We are culturing A549 cells from last 3 months, but recently a few discrepancies are being observed. Cells are revived well (Figure-01), but from the first passage there is a change in the cell’s behaviour and appearance also. Unable to comprehend it, please help us to know whether it is the mycoplasma contamination or something else.
Following issues were noted:
1. Trypsinisation of cells is taking much longer time (previously it was between 1-2 minute, nowadays it is more than 8 minutes; Trypsin-EDTA 0.25%).
2. Cells morphology has changed from less elongated to more elongated and less elevated (Figure-02 & Figure-03).
3. Little bit patchy growth and one more peculiar thing which we noted was that there is a significant increase in the stickiness of the cells (by appearance under microscope; Figure-04).
4. Prior to facing these issues, we have also experienced bacterial contamination twice of two cell lines NCI-H23 and A549 cells.
The photos are quite small so it's hard to say, but Fig 3 looks almost like HEK293T cells. Is it possible your cells are contaminated with a different cell line, and those cells are out-competing the A549s? There are protocols online you can find to test your cells for HeLa or 293T contamination. Although I would expect HEK293T cells to be trypsinized much more quickly, not more slowly, than A549s.
Just to clarify, you aren't culturing the same cells for 3 months right? Cells should not be passaged for that long, they will undoubtedly undergo changes. Also some of your photos look to be a bit high confluency; growing cells at extremely high or extremely low confluency will also cause changes in the cells.
Mycoplasma contamination is easy to test for by PCR. The previous two bacterial contamination issues that you've had have been with mycoplasma or a different microbe? Do you grow your cells with pen/strep?
Ciaran Daly Thanks Ciaran for your response!
Possibility of contamination with another cell line is negligible, as we culture only two cell lines, namely NCI-H23 and A549.
Yes, your speculation is correct, we are not culturing the same slot of cells. We do the cell revival with earlier passage number to refresh the cell's stock.
We are in the process of purchasing Mycoplasma detection kit.
Previous two contamination was surely bacterial contamination as the media of the flasks became turbid. Yes, we grow our cells in presence of both the antibiotics (pen/strep) and anti-mycotic solution.
It would be very kind of you if you can guide us on the increased stickiness (by appearance, Figure-04).
Munmun Kumari
Unfortunately I have never observed cells that change their stickiness over time, so I have no advice regarding that. The best solution to this issue would be to re-purchase a stock of A549 cells from a reliable source like ATCC, although I realize that is not an ideal solution.
Mycoplasma contamination can be easily detected with PCR, there's no need to wait for a detection kit. See this paper: Article Detection of Mycoplasma in cell cultures
Dear Dr. Munmun Kumari
The human alveolar type II cells are known for their role in innate immunity. They express surface molecules necessary for efficient antigen presentation. A549 which is a type II alveolar epithelial cell line, express cell adhesion molecules (CAMs) on the surface when infected.
As you mentioned that A549 cells experienced bacterial contamination twice, could be the reason for the abnormal phenotypic behavior. Longer time taken for trypsinization could be because of the increased expression of CAMs on the cell surface that experienced bacterial infection twice. Please note that CAMs help cells stick to each other.
As Ciaran Daly mentioned, you need to purchase a fresh vial of A549 cells.
Just a suggestion. Whenever cells get contaminated in culture, they should be discarded at the earliest. Even if you succeed by treating the contaminated cells with antibiotics, they are not fit for further culture and cell-based assays because they will no longer show the original characteristics.
I have attached a few references below which you may want to refer.
Article Microbial uptake by the respiratory epithelium: outcomes for...
Article More Is Not Always Better—the Double-Headed Role of Fibronec...
https://erj.ersjournals.com/content/erj/15/2/358.full.pdf
Regards,
Malcolm Nobre
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