medium together with the new. If you try different "lod/new" ratios, you could even understand if this could be a dose-dependent behaviour.

Moreover, please remember some simple notes that escape in the most complex works with cell cultures:

1) don't forget to check for the quality of your glutammin additive: absence, degradation or "ageing" of your glutammine stocks could induce similar effects on your cells;

2) check with different cells the effectiveness of heat-inactivation of serum;

3) be sure that temperature and pH of the new medium are not so far away from the old in your working/storing conditions.

Three suggestions (most already mentioned)

1) Warm new media (if you dont)

2) Live a bit of old media

3) Be REALLY careful when relplacing the media (operate very slowly)

Dear Ruchi,

I think that all discussed tips are very useful to avoid general culturing problems. So, you should be sure to follow.

In my experience I worked with HUVEC and I found that create many problems, temperature sensibility as well as the coating or glass o plastic dishes. Another problem is due to pH, you can use DMEM supplemented with HEPES as stabilizer and all buffer should be warmed at 37°C-

It is clear that it is not escluded that you have a contamination problem, so it is necessary to verify that or avoid the problem using a new cell stock.

Good luck :)

Hi Ruchi,

Many of the aforesaid answers look good to me. Go through the following points also (which might have been already mentioned above):

1) Use new lot of medium which works good for other set of cell lines in your lab or in neighboring lab.

2) Check whether you added much more Hams F12 or penstrep than the recommended concentrations.

3) Check if there is some contamination (e.g., mycoplasma). My experience says if there is mycoplasma contamination then the adhered cells get detached from the plate, morphology changes into round shape and cells look little bit crappy.

4) Do you generally use beta-mercapto ethanol (b-ME) in your DMEM medium? If so then check the quality of b-ME and concentration of b-ME added into the medium.

5) Do you use commercially available heat-inactivated FBS or you generally heat inactivate FBS before using it? If you heat inactivate FBS before using it then do it properly. I generally use to follow the heat-inactivation protocol attached herewith.

6) Add warm medium, very gently along side the wall of the petri plates or well plates, during changing the medium of your cells.

Hopefully these will help you (along with other given suggestions) to overcome your problem.

Good luck!

It looks like some sort of contamination. If you use a fresh batch of media, you would see the difference.

I suggest you do all the above, Please check the following

1.Change in the pH of your medium

2. Check does your cells need NaHCo3 or Pyruvic acid or any other aminoacid..Read the literature.

3. Please filter the FBS after purchase and add according to specifications mentioned in the publised literature or according to ATCC.

4. Circular shape of cells also due to they are not adhering to base of the dish. Check if you can coat the dish or plates with collagen or poly-L-lysine.

5. Adding Gentamycin antibiotic solve lot of problems @ 2- 5ug/ml. Most of the black small debries in the cell cultures might be due to dead cells or mycoplasma contamination. Adding Gentamycin will help tremendously.

Hope this helps

Good luck

Dear All,

I appreciate all your suggestions.

Firstly the media that I use is pre-warmed to room temperature so that it is not a shock to the cells.

Then I replace only 50% of the media with the fresh one so that growth factors are not removed entirely. And this is done very slowly.

pH of the media is between 7.2-7.4 and is supplemented with NaHCO3.

The problem has been occurring since 3-4 months and in between the lot of media has also been changed so it probably has noting to do with the medium.

The cells are very adherent and attach really fast to the base of the flask.

We already use Heat Inactivated serum from Sigma.

No beta mercaptoethanol is added to the medium.

Glutamine is already supplemented with the medium and the medium is commercial 1:1 DMEM and Hams F12. 10 mM HEPES is supplemented to make the final concentration of 25 mM.

Attaching the pictures for your perusal.

This pic is of the cells whose medium has been changed

Another pic of cells whose media has been changed

First and last pic are of cells in the flask

Looking forward for your replies.

What about changing to a transfection reagent, which does not need a change of media. e.g. TurboTransfect. This does not solve your problem with the detaching cells at all. But it could work anyway.

Just to know: will the cells become adherent again afte a while?

One more thing: could you please add the time scale on which things happen? I also guess, a contamination can be excluded: as you use a new batch of medium and also I do not think, a contamination that is not visible in first place will kill your cells in a very short time. At least, it is not very probable.

There is possibility that your cell line quality is degraded...try use same cell from different lab...what about incubator tempereature? Is it stable? Check it water reservoar for contamination

Dear Jan, as soon as i replace half the media, by the time i turn back to the microscope the cells become rounded.. that is the time that it takes.

Check pH of spent/conditioned media, fresh media and the 50% mix. Also, see this happens sometimes when High Ca / Low Ca mix for some of my primary keratinocyte cultures.

Hi Praveen, what did you mean by high Ca/Low Ca mix?

one good strategy which i successfully tried is remove only half of the medium from the culture well and replenish with new medium . Hope it will solve the issue . I tried this with endothelial cells.

Dear Ruchi, I have gone through your problem and want to assure you that it is nothing serious. I have faced this problem during bone marrow stromal cell culture. In my case, whenever I added fresh medium directly into that stromal (predominant population is fibroblast) culture the cells become round and even detached from the plate. It is also concurrent with macrophage culture from mouse peritoneal fluid. Actually most of the cultured cells are cold sensitive. That is why it is better to add culture media with ambient temperature or keep the media in incubator (37degree Centigrade ) for a while to bring the temperature at that point in which cells are incubated. Besides, you should not decant whole old media from the dishes. It some times become detrimental so you have to keep thin rim of old media (e.g. 1/4) and replenish the remaining part by fresh warm (37degree centigrade) media. If it works then fine otherwise you have to consider other factors as stated by other friends.

Bye. Malay Chaklader , CSIR-SRF, Calcutta School of Tropical Medicine , Stem cell wing.

Hi Anu, I do the same thing of replacing half the media with fresh one.. Still I see cell death

Hi Malay, If you could go through the pics and try to decipher what would the greyish black structures be?The media that I use is at room temperature and not 37 degrees. I can try your suggestion of using media at 37 degrees instead of room temperature. N ya i do not replace the entire media, but only half of it. Let me know if you can think of something else too.. Regards

Hello Dear Ruchi Kaka

As said your colleauges above, thare are many possibillity. u shoueld try step by step and first evaluated your media for PH, and temprature of icubator, if they are right, incubate your cells for some days and chek them for contamination,

Hi!

As the others there, I will say that something is wrong with your new medium. Do you use any feeders in your culture? do you cultivate them in enriched CO2 atmosphere? at what temperature do you cultivate them? Do you have any goodies in your medium (cf Rheinwald and Green, Cell, 1975)? I suppose you use commercial medium.

anyway, your medium has to be at the appropriated pH (if you have phenol red in your medium, the color is from yellow - acidic- to violet -basic- orange red is around 7.4), the medium has to be warm (37°C if you cultivate them at this temperature), if you are in flask and cultivate the cells with enriched CO2 atmosphere you have to put gas in your flask, the adherence is better wit Ca2+ enriched medium (it is necessary for cadherin junctions). Epithelial cells do not appreciate at all when pH is too basic and grow very slowly when it's cold. I have never seen my cells detaching like you describe, except when I had EDTA (disrupting the cadherin junctions) or when the pH is abnormal.

Regards

Steph

Hie Stephanie, The medium lot has changed too.. Also there are no goodies or anything else or any feeder layer as such on the plate. The medium at the time of change is at room temperature and not 37 degrees. the medium is also phenol red free. Lemme know if you have any other suggestion.. Thanks :)

In addition to all the above suggestions re 1/2 media changes etc., get in touch with the medium manufacturer to see if you can get a bottle or two of the medium lot number that cells were OK with. In addition, has the serum lot number changed as well?...If it has, do the same with the serum as a different lot of serum could definitely be a factor. If not, has the serum been left out overnight at RT? This could also give problems.

all the best

I agree with Bruce. Some manufacturers accept to save (x) bottles a year from the same batch, after you have tested the better one for your culture; the most critic being FCS (as they are built by pooling different serum, and some can contain some toxins for some cells).

Regards

Didier

I would only contact the supplier in case semi-conditioned medium shows the same effect. maybe the cells simply produce something essential to sustain viability.

josef

The "inclusions" you see in your cells may actually be vacuoles of water - they can form as a transient protection from an hypoosmotic shock - have you checked the osmolarity of the media? The other responses re pH, toxins, etc. are obviously worth considering. Good luck!

Hi Virginia, Have you gone through the pics?? you think those are vacuoles only? I have not checked the osmolarity of the media. Can you guide me on how to do it? Thanks

I agree with Virginia, you could have an osmotic shock. The fact is that if you seed your cells in the same medium that you feed them, this problem should not occur. Be sure that you have not a problem with the bottle. Sometimes it is easy to make a mistake between 10X and 1X. For me, you have to carefully check your medium (osmolarity, pH, Ca2+ concentration). In your incubator, do you have a constant CO2 concentration? what are the conditions you use to grow the cells? In your medium before feeding, what do you use as serum? the same as for the feeding?

Steph

silly question time - where are there photos & how would I access them?

Hi Stephanie, There is no issue with the bottle or the concentration. The incubator conditions are 37 degrees temp and 5% CO2. The medium bottle is the same that is used for seeding the cells. I use Heat inactivated fetal bovine serum, concentration 10%

Hey virginia, the pics have been uploaded and are somewhere in between the replies i am getting.. if you scroll through the above replies, U ll see them.. Any help is appreciated.. thanks :)

Would anyone be kind enough to supply me GMP certified EBV-LGL cells and/or K562mb15-41-BBL cells or similar cells to be efficiently used to expand human NK cells!

Did you find out what was wrong? I have the exact same problem with LLC-MK2 cells. I have tried several different medium flasks with no luck. I guess I will try to pre-incubate the medium in the incubator and see if that helps.

Are you using the right kind of tissue culture treated plates/flasks? Cells would sometimes detach if they don't stick well.

yes, I seed the cells and 24 h later they look perfectly nice. However, after I change the medium they look horrible (detachment, shrinking; necrosis?, lots of small droplets at the cell surface;apoptosis or lysis?) in several of the wells (24 well plate), but not all of them. I have been passaging the cells in T25 flasks for several weeks and never had any problem.

I don't know how sensitive these cells are to osmotic shock, but for neurons, which are sensitive, a straight 1/2, for example, medium change can make the cells unhappy. The reason is osmotic shock: some of the water in the original volume of media bathing the cells was lost to evaporation during the time interval between when the cells are seeded to the medium change. Thus, the medium following the change becomes hyperosmotic. The answer to this is to measure the volume loss in your incubator, using the same style of plate/dish you will be culturing in, over the same time interval using water as a substitute for medium (no cells in this experiment). Once you have the volume loss, just perform your usual % medium change on the cells after the usual time between plating and medium change (% based on original plated volume) and add to each well whatever the calculated volume loss is in sterile water. For 25 well plates, do not use the outer wells as these are susceptible to larger and inconsistent volume loss to evaporation

Hope this helps

I can see that the cell density has an influence on the effect of the medium change. After I doubled the initial number of cells they survive. However, if there are some places in a well with lower cell density (non-confluent) they die as before.

It appears that this is caused by an osmotic shock, but I think I might be misunderstanding something here, Bruce. Will not diluting the medium make it worse when you change the medium after evaporation as the difference in concentration of various compounds/ions increase even further?

I have started to measure the loss due to evaporation as you suggest, thank you for the advice! I have seen that the cells in the outer wells usually are in the worst shape, which supports your explanation.

Hi Ruchi, as suggested above while you change medium keep old medium too, I mean 1:1 old and New. Also watch for volume loss in incubator, if loss is significant noticeable, then check humidity. One more thing, did you leave the plate in incubator for a day or couples of hours and cells are attached as monolayer?

Hi,

If you do not replace the lost water, the solutes in the medium become more concentrated after evaporation...because the bulk that is lost during evaporation is water. If you add back the water lost to evaporation, then the solutes are brought back to their original concentration.

I assume that you are not doing a complete medium change but are only replacing some percentage, lets say 50% for example, of old with new?

No, I am trying to replace all of the media.

So, you mean that you seed the cells, wait 24 h and add the evaporated amount of water, remove 50% of this medium and replace it with the same amount of fresh media? Will they not still be "shocked" when the water is added if they have gradually adapted to the more concentrated environment? Anyway, good idea, but it will still not work if I have to change all of it...

You should check the humidity level in your incubator...

this is how to do a partial replenishment with neurons, I dont know how your cells will respond....

Lets say you are starting with 1 ml volume at plating, are performing a 50% change

and for example have during the interval of plating to medium replnishment measured average loss of 10%, or 100 ul

You would at your medium replenishment:

remove 50% of original volume, 500 ul, so you would then have 400 ul of "evaporated" medium left

You would add 560 ul fresh medium, together with 40 ul of water to the cells.

By adding 40 ul of water you would effectively replenish the remaining conditioned medium with the ~10% evaporative water loss that it had experienced

Neurons seem to respond well to this. You could try this out on your cells to see how they respond.

Hope this helps

Hi Jostein, Its the same problem.. even my cells when they are less confluent die more when compared to cells which still are confluent.. now i replace the media pre warmed to 37 degrees and then change it one by one.. somehow they are surviving but i still feel its not the same as it used to be previously wen i cud replace with room temperature media and replace in all wells together.. lemme know if it helps u

Increasing the seeding density (apprx. 100% confluent at 24 h), incubating for 48 h before media change and using 48/96 well plates without using the outer wells solved the problem for the LLC-MK2 cells.

Hi Jostein,

Alot of good suggestions so far: Certainly carrying over some old growth media may well help. You may also like to pre-equilibrate your media in the CO2 incubator prior use to stabilise the pH and add this to your cells since a pH shock may well cause problems with yours cells

all the best

Ian

I'd add to the same line of problems the experience I had this morning - 3t3-L1 were seeded three days ago and today became 90% confluent, so it was time to change media and keep them confluent for 2 more days. I took out 80% of the old media (in T6 well plate and 25cm2 flasks) and added pre-warmed media from the very same bottle, from which growth media was taken and used for the initial seed. Then, after the change, the plates stayed for some 15min in the incubator and then, for "just in case" I had a look at them. What used to be a nice almost confluent monolayer, now became a battle field with empty spaces, small quantity of detached cells and confluency of about 60-70%. I have no logical explanation what have caused that and where the cells just "dissapeared" - I am always very careful when changing media, never pipet onto the cells, but on the wall, etc. Hypoosmotic shock and cell burst ? But the media was exactly the same in which the cells used to grow until formed monolayer.... Suggestions ?

Patar - Ian's point about equilibrating with the CO2 to achieve the correct pH is actually worth considering as you can get osmotic transients from the hydrogen ion flux that would/could induce lysis. There were papers on volume regulation in nucleated erythrocytes from necturus by Peter Cala showing regulatory volume increases & decreases depending on whether it was a rise or fall in pH.

Petar, The 3T3L1 line is adipogenic. So, these cells can differentiate into fat/adipose cells which are light and can float. There will be some sponataneous formation of fat cells as the cells become confluent. Its best to passage them BEFORE they reach confluence. Also, insulin concentration in media must be under control-more insulin means more fat cells! I hope this helps.

Dear Venil, as you said that it,s best to passage 3T3-L1 BEFORE they reach confluence? do you have any experience to passage them AFTER confluent? and is that passage available to analyze the expression of gene? because i have some passages which are from the confluent subculture.

Sorry, R. Sitepu. I do not have access to these cells. Since you have some Passages from confluent cultures-you can try replating them at low density. You can check papers and find what media people use. Try these media, and see how fast your cells grow. Now, you can re-passage them before confluence--it is tricky, but may work. Hope this helps!

Thank you very much for your answer. I have tried to find out papers about what media people use for passage confluent culture. But, I haven't got it until now. so, for this issue, what is the term or topic that people talking about so that i will try to find it again? appreciate for your answer. Thank you.

R. Sitepu, You can use the regular 3T3L1 media for growth and passage. But insuolin and other factors are added to the media--that is what you should check from papers. I already explained about too much insulin.

You may spend much time and money on these confluent cells-and still not get good data. So, It maybe better to start with new early passage 3T3L1 cells from ATCC.

Hi, is anyone has a solution for this? I just experienced this problem too, but only in wellplates. The nucleus detached shortly after I changed the medium.

Dear Ruchi,

I would not exclude an osmotic shock...

Did you check for excess of evaporation in your incubator? this could lead to actual increase of solutes in the growth medium. cells may undergo a continuous "silent" adaptation while growing, but the "new" medium added may suddenly represent an hypo-osmotic, stressful situation. 

anyway, please consider other issues possibly inducing osmotic stress.

Anyone found a solution for this.

I have the same problem with all cells I work with and it has been years working with them without ever experiencing this

Ifsudden detachment of cells persist, its best to check with a fresh, low passage batch of the same cells and culture them on plates with/without coating suggested by ATCC or the original developer of the cells...sometimes these original papers have valuable clues!! Media should have correct pH, osmolarity, and FBS as mentioned by others earlier.

Good Luck..I know how annoying it is to loose cells in the midst of a series of related experiments!

Hi Ruchi, Did you solve the problem?

I'm culturing iPSC, recently after I thawed my cells, I got many round, grey, oil droplet-like things attaching to the cells or in the medium, exactly like the ones you circled in your pictures.

And most cells didn't attached, the attached ones are also dying.

I changed new medium and coating material, but the same problem persists.

I'm wondering what are these?

I am facing similar kind of problem in my MSCs culture although the detachment started after 5-6 days (with old media though). Can you give a piece of advice how to curtail it in my culture?

I am also interested into any additional insights on this or whether those of you having problems found a solution. For the past month, any cell line I grow shows signs of clumping and loss of adhesion within the first 24 hours of bringing them up from freeze. I have tried new lots of media, flasks, and FBS. I have tried different cell lines from both recent and several year old freeze dates, and the phenomenon recurs without fail. There are no visible signs of contamination - I do plan to test for mycoplasma soon, but the rapid onset of this noticeable effect in every culture within a day of being set up does not seem consistent with what I know about mycoplasma contamination. Any thoughts are greatly appreciated!

Thank you, Tanya, I had a new tank arrive late today and plan to try that tomorrow - I'll keep you posted here. Do you recommend airing out the incubator for some period of time before installing the new tank - just wondering how long I might need to rid the chamber of any toxic gases that may have come in. Thanks for your insights!

Thanks, does the incubator door need to be left open during that 6-12 hours? Do you just clean with 70% ethanol? Sorry for all the questions, just want to try to do this correctly so we can get things back up and running!

I meant prior to the cleaning for the 6-12 hour gas exchange. I've had the incubator turned off (with door closed) overnight. Should I leave the door open for 6-12 hours and then clean before rebooting the unit?