Recently, we've had our NIH 3T3 cells dying at a high rate in a very short span of time (1-2 days) of culturing. We suspected contamination in the incubator/biosafety cabinet at first, and performed decontamination procedures, which didn't seem to help. We've reviewed our other operations etc to identify a possible change which may be contributing to it, but we're unable to do so. We tried starting new cell cultures from different frozen cell samples, and they all have a fairly similar rate of cell death. We've never had this issue happen to us in the past. Can someone offer possible solutions/suggestions?
For reference, we use DMEM supplanted with 10% BCS and 1% Antibiotics.
Have you examined them at higher power? Looked for tiny dots, or media turbidity?
Contamination (to my mind) remains the most likely culprit, but this can be fiendishly difficult to troubleshoot. When you say "decontamination procedures", what do you mean?
In my experience, it is usually necessary to switch out almost everything. Throw away your media. Throw away any open tip boxes, any open boxes of gloves. Change your pipettes. Use fresh bags of tubes, plates etc. Use fresh stocks of antibiotics and serum.
If possible, use a different incubator and a different flow hood just to be on the safe side.
The other (dreaded) possibility is that this is an infection that set in before you froze the cells down, and which is only manifesting now because cells are typically weaker and more vulnerable immediately after defrosting. Do you see the same if you defrost cells from a different batch (frozen at a different time), or cells from another group, ideally grown elsewhere?
Like Sandhya, I would suspect mycoplasma contamination. Regularly used antibiotics are of no use against mycoplasmas. Your media controls and other cultures do not have this problem? Throw away this batch and order a new one of 3T3.
Srikumar,
3T3 cells are usually fairly bulletproof, so there is probably a simple, fundamental problem causing this.
There are several possible sources for this type of problem.
1. As suggested by others, microbial contamination is possible. The images you provided are not sufficient to entirely rule this out.
2. An incubator problem, i.e., temperature, CO2, humidity regulation, residue from agents used to clean incubator. Do other cell types grow well in this incubator? If you or another investigator are using this same incubator, same flasks, same batch of media to grow another cell type and it is healthy and growing well, then many of these possibilities can be ruled out. Have you obtained a growing culture of some cell type from a colleague and put it into your incubator to see if it remains healthy?
***3. There are multiple steps involved in freezing, liquid nitrogen storage, thawing cells that could be the source. Also, cell damage may have occurred prior to freezing and the damage would not be seen until thawing is performed. If the frozen ampoules are all from a vendor like ATCC, then this is less likely, but still possible. Problems can occur during shipping, after arrival while ampoules are stored in liquid nitrogen (I assume that cells were never held at -80C for more than a day or two), or during thawing. Considering the timing of the problem you describe, freezing, storage or thawing seem likely to be involved. Do you check the cultures immediately after thawing and plating? Are the cells rounded and floating initially or irregular shaped, blebbed, or otherwise damaged? Do they settle and attach after an hour or so? Do they spread normally and look healthy after a few hours? When does the damage and death begin?
4. Problems with culture media including possible dilution errors when adding supplements, use of improperly or non-heat inactivated serum, etc. Problems with flasks used...are they tissue culture treated or are they bacteriological flasks? Flask in photo has a blue cap, blue capped flasks may be bacteriological (depending on vendor). Tissue culture cells will not attach and grow in bacteriological flasks.
This type of problem is very frustrating and you will probably want to kick yourself (or someone else) when you finally discover the source of the difficulty. Double checking everything is usually the only way to find the solution.
Good luck
RW
Srikumar,
Just noticed that the flask in the picture says “NUNCLON” on it. NUNCLON flasks are low adhesion flasks that are used for spheroid cell culture. This is a problem. Use regular tissue culture grade flasks for adherent 3T3 cells.
RW
Just to clarify, NUNCLON delta should be used not NUNCLON spheroid flasks.
RW
I second the recommendations of Robert J Walter. In particular, are other cells in the same incubator growing okay? If not, it could be the incubator CO[2] getting out of calibration -- this will hose even robust cell lines.
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