Problem with HepG2 cell culture

[evilhamster]

i think this is the second time i tried posting this- there seems to be a problem with the last time i tried.

uh, ive been trying to culture hepg2 cells for my thesis, but they dont seem to adhere very much, and the floating cells seems to grow, and then eventually die. i thaw them and add 10% FBS in MEM dropwise and replace the media with DMSO a few days later, and they still die. please help. i don't put any pen/strep and am under the assumption that these hepg2 cells should be easy enough to culture, but damn they form aggregates/clumps and just float. the attached cells dont seem to multiply much. in some of my trials the floating cells multipled after a few days, but then almost instantaneously die, without the media changing colour. i tried tranferring the floating cells to another flask, but the previous flask's cells died too.

any advice on how to properly grow the cells? im in dire need of assistance


please please please help.

[admin]

Hey there do you have some in liquid nitrogen?

I would take a fresh stock out and start culture from there. These cells do not require any special fibronectin or coating on the plates, and actually grow and adhere very well.

There must be something wrong with the cells, or incubator conditions. What passage number were your cells? Is the CO2 incubator working well and other cell lines in it growing well?

[evilhamster]

Originally Posted by admin

Hey there do you have some in liquid nitrogen?

I would take a fresh stock out and start culture from there. These cells do not require any special fibronectin or coating on the plates, and actually grow and adhere very well.

There must be something wrong with the cells, or incubator conditions. What passage number were your cells? Is the CO2 incubator working well and other cell lines in it growing well?

unfortunately the vials do not have a label as to what passage number they were.

other cell lines seem to grow quite fast, much faster than the HepG2. we have DU 145 that grow in 24 hrs or less...

i've tried using different media (DMEM:HAM, MEM, RPMI, DMEM, media with ITS, i've yet to try media with HITES; all in 10% FBS) and the HepG2 still seem to die. strange though, i was under the impression that they are not hard to culture.

[evilhamster]

by the way, i can't seem to delete this other post of mine. there doesnt seem to be a delete option =/ i think there was an error in posting. help? XD

[evilhamster]

no idea on the passage #. other cells (DU145) seem to grow quite well. i've tried quite a lot of media (DMEM:HAM, DMEM, MEM, RPMI, media with ITS, but not yet HITES)

oh and also, i've tried separating the floating cells into another flask, but both the original and transferred cells eventually died. those that do attach, however, dont really grow as much. i've also tried culturing the cells at 20% FBS to induce growth, to no avail . i have cells in a T25 presently, with floating cells that seem to multiply.

[robinshi]

I think the problem may be in the thawing process.
The DMSO should be removed immediately after thawing, not after several days of culture, cause it may be toxic to cells.

[evilhamster]

hah, the old hepg2 were mycoplasma contaminated. XD we ordered new ones, theyre growing fine. such beautiful cells.

[Juji]

Hi there,

seems a bit strange to me too... you will find that almost all cells will have some kind of mycoplasma contamination... and they will still grow (from my experience with primary cells, HeLas, 3T3L1s etc etc). I'd agree to 'robinshi' or I'd suggest they were frozen away the wrong way... doesn't matter in the end you'll get your new cells in anyway. Enjoy

[wr_1a]

thanks

[shaarawy]

To avoid clumping don't agitate the cells by shaking the flask while waiting for the cells to detach.

Too late just two years later.