HepG2 cells viability checked by Trypan blue dye was 855 before plating cells. After 24 hours of adhesion, I incubated cells for another 6 hours, 12 and 24 hours. Viability of cells reduced to 50% after 6 hours and remained the same untill 24 hours. I am just wondering that If it is normal or some contamination is there.
As per contamination, I have seen that cells have intact walls and growing in clumps, but they are very bright from inside as well.
What was your split ratio? HepG2 can grow to clumps if your experiment time frame is a little longer. If that is not the case I would suggest you change your stock.
Hi,
Let us put away contamination problem for a second... HepG2 cells are easily to grow in a number of media if you start from the properly prepared (frozen) sample. If you are using the cells from a vendor, just follow the instructions and let the cells to make several divisions before you start doing experiments. They may start looking OK after a couple of divisions. If the cells are home-prepared, there is always a chance their viability is challenged. Very often, one may easily damage the cells (during passaging them or for freeze-down stock) by overincubating them with trypsin. Make sure Trypsin stock has the right pH and put the cells in suspention immediately as they got detached from the plate. If preparing the stock - wash out all the Trypsin but do it fast.
So, let the cells grow for a couple of generations and if the problem persists, try and use a different batch of HepG2 cells.
Good luck.
Hi Abbas, I split cells in 1:4 ratio as per instructions, but sometime when I need cell early, then I just split them 1:2 so that they grow faster and I can use them. Can this be a problem?
Hi Oleg,
I donot leave my cells in trypsin for long. I incubate for 5 minutes at 37 degrees and then immediately put media into it. So I assume this is not an issue.
Splitting ration 1:2 is not a problem. Most of the cancer cell lines can be split between 1:1 and 1:9 without any problems (except it takes longer to obtain confluent plate with 1:9 ratio). If you state the cells are not damaged, and you have continuous problems with their viability I would purchase a brand new stock or ask for a new stock from another lab.
One more question: did you try and check cell`s viability 24 or 48h post splitting?
I mean, if they recover in 24 hours, then there is nothing to worry about. Splitting should give you certain reduction in cells viability, simply because it is stress, and after 6 h to 12h post splitting you should see some (though not many) dead/not attached cells in the media. Try and change the media after 12 h (do not split just remove the old media and add the fresh one) and see how it affects the cells.
Good luck.
Hi Monika,
The HepG2 are very tricky cells. Once splitted the cells need to recover (usually 48h)
The problem can be:
1) Diluition (i.e. few cells in a 75cm2 flask does not growth very well);
2) Medium cold, expired (i.e. opened for a long time), contaminated (myco or bacteria) or not well prepared (FBS, Earl's salts, Sodium Pyruvate, mix essential aminoacids and so on);
3) Using different medium than before (the cells have to adapt at the new medium);
4) Incorrect protocol for freezing (20% FBS- 10% DMSO on ice, mr frosty at -80°C ON and at -80°C for a long time);
5) Changing the medium containing DMSO after 24h;
6) Cells-thawing rapidly and changing medium (for DMSO diluition);
7) Trypsin-EDTA (0.025% in the same medium);
8) Cells mycoplasma contaminated.
And that's it!! XD
Good luck
Hi Oleg,
I donot see death problem in flask, I see this problem in fLASK. But, when I plate cells in 24 well plate, I see this problem, I change media after 24 hours once they adhere.
And viability remains the same untill another 36 hours.
Dear all,
I am a cell culture beginner and have one question?
What is the best splitting ratio for HepG2 cells if you are just intended in maintaining them in culture?
- Badges
- Science topic