hep-2 & Hep-G2 cell line - difference between hep-2 & Hep-G2 cell line (Dec/15/2004 )

Thanks stefan. Maybe I should also try to grow them without antibiotics.
I have transfected HepG2 with Fugene6 (Roche), but not very successfully. I think they are in general very difficult to transfect (20-30% of cells) or so I have heard.

If anyone has optimized transfection to HepG2 cells, please let me know too!

Thanks!

Thanks stefan. Maybe I should also try to grow them without antibiotics.
I have transfected HepG2 with Fugene6 (Roche), but  not very successfully. I think they are in general very difficult to transfect (20-30% of cells) or so I have heard.

If anyone has optimized transfection to HepG2 cells, please let me know too!

Thanks!

Hi, I am new in cell culture but I did have some experience in transfection with HepG2. It is true that HepG2 is a difficult cell line to transfect. The best transfection efficiency I got with HepG2 using SuperFect (from Qiagen) was 10% (use GFP as a reporter), while it was 40% with 293 cells.

However, I did have several troubles with HepG2, namely

1. follicles or bubbles inside cells.

2. So adhesive to plates that it is impossible to detach by trypsin. With only one vial at passage 5 and there after to passage 7. Discarded.

3. Recently I have another problem: clump formation.

Normal HepG2 cells have their clear cell borders and nuclei with darker cytoplasm. These problematic HepG2 cells lost their cell borders and merge into growing clumps, which look brighter under microscope. Trypsinization and pipetting do make them smaller but I have never completely dispersed them into individual cells, which make cell count impossible. These cells seem to grow into clumps instead of spreading away, so they seem never to grow into 80% confluent or higher.

I took some pictures, so I can send you if you are intereted in.

I bought this HepG2 from ATCC. Media is MEM with 10% FBS and other stuff recommended, including p/s. I seeded them at 1x10^6/100mm plate, and subculture in about 5 days without changing media during culture. ATCC recommended not to hit or shake the cells during trypsinization, but I did do so to detach the cells. The first 4-5 passages seem OK, but then they began to form clumps.

Any one who knows how to solve these problems is highly appreciated!

Thanks!

Rosi

I just read about a technique called 'reverse transfection' for getting siRNA into those difficult-to-transfect cell lines. Basically this means that instead of plating the cells and leaving them alone for 24 hours before transfecting, you resuspend them after trypsinisation in siRNA/liposome soup and *then* plate them.

Haven't tried it, but it was in a newsletter from Ambion - you can find the details at the following link:

http://www.ambion.com/techlib/tn/121/9.html

They specifically say that this is a great way to get really good delivery/silencing in HepG2.

Hi, guys:
I have cultured HepG2 cells for years and I have some experience to share. If you use purchased hepG2 cells from ATCC, I never found any problem with antibiotics. No problem if you use P/S, gentamycin or even tetracyclin. The medium is the key. I have tried low glucose DMEM, MEM or William's medium E. I found that William's medium E, which is used for primary hepatocytes, is the best. I used 10% FBS, because 5% FBS makes cells grow very slowly.
hepG2 cells will look clumpy. Try to pipette more. Cells cultured in william's medium E for several passages will grow more like epithelial cells. Coating is absolutely not necessary. But if it is desirable to make cells attach better (such as for virus infection), collagen could be used. collagen coating will make cells spread very well so that the morphologic feature looks different.
hopefully my humble opinion helps.
good luck

by the way, in terms of transfection efficiency, hepG2 cells are not very easy to work with. I used double amount of DNA for transfection of hepG2 than that of 293 cells. for example, 1 ug DNA for each well in a 12 well plate. Use more lipofectamine than 293 cells. fugene is fine too.

Hi everybody,
I am working with HepG2 cells, I grow them in RPMI1640 with 25mM L-glutamin, 10%FCS and 1%P/S. They are slow growing but they form more or less nice layers if you trypsinize them often and long enough. I trypsinize them for 15 min at 37°C in the incubator without touching the fleak to avoid clump formation. But even after 15min cells can stick to each other but this can be overcome by pipetting them up and down..
I am transfecting HepG2 via elcetroporation wich works quite well (up to 40% efficiency)..
paul

Hi!

May I ask your optimization for transfection of HepG2 via electroporation (cell density, electroporation controls etc.)? I have never tried it but I think it sounds really promising!

Thanks in advance!

Joana

Hi Stefan!

My transfection efficiency has also been under 5%. I have heard that it could be somewhere near 20-30% but never managed to get that myself. I haven´t tried Effectene but I´ll try oligofectamine (invitrogen) soon. I´m also interested in electroporation. Have you tried it?

With best, Joana

QUOTE (Joana @ Feb 17 2005, 01:00 PM)

Thanks stefan. Maybe I should also try to grow them without antibiotics.
I have transfected HepG2 with Fugene6 (Roche), but  not very successfully. I think they are in general very difficult to transfect (20-30% of cells) or so I have heard.

If anyone has optimized transfection to HepG2 cells, please let me know too!

Thanks!

Hi Joana,

this week I also tried to use Fugene6, but the effeciency was below 5%.
May I ask you for your optimization?

Have you ever tried Effectene by Qiagen? This sounds promising and therefore I wanted to test next.

Bye,
Stefan