18 replies to this topic

[pankaj]

if someone could help me clear the difference between hep-2 & hep-G2 cell line, do both of these are hepatoma cell lines?
  i have been trying to grow hep-g2 cells with 10% FBS in DMEM but cells do not stick & dont divide i have been waiting for almost a week but there seems no response. i m using 25mm sq flask, even if few cells stick it seems as if they will take years to be cofluent. plz help me to solve this problem, thanks in anticipation, bye

[Daniel5306]

I don't know the difference between the two cell lines. But I had cultured the two cell line. It is very weird that your cell could not attach to the wall of the plate. Are you sure that your plated is coated?

[DevGrp]

By coated do you mean something like sarstedts cell+ (yellowcap) "for adherent cells"?

I've mainly used the standard sarstedt (red cap) flasks for cell culture and had a lot of problems growing HepG2s.

Think you might just have solved it.

Pity I that was two years ago!

Thanks

Ian

[LabGirl]

I don't know about the hep2s, but HepG2 information is listed by ATCC. Check out www.atcc.org for information on cell derivation, culture conditions, etc. About coating, although I would not expect the cell line to require this,  I have grown primary rat hepatocytes and they require collagen-coated plates.    Good luck.

[DevGrp]

Some primary cells need it, some don't. We used gelatin for HUVECS but nothing for human primary breast or endometrial epithelial cells.

[kalyani]

I have had problems growing HepG2 cell line. They do not grow very well as a monolayer, instead they tend to clump up. I use only T-25 plates and not any coated plates. Can anybody tell me how to avoid this problem?

I have had similar problems with MCF7, at low passage numbers (from ATCC), but as the passage number increases, they grow as monolayer than as clumps.

Any help is appreciated.
Thanks.

[Osiris-gdw]

pankaj, on Dec 15 2004, 07:04 AM, said:

if someone could help me clear the difference between hep-2 & hep-G2 cell line, do both of these are hepatoma cell lines?
  i have been trying to grow hep-g2 cells with 10% FBS in DMEM but cells do not stick & dont divide i have been waiting for almost a week but there seems no response. i m using 25mm sq flask, even if few cells stick it seems as if they will take years to be cofluent. plz help me to solve this problem, thanks in anticipation, bye

Hi!
HEp-2 is a cell line derived from HeLa (they were are contamination in an originally hepatic cell line --> see ATCC for details) and so are a cervix carcinoma cell line, most useful, because they have a rather big nucleus.
HEP-G2 are hepatic cells.

So, if you need liver cells, HEp-2 are a bad choice!

If you need attached HEP-G2, just use poly-L-lysine to coat your dishes, that works quite well!

I hope, this helped...

Best wishes, Gerhard

[Hooly]

HEP-G2 are as clumpy as hell, that's just the way they grow. You'll see that a thinly-seeded culture will tend to show the cells forming piles or mounds as they grow, rather than making nice monolayers - and even when they get confluent, the cell are still very piled-up. They are also !very! adherent, so trypsinisation will take longer than you might expect.

The kind of plastics you use can really make a difference - our stores regularly change our supplier of culture flasks for a cheaper option (without telling us), then change back real fast as the complaints flood in from all the users. Makes a big difference, so if your cells aren't attaching, you could try using flasks fom a different supplier. Your culture conditions sound okay, are you adding L-glutamine to 4 mM (I don't care if the media is supposed to have this already, it goes off so fast you should probably try this, and excess L-glut won't harm the cells anyway so no loss)? Lack of L-glut in media generally causes highish levels of apoptosis - particularly with cancer-derived cell lines -  plus cells don't attach well and grow very slowly.

You shouldn't need any coating to grow HEP-G2, if the plastic's okay and you're using the correct media.

Ditto for MCF-7. Different cells grow in different ways, some are very clumpy early on, then start making nice monolayers as they get more confluent, others display the opposite. Generally, I don't make any descision about a cell line taken from liquid nitrogen for 2-3 splits, because it's only by then that you can decide if it's behaving 'normally'. MCF-7 is a clumpy cell line (when trypsinised), but will spread out to monolayers until it gets near confluency, at which point masses of cells will detach and float...and the remainder will get stickier than ever. You want to make sure you split 'em regularly, and don't let 'em get confluent before you do so. If you really need non-clumped MCF-7s, best strategy is to split 'em 1:10 last thing the night before you need 'em then detach 'em first thing next morning - should be pretty non-clumpy.

[Joana]

I have had the same experience as Hooly. I have grown HepG2 cells for years and every time I try to start growing them I have problems.
The majority of cells die or don´t divide normally.
The seeding need to be done very carefully, because if you put too much cells, they´re already forming 'mini-livers' after couple of days and you can´t separate them anymore. However, if you put too few cells there, they´ll stop dividing and eventually die.

I´d advice you to start with new cells. I have grown them in filttered 75cm2 flasks. Seed them twice a week, but only 1:2 or 1:3. And use the appropriate medium (ATCC).

Hope it works!

[steh-fan]

Hello!

I am culturing HepG2 for several years now and I newer had any serious problems with them.
They grow well on normal tissue ware and there is abolutely no need for coatings (even not when grown on glass cover slips)

However, I detected some limitations:
1. They do not like Pen/Strep in the medium. Whenever I enclosed it, they stopped growing.
     So I rather culture the HepG2s w/o any Antibiotics .
2. Sometimes when these cells were passaged too many times they form a lot of vacuoles
    and do not look too healthy anymore. So discard old HepG2s!  
3. You may not expect that there will be a confluent monolayer. They stop proliferating before!
    So split them when they look like 80% confluent.

Unfortunately what I do not know and would be really interested in:
Has anybody successfully transfected these cell line?
If yes, I would appreciate any help!

Bye,
Stefan

[Joana]

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!

[steh-fan]

Joana, on Feb 17 2005, 01:00 PM, said:

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

[Rosi]

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

[Hooly]

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.co...b/tn/121/9.html

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

[luminb]

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