Question

Cells become round and detached after changing to fresh medium in plate

I have been trying to transfect an endometrial epithelial cell line with a plasmid. But before transfection, when I change the medium of the cells and replenish with the same growth medium in which the cells have been seeded, the cells turn round after addition of fresh medium. I see round structures that are greyish black in colour. The cells start losing their shape, become round and start to detach and are afloat. The structures that I see look like lipid droplets and some such structures have a shrunk debris kind of thing attached to it. The media I use is DMEM+Hams F12 and supplement with penstrep and heat inactivated FBS.

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  • Ratneswary Sutharsan · Griffith University
    It looks like some sort of contamination. If you use a fresh batch of media, you would see the difference.
  • Rajendra Gangalum · University of California, Los Angeles
    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
  • Ruchi Kakar · Indian Council of Medical Research
    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.
  • Ruchi Kakar · Indian Council of Medical Research
    This pic is of the cells whose medium has been changed
  • Ruchi Kakar · Indian Council of Medical Research
    Another pic of cells whose media has been changed
  • Ruchi Kakar · Indian Council of Medical Research
    First and last pic are of cells in the flask
  • Ruchi Kakar · Indian Council of Medical Research
    Looking forward for your replies.
  • Jan Bandemer · University of Münster
    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?
  • Jan Bandemer · University of Münster
    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.
  • Indra Kusuma · Universitas YARSI
    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
  • Ruchi Kakar · Indian Council of Medical Research
    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.
  • Praveen Arany · National Institutes of Health
    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.
  • Ruchi Kakar · Indian Council of Medical Research
    Hi Praveen, what did you mean by high Ca/Low Ca mix?
  • Anu Jose · Union Christian College, India
    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.
  • Malay Chaklader · Children's Health Research Center, Sanford Research, University of South Dakota, Sioux Falls, SD, USA
    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.
  • Ruchi Kakar · Indian Council of Medical Research
    Hi Anu, I do the same thing of replacing half the media with fresh one.. Still I see cell death
  • Ruchi Kakar · Indian Council of Medical Research
    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
  • Ghasem Bagherpour · Pasteur Institute of Iran (IPI)
    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,
  • Stéphanie Claudinot · École Polytechnique Fédérale de Lausanne
    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
  • Ruchi Kakar · Indian Council of Medical Research
    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 :)
  • Bruce Lynn · University of Manitoba
    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
  • Didier JAMBOU · Centre Hospitalier Universitaire de Nice
    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
  • Josef Gotzmann · Max F. Perutz Laboratories
    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
  • Virginia Huxley · University of Missouri
    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!
  • Ruchi Kakar · Indian Council of Medical Research
    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
  • Stéphanie Claudinot · École Polytechnique Fédérale de Lausanne
    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
  • Virginia Huxley · University of Missouri
    silly question time - where are there photos & how would I access them?
  • Ruchi Kakar · Indian Council of Medical Research
    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%
  • Ruchi Kakar · Indian Council of Medical Research
    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 :)
  • Shuen-Kuei Liao · Taipei Medical University
    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!
  • Jostein Malmo · Norwegian University of Science and Technology
    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.
  • Nazish Abdullah · Oregon State University
    Are you using the right kind of tissue culture treated plates/flasks? Cells would sometimes detach if they don't stick well.
  • Jostein Malmo · Norwegian University of Science and Technology
    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.
  • Bruce Lynn · University of Manitoba
    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
  • Jostein Malmo · Norwegian University of Science and Technology
    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.
  • Vinita Singh-Gupta · Henry Ford Health System
    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?
  • Bruce Lynn · University of Manitoba
    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?
  • Jostein Malmo · Norwegian University of Science and Technology
    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...
  • Bruce Lynn · University of Manitoba
    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
  • Ruchi Kakar · Indian Council of Medical Research
    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
  • Jostein Malmo · Norwegian University of Science and Technology
    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.
  • Ian Wilkinson · The University of Sheffield
    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
  • Petar Petrov · University of the Balearic Islands
    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 ?
  • Virginia Huxley · University of Missouri
    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.
  • Venil Sumantran · Indian Institute of Technology Madras
    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.
  • Rehmadanta Sitepu · Bandung Institute of Technology
    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.
  • Venil Sumantran · Indian Institute of Technology Madras
    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!
  • Rehmadanta Sitepu · Bandung Institute of Technology
    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.
  • Venil Sumantran · Indian Institute of Technology Madras
    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.

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