I have cultured these cells on collagen-coated plates. This has helped quite a bit for me. This may also affect their phenotype slightly, but in my opinion, this is more realistic of how they grow in the liver (surrounded by a matrix of collagen).

First of all, try to implement serum concentration to 20%, in order to reduce vacuole formation. It's also important to avoid high cell concentration/well or flask (I used to seed less than 1 million of cells/T75 flask). Try to refresh the medium daily, completely or partially. The clumping could be minimized using EDTA instead of Trypsin for cells off.

I use to grow them using double amount of Glutamine, but this was before the more stable DMEM+glutamax was available... I do not recall major problem with the vacuole formation, but I remember that they were very clumpy. I use to pass them through a needle at every split and not plate them to sparse. Even if this would not preventing the clumping, starting for a single cell will require more time for the clump to be problematic. I use to split them 1:4 and even 1:8 and the clumps were not so big.

hope this will helps

To Marika

"The clumping could be minimized using EDTA instead of Trypsin for cells off"

Please could you explain how this could be done

Thanxs in advance

Dear Meera,

EDTA appears to be more effective than Trypsin in preventing clumping due to its effects as Ca++ ions chelating (Ca++ is an important co-factor for many adhesion proteins which can be involved in cell clumping). Moreover EDTA acts more quickly than Trypsin and this is critical as well to reduce the damage of cell membrane which represents another hot point in clumping. Passing HepG2 using EDTA instead of Trypsin clearly reduced clumping phenomenon...

Dear Meera,

Are you using Trypsin + EDTA to detach the cells? Because trypsin without EDTA will produce clumps. Exactly determine by microscope inspection that you are incubating the cells with trypsin/EDTA for the required time because the incomplete incubation will also cause clumps. Alternatively, I agree with Marika Crescenzi that EDTA alone is better to avoid clumping.

If you are changing from DMEM/F12 to DMEM high glucose it is advisable to do it step wise, for example mixing old/new media in changing proportions: 3:1 for a few days, 1:1 next, then 3:1 to finally arrive to DMEM High glucose alone.

You also should be sure that your culture is healthy. If you use antibiotics routinely and you freeze your stock cells without having withdrawn antibiotics for at least 2 passages before freezing you can have cryptic bacterial contaminations that are difficult to see under microscope because they remain in very low concentration. Also check for mycoplasma contaminations.

Besides the use of collagen-coated surfaces and to increase the percentage of FBS, to avoid clumping I recommend you to disgregate cells with an insuline needle after washing the cells and before seeding them. Resuspend cell in 1 ml of complete medium with an automatic 1mL pipet (blue tip) several times. Take all the volum with an insuline 1mL needle and leave it in a new 10 mL tube containing 9 mL of complete fresh medium. Seed cells at a density of 5000cells/cm2 and split them again once a week. Do not let them to reach confluency.

I hope this can help

If trypsination is well done you must obtain a single cells suspension and you shouldn´t need to disaggregate any clumps by passing it through a syringe. Besides, passing cells through a needle at every passage you perform will result in a stressed culture that can change over time and passages.

I have worked with HepG2 in the past and never had problems with the trypsinization step. I insist that changing media requires step by step adaptation of the culture as many cell lines cannot be directly changed.