Good morning everyone,
I've been trying to keep HepG2 cells in colture for weeks now but they keep dying and I've no idea why. We use:
- Minimum essential Medium-Eagle EBSS, with L–Glutamine, supplemented with 10% FBS + 1% Sodium/Pyruvate + 1% Penicillin/Streptomycin;
- 1X StableCell™ Trypsin Solution (diluted from 5X, sterile-filtered, BioReagent, suitable for cell culture, 2.5 g porcine trypsin and 0.2 g EDTA • 4Na per liter of Hanks′ Balanced Salt Solution with phenol red).
The protocol I follow is:
1. Remove the old medium;
2. Rinse gently with warm (37,0°C) 1X PBS once;
3. Add 1mL trypsin and wait for detachment (it should be 5-7 minutes but with our trypsin it takes ~20 minutes. I think this is the main issue); [EDIT: I put them back in the incubator after adding the trypsin]
4. Neutralize the trypsin by adding the same amount of fresh medium;
5. Centrifuge for 5' at 1000 RPM;
6. Remove the surnatant;
7. Resuspend in fresh medium and plate.
In the attached image you can see how bad the cells looks after 1 week being plated. The medium has been renewed 2 days ago.
Thanks in advance for any help.
Hello Simone Fiorilla ,
I have worked with HepG2 in the past and they are not the easiest cells to deal with if you are learning cell culture. However, your cells do not look extremely bad, so probably with some advices you could salvage them.
First of all, in my experience, these cells like to not be far away one from the other. In your picture I see many clusters but also single cells, this is generally something that slows down their growth and ultimately leads to cell death. Try to dilute them a bit less when you split the cells.
Second, as I mentioned these cells like to group up. When they form clusters they are difficult to separate but it is crucial to do so. Indeed, moving cell that are already aggregated will reduce their growth and potentially change their metabolism.
Third, it is normal to increase the time of exposure to trypsin when working with these cells as they are difficult to deattach from the flask and from the other cells. However, 20 minutes is surely an overkill. I was using typically 5 minutes trypsin exposure for other liver cells and around 10minutes for HepG2. A trick to help deattaching the cells from the dish/flask is to hit its side against something (the wall of the hood or incubator for example, or the side of a desk). Of course do not do it too strongly as you ideally don't want to splah liquid around inside your flask. Also, since it is not specified in your pipeline, once you put trypsin, you should place the cells back in the incubator until the cells are deattached. Keeping the cells 20+minutes outside of the incubator can affect their behaviour.
Finally, I have always deactivated the trypsin using larger volumes of medium. Typically 1:5, meaning to deactivate 1mL of trypsin I would use 4mL of medium. This can potentially affect cells viability.
I hope all these tips can help you, good luck!
Mario
Hi, I have worked with HepG2 in the past and the following protocol worked well for me.
- Wash cells with PBS without Ca, Mg
- Wash cells once with Trypsin-EDTA (0.25%) (LifeTechology). 2ml in T75 is sufficient.
- Add Trypsin-EDTA (0.25%) to coat the cells and let it sit to detach (RT or 37c)... I usually use ~2ml to a T75 flask. It usually takes less than 5min to see them lifting off.
- Neutralize the trypsin by adding complete media with FBS to flask. Pipet up and down to break up the cell clumps. Split/passage to a new flask.
Hope this helps
** Try adding different antibiotics such as special anti-mycoplasma, etc. (Gentamicin is also good for mycoplasma and others)
** Therefore, it is preferable to use 0.22(μm) filters to pass the components of the medium before adding them to the cells.
** Try using another company's trypsin and double the amount of trypsin .
** Before adding trypsin, do not forget to wash the cells well with PBS because the remnants of the medium hinder the activity of trypsin .
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