I have read a lot of great ideas here. What troubles me is that 3T3 cells are usually pretty hardy. I've not cultured human embryonic kidney but I've worked w/ BMKs. Same scenario: hardy cells. You're telling us that of late EVERYTHING has bombed; cells are floaters. Unless you've done something really out of the ordinary lately with your plates (like UV irradiation), allowing solvent/cleaner fumes in the incubators, or got some mechanial vibration going on, you may well have contamination. (You've already tried a different media source that a colleague gave you--that didn't work.) Sure, you could test for mycoplasma (other infections you can often smell; myco you can't), but that's labor-intensive, and may not be cost-effective. My tack would be to ditch everything (if you can, that is), clean that incubator (be careful of fumes when you do this!!), start with fresh media for each of your lines, pay close attention to shelf-life of nutrients (gln) in different media, buffering capacity (but you may have had that worked out). My mantra: when in doubt, throw it out, clean up, start fresh. Best of luck to you.

You might want to try a different source for your plates and dishes. Some companies treat the surface of their plastics differently. Or if you want to stay with a certain brand of plasticware, you might want to try a different lot.

Susan Ross

We coat flasks with gelatin 0.2 %. You can buy a solution of gelatin 2% for culture (either Sigma or gibco). You can also prepare 2 % solution form powder porcine or bovine gelatin for culture (MP or sigma) by autoclaving the gelatin and then prepare a 0.2 % in autoclaved water. Coat 30 min the flasks at 37 C and aspirate the gelatin. No need to wash. We regularly use this for HEK 293 and NIH 3T3 cells. I don't know for the others.

We thought about gelatin because we worked previously with endothelial cells and we used to use gelatin as an cheap way to coat. Indeed you can use also fibronectin but this expensive.

The only limitation will be if you are studying matrix effect.

Hope this will help

Check that the incubator has water and is fully humidified.

Susan Ross is rigth. Perhaps could you also verify that your incubator works well and to be sure you could also ascertain that there is no contaminations (such as by mycoplasms)

If other people using the same incubator? Mb smth wrong with oxygen concentration? Are you sure that medium did not expire or it has all nessesary components like FCS, glutamin ect.?

Did you check if these cell lines need a specific subtrate in order to get attached to the surface? (i.e. collagen, fibrinogen). For how long do you tripsinize the cells? I know that cells get detached when are over tripsinaze. Another thing it could be is mycoplasm contamination, but you have tried with cells from different sources, so, maybe this is not the case. Does you medium contain attachment factors (usually in FBS)?

That's all I can think.

Gook luck!

have you changed your FCS batch?

I had this problem with a new batch of FCS

Check that there are no vibrations in your incubator

If there is no problem about heat and pH, it may be the CO2 level. I think % CO2 level inside incubator is the problem.

you may use gelatin (0.1-0.2%) coated plasticwares. Also I am curious to know if the confluency of your cultures is too high or if you are seeding cells having clumps. Because my personal experience with HEK293 and NIH3T3 has been that in high confluency these cells tend to lift off.

Have you determined the presence of Mycoplasma in the culture?. Often cells die due to the presence of mycoplasma. In addition, It would be interesting that a technician check your incubator.

please check the humidity and CO2 level

I think Melissa could be right. Usually CO2 is the problem. Check also the water. Mycoplasma contamination should be routinely checked, if your cells are positive you can have a big problem.

I have to agree with the others..problems occured in various type of cell...its got to be the incubator..check the water, Co2 and temp level...

I agree this could be a humidity or CO2 level problem.

I think is defenitely a CO2 problem, measure it adjust it and it will disappear.

How much serum do you add to your medium (10% or lower??). Serum is crucial to keep these cell attached. Have you cultured any other cell line that did not detach...

Are you using all disposable material?. If not check the quality of washing. One of major contributors to this kind of effect is cell culture medium. Please check the batch you are using, the serun (as somebody recommended already), the quality of reagents you add and more important the quality of water you are using (conductivity and TOC)

I also think that could be something with the incubator since you can see this sort of "gradient effect" on flasks from the neck to the bottom. After checking fr some contamination (mycoplasms first) try to find some space in another incubator just to test if something changes.

It may be the CO2 and it should be measured. Again, you have to check the cell contaminants (ie, mycoplasms) by using appropriate kits or DAPI staining and the cell medium, including FBS or other components.

Another question: do you accurately remove the trypsin from your cells?

However, you could try to maintain the cell lines in another cell incubator and then change completely the components of cell medium.

What do you use in the water pan to control fungal growth? I have had problems with chemical toxicity (from fungi that grow in the water for humidity or from fungal suppression chemical) killing cells in my cultures. One was a commercial product that a student put at 10X in the humidity pan. Not a good end

Dear Neeraj,

First of all you should check microscopically that is there any contamination in your culture. you can inoculate the discarded medium into the agar plate at the time of passaging, then check, Is there any bacterial growth in agar plates?

If there is no contamination please read the literature about the medium constituent of that particular cell line. If any ingredient is not available in your medium, you may face these problems.

you can also check growth factor concentration and its sterility......

check all these things and then tell me what are the results...........

Thank you all for your suggestions. I have been using the same lot of plasticware, though i have changed FBS...so I need to check if thats causing the problem. I have not checked for mycoplasma but there are no signs of bacterial or fungal contamination. Anyhow, have got the incubator recaliberated for temp and CO2 levels.. Hope the cultures will survive now. Will update you all. Thanks.

Dear Neeraj,

It seems there is fungal contamination in the incubator along with Co2 issue, so, fumigate you hood and use fresh cultres and fresh media, it may solve the issue.

i agree this could be a humidity or CO2 level problem as well may there is a fungal contamination

Dear Neeraj, check the incubation conditions, FBS and mycoplasma contamination as suggested by Anna, Enrico and others. Try to change the batch of plates/flasks you are using at the moment. As you said it is all of a sudden I hope you have good handling and better understanding of these cell lines also keep in mind the suggestions given by Pankaj. Good luck.

You can try by adding extra amount of serum & also check the CO2 level in your incubator. I should be 5%.

1. Check the water in your incubator whether it has any contamination.

2. Check the quality of the flask and petriplate, it all comes with precoated for adherent cells, some time it may be bad

3. What is your cell line passage number?

Suggestion:

1. Get a flask from other lab and grow your culture in it, and also give your flask to check them

2. Calibrate the CO2 levels in incubator

It's quite obvious the FBS is the reason... It's there to bring the principal first ECM component so the cells adhere and will then synthesize their own ECM. Make sure to heat-inactivate it before use, that could be the reason why it's going less efficient.

If you have some, try to coat your plate with fibronectin, collagen vitronectin or any ECM protein and look if it adhere better!

Good luck

I think if the pH and temp. and co2 is OK, maybe the media components including L-glutamine may be checked. Why would it start detaching from the neck side when the cells are supposed to be plated on the flat side of the flask ?

Hey Neeraj.

I have experience of NIH3T3 cell culture.

1- These cells grow better in T25 or 75 instead of 35 mm disc or 100 mm culture disc.

2- Change medium after 48 hr( not after 72 hr)

3-Use of NEAA or Glutamax is not effective for 3T3 cells

Good Luck.

Be sure that all environmental conditions such as temperature and CO2 are correct. Check that the number of cells you plate in each flask or plate is adequate for the type of cells you are working with. As last point be sure that the cells you are using can grow directly on plastic or they need another support such as gelatin, collagen or similar. Hope you can solve your problem as soon as possible!

If you are sure that all parameters of your incubator are OK, you have to test all parameters of your media especially decomplementation of your FBS

Dear Sir, Did you use in your media composition non essential aa? Last week I have face the same problems with my bWJ-MSCs and it was non-essential aa... Lets try

Are these cells that you have grown before without any problems? If so, what have you changed since then - new source of plasticware, new batch of serum etc.

I have no further comments. I would nevertheless suggest to try another incubator, in a nearby lab and to test another FBS batch (and by the way, why FBS instead of FCS?).

FBS (foetal bovine serum) and FCS (foetal calf serum) are the same product - as distinct from newborn calf serum (NCS) - if that is what you mean by your question Bruno.

Chek the amount of iron ions on your medium formula, generally low concentration of it can cause this. By the way, sometimes, specially for fibroblasts, I use 10% of FBS and 5% of BF (bovine serum) in the first two passages.

As Annemarie Koch was saying, I would also check for mycoplasma contamination if the problem persist with new plate/medium.

Hello,

Are you using the same incubator for viruses for example?

I think that you are the only one able to solve this problem!

actually, you are familiar with your cells. So, you should analyse all the products used and define what you changed.

If many factors were changed, test them one by one.

GOOD LUCK

Be sure 10% FBS is added to the media, also you can try glass petri dishes, glass some time is better for binding. Actually you can add an sterile coverslip to your plastic petri dish to see if this improves the attachment.

Please check your cells for mycoplasma or any bacterial contamination. Please check heat and pH,and CO2 level again. Use 10% of FBS and 5% of BF (bovine serum) in your media. At the final you might want to try a different source for your plates and dishes.

If it was pH or CO2 the the media would be obviously a different color if you use phenol Red if you don't use phenol red media then find some and put some in a plate or flask and leave for an hour. Heks are often not very adherent but 3t3 should be well attached. If you buy comercial media then make up is undlikely to be a cause 3t3 will gow in just above anything and are very resitant to changes in serum conc. I would have said check that someone has not ordered bacterial plates by mistake but you say flasks are showing signs, which is highly suggesitive of infection. Look for fine hairs in media fungi, very some dithering objects at high power early bacterial intection niether of these suggests micoplasm as mentioned above. All cell lines also suggests there is an infection do you the same botle of media for each line. The pipettes can also habour infection and deliver as an airosol between plates so if infection proves to be problem and you are not sharing media you will need to discards cells, unused media, the 0.2µm filter from pipette and clean the hood. Check with other users for there issues. Incidently have you check the water in the bottom of the incubator, is there any?

My initial thought on reading about your cells is a mycoplasma contamination. You can get a get to detect or even run PCR.

1- chek your CO2 level

2- chek youe incubator for any contamination( viruses,mycoplasma,..)

3- try to use anotherr labware sources (petridishes, plates,..)

4- chek your cell line for having excessive passage number

Contamination

I agreed with Susan. the problem could be the plastic plates and dish lot

Try using poly-L-lysine or collagen.

In addintion to all others' comments, pls check your incubator filter. It might be very old and dirty.......also the water level, if it is dry, the temp. will not be consistant. Of course, you can change a incubator....:).

ZYX bioreactor provide you a reliable culture condition by its computer controlled system and complete culture analysis data, but it is too expensive.

Good luck.

Sounds loud like contamination as many already suggested. In general contaminations can be avoided if all your work surfaces including hood are working properly and cleaned before and after use. In addition the researcher is the most likely source of mycoplasma (for instance from finger nails etc...) to cell cultures and is supposed to wear gloves and spray with 70% EtOH as many times as necessary while working in a sterile environment.

First of all, you should transfer your plates and flasks to another incubator in a nearby lab....This would give you a key indication with respect to your own incubator.

Solutions.

1. check CO2 levels in incubator.

2. Check mycoplasma contamination by PCR

3. change incubator used for incubating the cells

4. change the plasticware used for seeding the cells

5. Coat the plates with poly-L-lysine or fibronectin

Good luck

Just to be sure,,, add more serum,,, 20%

that also helps!!! : )

did you check the CO2 concentration?

If the pH readings are acceptable, the CO2 levels have to be accurate (assuming you have not changed the buffer). No one has yet mentioned osmolarity. If your incubator has a low relative humidity (there is no readout for that), then you will have an osmotic shock every time you feed. That will detach cells. Monitoring the osmolarity in a test flask over 7 days will give you a good idea of osmo drift. Cell detachment in flasks from the neck side with progression to the rear is an interesting clue. If you generally add medium near the front of the flask during feeding, I would suspect osmotic shock. Most contamination (except for myciplasms) should be visible microscopically, especially if you keep a test flask or dish in the incubator for a few days. You could have a general contamination that may originate with the feeding medium.

Did you check the percentage of CO2? Or the ventilation system? Some cell types are sensitive to both the parameters, especially when they resulted altered together.

Some time ago, we have had problems in our incubator, concerning both CO2% and ventilation, and the cells grew unevenly.

Did you introduce in culture materials that could exert toxic effect on the cells?

Many years ago, in studying the cellular response to some biomaterials, we observed that seeding the cells on a biomaterial in a Petri-dish, the cells near the material detached and died, while far from the material (peripherally in the culture) the cell were viable. The investigation showed the toxic property of that biomaterial.

Have you recently changed the supplier for disposable plastic? Some cheaper materials are less suitable for cell adhesion.

Did you exclude any incubator contamination? Some fungal contaminations are not clearly recognizable.

Are the detached cells alive or death? In the first case, the problem could be related to the plastic holder.

Best wishes for your cell cultures

Check your cells shape. if their shape are abnormal , change your cell sours. check virus contamination in incubator, and another contamination

I dont think mycoplasma contamination would detach the cells, obviously its affect ur results..... I am using same 2 of ur cell lines HEK and NIH3t3, better u check ur CO2 con. I think it should be 5%, apart from this check your medium thoroughly, may be a contamination in the medium or contamination in the incubator... Starting from fresh resources, could solve ur problem... gud luk

May be due to unproper sterility during washing or unwanted contamination

All of the above recommendations are good, (I suspect the plastic surface treatment for this batch or this company's TC plasticware, as we have had that problem before) but a few other steps might help if the problem has not been eliminated by correction of the already dicussed possible causes. 1) Add HEPES buffer to your medium. This will damp out the CO2 dependent pH changes in your medium (particularly helpful with Petri dishes if taken out of CO2 incubator for extended times). 2) If the medium has been stored in liquid form, even at 4 degC, the glutamine tends to be the most labile component and addition of a 1% supplement of InVitrogen/Gibco's Glutamax (a glutamine dimer that is very long-lived) could solve that problem. 3) If this is a new problem in this incubator, there could be a problem with vibration (often the fan is the problem) causing cell detachment.

I also think you have to check for mycoplasma contamination.

Calibrate the incubator CO2 levels with any of the available calibration kits in the market and see if the problem persists. It is trivial, but the water level in the reservoir is also very important.

Mycoplasma contamination can induce detachment of adherent cell lines although it is unlikely that all your cell lines have become contaminated at the same time. Poor quality serum or low concentrations can also induce detachment. In fact low concentrations of serum is a technique used to encourage the growth of adherent cell lines into suspension. Sounds most likely a common denominator such as plastic quality or a serum problem. Have you changed to a new stock of serum lately? Have you used a different serum in your borrowed medium? You could also try a different brand of flask from the other lab.

Good luck,

Lisa.

I agree with Lisa. I would say it's a serum problem, or mycoplasma. If it will not interrupt your assay needs, perhaps consider adding 1% anti-anti to your medium, at least until the problem is alleviated.

I agree with all comments. Sometimes less percentage of serum also will cause this problem check the batch and expiry date of the serum and also the Co2.

It depends on your cells, some cells will no tolerate rough treatment, such as trypsin treatment, though short exposures won't hurt generally. I incubate cells with trypsin either at room temperature or at 37 until they start to detach with a bit of tapping. This usally occurs within 5 minutes. Never go over 10 minutes with trypsin as it does horrible things to the cells (expression, morphology, you name it) if you repeatedly do it.

Check whether the Co2 level is maintaining at 5% otherwise better you would use another Incubater for a week. As your all cell lines and primary cultures get affected means due to contaminated by slow poison (mycoplasma). You could not find this contamination as much easy. So, Please check the contamination with fluorescent dye (Hoechst) in a flask.

I have read a lot of great ideas here. What troubles me is that 3T3 cells are usually pretty hardy. I've not cultured human embryonic kidney but I've worked w/ BMKs. Same scenario: hardy cells. You're telling us that of late EVERYTHING has bombed; cells are floaters. Unless you've done something really out of the ordinary lately with your plates (like UV irradiation), allowing solvent/cleaner fumes in the incubators, or got some mechanial vibration going on, you may well have contamination. (You've already tried a different media source that a colleague gave you--that didn't work.) Sure, you could test for mycoplasma (other infections you can often smell; myco you can't), but that's labor-intensive, and may not be cost-effective. My tack would be to ditch everything (if you can, that is), clean that incubator (be careful of fumes when you do this!!), start with fresh media for each of your lines, pay close attention to shelf-life of nutrients (gln) in different media, buffering capacity (but you may have had that worked out). My mantra: when in doubt, throw it out, clean up, start fresh. Best of luck to you.

I agree with Lisa. When mycoplasma is causative, tetracycline might be effective to remove it. Wash cells with a medium or buffer with a high concentration of tetracycline once or twice and then culture in a normal concentration.

I think all of the above comments sound the case. CO2 and mycoplasma contamination are the big problems to be solved for all researchers who are involved in cell culture works. Good luck to you

I agree with above comment, this problem in old age culture also seen

I agree with Lisa , the Myoplasma coild be the casautive.

First maintain extreme sterile condition during culture. Increase the concentration of serum (sure that its procure from good brand).coat ur plates with gelatin.gud luck for ur experiment.

I agree with Nancy. Clean the incubator or use another Incubator for a short period of time. Good luck!

Did you use the same amount of FCS or BSA? I've always problems with cells deteachment when I try to use medium without any serum support. Besides the Mycoplasma might be a reason of that situation.

We also meet this problem. And we also think the reason is 6 well cell culture plate, which come from costar(3516). So I would like to know what kind of 6-well plate to culture adipose-derived stem cells?

thanks!

If possible check the carbon dioxide percentage in your incubator.

Do you use the same medium and serum? We had such a problem with liquid medium, deficiency in glutamin was the reason, addition of 0,3 g/l glutamin solved our problem (we used primary culture of thyroid cells).

You can also check the PBS, Media solution (pH/ concentration) and serum (LOT) but, I really agree that a clean up on the incubator may help.

If cells from other lab, was good, "also detached once kept in our incubator", have some one come to check the CO2 level at first. But you can see the difference from the color of medium if the CO2 if not right, normally. Then do a thoroughly cleaning of your incubator. I mean really thoroughly!!!

Are you using trypsin.

Based on what you said...did you try another incubator?

I would definately do what Nancy suggested, Chuck everything and start afresh. This is the most easy and economical way to go about this. Clean your incubators and hood. Start with new media for each cell line. I may also suggest aliquoting 50mls of your media so your avoid contaminating the entire batch. Seems had but it's actually very easy and safe. Good luck!

One other reason caused cell detach is the cells over grown. They overlap each other. The bottom layer of cells which sticking to the flask or well can’t reach the culture medium and die. To prevent this happening one is don’t let them over grow, the second is pipette the cells up and down to separate them into single cell before plate them into the flask or well after trypsinized.

See, firstly, the media for cells like 3T3 and hMSCs would be different. You should check that first. Secondly, there is a definite proportion of the tissue culture flask and the corresponding media volume. Like for T-25 flask, the average amount of growth media should be around 6-8 ml. For T-75, it should be around 25 ml, for good growth. The, for other parameters, I would suggest that you should clean the CO2 incubator completely with sodium azide to counter any possible fungal infection. And as passaging step, after you put trypsin, do remember it to wash with incomplete PBS, so that trypsin remnants may not cause detachment problem. Do check for the cell density before seeding the next time. All the best.

When adherent cells suddenly start to float, these could be the potential reasons: MAINLY REMEMBER THAT WHEN SOMETHING GOES WRONG, YOUR NEED TO DO A THROUGH POST-MORTEM TO IDENTIFY THE CULPRIT.

1) Incubator problems: CO2 concentration, humidity, mycoplasma contamination (check with other users of the incubator if they face similar problem. If you are the only one facing this problem, then it’s not the incubator issue). A cell culture incubator should only be used for cell culture. I hope you don’t share it with some bacterial/ viral culture.

2) Media problems: Lack of serum/ glutamine/ supplements (check its expiry date too. You can pass some of your culture media to someone else to try with their adherent cell lines, ensuring that your culture media is suitable for their cell line.)

3) Culturing problems: Improper washing of typsin residue post tripsinization ; culture confluence (at what confluence does the cells start to float??); insufficient support for adherence (coat the plates with fibronectin to support cell adherence ); be gentle, especially when changing media.

4) Has anything changed recently?? Ie. New type of petri dish or different brand/ new batch of media or media supplements / change of serum concentration / change of supplement’s brands / etc ?

5) You said that the cells are detaching from the neck side of the flask and gradually progressing down the flask. Is that the place where you pipette in the new media?? Be gentler, if the cells have weak adherence, it’s best for u to drip the media drop by drop.

Hope it helps.

Check the quality of CO2, sometimes it contains CO.

Thank you everyone for your suggestions....my cultures are doing much better now....got the incubator temperature and co2 recalibrated...cleaned the incubator....installed new CO2 cylinder. Thank you once again.

I think you have to think about seeding density of your cells

I have my own experince with NIH-3T3. cells.these cell start deattaching when they are near the confluncy. split before This stage.

For hMSC after a limited passage they gain "fried egg morphology" So check the passag no of hMSC..

I agree to Huika Li suggestion. "One reason caused cell detach is the cells over grown. They overlap each other. The bottom layer of cells which sticking to the flask or well can’t reach the culture medium and die. To prevent this happening one is don’t let them over grow, the second is pipette the cells up and down to separate them into single cell before plate them into the flask or well after trypsinized."

There might be several reasons that resulted in the detachment of your cells.

1. Culture culture medium are contaminated by bacterial, yeast, or mycoplasma .

Suggestion: thoroughly clean incubator, hood. Toss all old medium including FBS, P/S, Glu.

2. Flask is not appropriate.

Suggestion: try the flasks or dishs that work very well for other lab.