We noticed that our HepG2 cells have been growing in large clumps lately. Cells have been cultured in DMEM plus FBS and trypsinized according to regular protocols. They don't seem to be contaminated, although no further testing has been performed. Suggestions?
Hey Pedro, HepG2 cells do tend to 'clump' or produce 3D structures, more so when confluency approaches >70%. You could trypsinize your cells once they reach ~70% confluency; using slightly aggressive pipetting to remove the clumping, then simply add more media and wait for them to reattach (roughly 6 six hours), then change the media with more fresh media (this will remove a lot of the dead cells from the aggressive pipetting). Usually this results in unifrom monolayer growth.
Another method I have read about but never tried myself is to coat your flasks surface in a 1% collagen solution using a syringe and cannula/ long needle.
Hi Pedro,
I think you need to trypsinize and make sure every single cell separated before subculture. I havent experience yet such clumping. even if 95% confluence, my culture was okay.
regards,
mathipi
Pedro,
As we discussed previously, and as pointed out by Craig up there, HepG2 cells tend to clump. DMEM + FBS is a suitable media for cultivating HepG2, and trypsin should be used for spliting. Though it may not be contaminated, just remember to check for mycoplasma contamination every once in a while and create viable cell stocks as a backup. I don't know what you plan to do with these cells, but unless you are doing imaging, clumps should not be a problem.
Best,
Rafael Elias
Hello dear colleague
You should add trypsin 0.25% plus EDTA 0.05% to your culture, after detaching them , centrifuge 800 rpm/10 min. then wash them with PBS and re-suspended the cells with completed medium (DMEM+FBS, 10%) in new culture vessel (Plate/ Flask). It is recommended a gentle pipeting before adding the culture medium
Be SUCCEED.
M currently working on HepG-2 cells. Every time i subculture, i make sure, i re suspend the centrifuged cells properly before adding them into new flask.And its growing just fine.
Hello people, quick update here.
I got back to working with HepG2 cells and experimented with poly-L-lysine coated coverslips. To my surprise, the cells were very stretched out, similarly to HeLa and endothelial cells, and no clumping whatsoever.
I am not sure if it will be of any use, but good luck with your experiments.
Hi Pedro,
May I ask what protocol you used to coat your coverslips with poly-L-lysine? I am experimenting with different conditions/protocols to see which works best. Thank you.
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