8 replies to this topic

[Neanderthal]

Hi everyone,

I am working with Hep G2 cells obtained from ATCC. I am growing them in DMEM (with 10% FBS, pen/strep, Sodium pyruvate, MEM-Non-essential aminoacids, sodium carbonate) without any problem.

For splitting these cells, I wash the cells with 1 ml PBS (made in my lab), and then 1ml trypsin-EDTA (0.025% from Invitrogen, with an expiry date of April 2012, and stored in 15ml alliquotes at 4-degrees) and incubate at 37 degree CO2 incubator for around 4 mins.

This worked fine for me till 3 rounds of 1:2 splitting. But after that, even with the same amount of trypsin the cells seem not to come out of the plate even after 8 mins of incubation, which is double the time that I usually get the cells out of plate!!

Can anyone tell me what is going wrong here - the trypsin expiry, its storage, problems with cells or something else?

Please help.

Thanks a lot

Neanderthal.

[leelee]

How confluent are the cells when you split them? In my experience cells can become more resistant to lifting with trypsin when they are grown to 100% confluent. You should aim to split them at about 80% or so.

Could this be it?

[Neanderthal]

leelee, on 15 November 2011 - 11:35 PM, said:

How confluent are the cells when you split them? In my experience cells can become more resistant to lifting with trypsin when they are grown to 100% confluent. You should aim to split them at about 80% or so.

Could this be it?

It is about 90% confluent. But I do not think this is the problem as we were going well with splitting at 95% confluency (that we follow in our lab).

An interesting thing I noted about these cells is that even at 95% confluency, they do no cover 95% of 90mm petri dish. Instead, they form a network of 'islands' with clear gaps between them (just like sea/rivers). But the cells inside the 'island' are touching each other and are bursting at the seems. My labmates and PI said it is not the kind of morphology they are familiar with Hep G2. But the trypsinization problem started after few passages (3 to 4 ).

Any thoughts?

[leelee]

I'm confused- how can your cells be 95% confluent, but not cover 95% of the dish?

Anywho, I would say that given your cells are not exhibiting morphology consistent with what Hep G2 cells usually look like, I'd say that inability to trypsinise is the least of your worries.

Do you have another vial you could thaw and start again? I'd be reluctant to use cells that have strange behaviour and morphology for experiments.

[bob1]

leelee, on 16 November 2011 - 01:38 AM, said:

I'm confused- how can your cells be 95% confluent, but not cover 95% of the dish?

Anywho, I would say that given your cells are not exhibiting morphology consistent with what Hep G2 cells usually look like, I'd say that inability to trypsinise is the least of your worries.

Do you have another vial you could thaw and start again? I'd be reluctant to use cells that have strange behaviour and morphology for experiments.

I second getting some more up.  If there are changes in the morphology and behaviour then you are probably working with cells that have undergone some form of lab evolution, and as such are no longer the cell line you think they are.  You could compare the cells with the images on the ATCC website to see if the morphology is actually strange.

Confluency is not necessarily ac direct function of the observable contact over a surface area, many cell types, but particularly brain cells like neurons and glia, will be confluent at about 50% based on area covered.  This is because the cells are contact inhibited and are in contact through small prjections of the cell body, which causes them to stop growing.

[Neanderthal]

bob1, on 16 November 2011 - 02:39 PM, said:

leelee, on 16 November 2011 - 01:38 AM, said:

I'm confused- how can your cells be 95% confluent, but not cover 95% of the dish?

Anywho, I would say that given your cells are not exhibiting morphology consistent with what Hep G2 cells usually look like, I'd say that inability to trypsinise is the least of your worries.

Do you have another vial you could thaw and start again? I'd be reluctant to use cells that have strange behaviour and morphology for experiments.

I second getting some more up.  If there are changes in the morphology and behaviour then you are probably working with cells that have undergone some form of lab evolution, and as such are no longer the cell line you think they are.  You could compare the cells with the images on the ATCC website to see if the morphology is actually strange.

Confluency is not necessarily ac direct function of the observable contact over a surface area, many cell types, but particularly brain cells like neurons and glia, will be confluent at about 50% based on area covered.  This is because the cells are contact inhibited and are in contact through small prjections of the cell body, which causes them to stop growing.

Thank you.

I still do not think there is any problem with the cell lines. I didn't mean that they look different in morphology, but only in how they become confluent without covering the petri plate area.

Is there any other possibility apart from the morphology issues?

Thank you

[zienpiggie]

hi there, how long does it take for the hepg2 to go to 'confluency'? My experience with Hepg2 was that it is indeed difficult to trypsinize and when it does trypsinize it clumps if not tended properly. I find it helpful to subculture prior to confluency, which is usually about 2-3 days after the prior subculture. I also usually subculture 1:4 or so.. as recommended by ATCC. So I can imagine if you have subcultured at a 1:2 ratio three times, I can imagine you may have a lot more cells than what you had initially. The hepg2 also, if I remember accurately, was never confluent as defined by covering all surfaces of the plates. I hope it helps, if you haven't resolved this issue yet.

[Neanderthal]

zienpiggie, on 01 December 2011 - 02:22 PM, said:

hi there, how long does it take for the hepg2 to go to 'confluency'? My experience with Hepg2 was that it is indeed difficult to trypsinize and when it does trypsinize it clumps if not tended properly. I find it helpful to subculture prior to confluency, which is usually about 2-3 days after the prior subculture. I also usually subculture 1:4 or so.. as recommended by ATCC. So I can imagine if you have subcultured at a 1:2 ratio three times, I can imagine you may have a lot more cells than what you had initially. The hepg2 also, if I remember accurately, was never confluent as defined by covering all surfaces of the plates. I hope it helps, if you haven't resolved this issue yet.

You are true, it is never confluent as defined by covering the entire surface of the plate.

[Monika Gluch-Lutwin]

Hi,

I think that it is independent of the confluence  of cells, but it depends how correctly were FBS washed. Washe the cells twice with PBS and then add trypsine, and put the cells for a few minute to incubator.