Hi, I am conducting experiments on growing A549s on agar gel. I am using RPMI with HEPE as my media. My problem is the A549s seems to proliferate much slower compared to those grown on tissue culture treated flasks. Media(phenol red) on the gel tends to turn magenta in less than 24 hours possibly due to lack of growth. What are some methods I can try to maximize growth condition on agar?
Yes, we do have DMEM. However, our CO2 incubator is broken so we switched to a media with HEPE.
Hi,
I obtain excellent aggregates of A549 cells when grown on 0.5% agarose gel prepared in plain DMEM. The number of cells as determined by the required seeding density (depending on the plate/petri dish) are suspended in DMEM supplemented with 10% FBS and poured on top of the gel.
We can obtain either floating aggregates or partially embedded aggregates of A549 cells on agarose gels by changing the % composition of the agarose gel.
Hi Maddaly,
Thanks for the advice. How were you able to change the medium without taking out the floating cells? and also, were the partially embedded cells in their normal triangular morphology? (when we normally grow them on cell culture-treated flasks)
Finally, did the cells grow exponentially? because what I'm seeing now is that cells grown on 0.7% gel tends to die out slowly, with minimum cell division..
Thanks
Hi,
I harvest the aggregates once they grow to a significant size and use them to study protein profile differences. So, I did not have to change medium when floating aggregates develop. But, we can visualize the aggregates quite clearly and should be able to change medium partially without disturbing the cells.
The cells grown on agarose hydrogels have a markedly different morphology compared to their monolayer counterparts. They do not have the spindle shape as they do not attach to a firm substratum (as with flasks) and tend to adhere to each other, forming aggregates.
Yes, cells do grow exponentially, but the cell culture stages (lag, log and decline phases) are typically much extended for 3D aggregates compared to their 2D monolayers grown in flasks. Monolayers tend to reach the decline stage much faster than their 3D counterparts.
Hi Maddaly,
Thanks for the detailed information. I have been seeing these aggregates since I started culturing A549s. My research goal is to create a 3D culture that mimics bile duct tube, I chose agar scaffold because I can easily shape them into tubes with lumens. Would the cells grown on agar (aggregates) best represent live A549s developed in human? Would adding poly-d(l)-lysin help with morphology?
Hi,
I am not too sure about agar scaffolds and their uses to shape body parts; I have not worked in that angle.
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