Cell lines can change their phenotype as they get progressively older in culture (i.e the more passages the more likely that there will there be phenotypic changes). This drift can be due to genetic or epigenetic changes or alternatively to the selection of subpopulations within the global population of cells that display an alterered sensitivity and therefore IC50 to whatever drug is being assessed for cytotoxicity. To overcome this potential problem you are advised to create both master and working cell banks for your SKOV-3 cell line. A useful indicator might be 50 or so frozen vials for a master cell bank (MCB). You should then create a working cell bank (WCB) by growing up a large number of cells (in one go, NOT from multiple pasages) from a SINGLE vial taken from your MCB and create a WCB of a few hundred frozen vials from this. You should have around 1E6 cells/vial. I would recommend freezing in a mixture of 90%FCS containing 10% DMSO though you may need to experiment here as some cell lines require more or less DMSO in the freeze mix. You would then replace your cells for experimental use regularly by pulling a vial from LN2 for your WCB (we do this every 28 days with our leukaemia/lymphoma cels lines). This way you avoid multiple passages of your cells and with it hopefully the drift in sensitivity you are currently encountering. As the number of vials from your WCB becomes depleted you would replace these by recovering a vial from your MCB and creating a new WCB from these cells.

Cell lines can change their phenotype as they get progressively older in culture (i.e the more passages the more likely that there will there be phenotypic changes). This drift can be due to genetic or epigenetic changes or alternatively to the selection of subpopulations within the global population of cells that display an alterered sensitivity and therefore IC50 to whatever drug is being assessed for cytotoxicity. To overcome this potential problem you are advised to create both master and working cell banks for your SKOV-3 cell line. A useful indicator might be 50 or so frozen vials for a master cell bank (MCB). You should then create a working cell bank (WCB) by growing up a large number of cells (in one go, NOT from multiple pasages) from a SINGLE vial taken from your MCB and create a WCB of a few hundred frozen vials from this. You should have around 1E6 cells/vial. I would recommend freezing in a mixture of 90%FCS containing 10% DMSO though you may need to experiment here as some cell lines require more or less DMSO in the freeze mix. You would then replace your cells for experimental use regularly by pulling a vial from LN2 for your WCB (we do this every 28 days with our leukaemia/lymphoma cels lines). This way you avoid multiple passages of your cells and with it hopefully the drift in sensitivity you are currently encountering. As the number of vials from your WCB becomes depleted you would replace these by recovering a vial from your MCB and creating a new WCB from these cells.

Is this a new cell line (SKOV-3), or an established one? If it is an established one, shift of IC50 can be a simple loss in activity of drug, or anything else. IC50s is depending on circumstances always. Surely, the method suggested above is correct, but probably sophisticated.

I faced the same problem before when I was testing my drug against HCT-116 colon cancer cell line, I observed the IC50% value decrease with increase no. of passage. So I am try to use cell in passage between (3 - 20 passage), I am trying to test the significance of this difference.

if the number of passage is more than that I try to thaw it again.

But I am still wondering about why this difference happened?

There really is only one way to do this properly and that is to create master and working cell banks to work from. This is an industry standard way of doing this sort of thing. Anything less is a poor compromise I'm afraid. It needs quite considerable work to make the cell banks but well worth it once you get there! If we are to aspire to high quality research using defined cell lines then it has to be done.

Bashir, it's anyones guess why this occured but it could be one of two things; either your stock of drug is deteriorating with time due to stability during storage as reflected with decreasing sensitivity with increased numbers of passages. How do you store the stock of drug you work with, 4oC, repeated freeze thawing or something else? What you should really do is only work with small aliquots of drug from a large batch with defined activity deep frozen (-80 or even LN2) and pull a vial each time you do an experiment. Of course you may not be able to freeze your drug, I don't know what it is so I can't say for sure. Alternatively the cell line is shifting in its sensitivity as I said earler because you're selecting for a subclone of cells during passage that are less sensitive to the drug due to epigenetic or genetic changes in that subclone. The only way to control for this is through the creation and use of cell banks as I've described. I hope that helps.

To Bashir: HCT116 cells are mismatch repair deficient. They are rather unstable in culture and tend to lose expression of certain genes spontaneously, if there are short tandem repeats in the sequence somewhere. I once had a batch that had lost expression of the important pro-apoptotic gene BAX in that way. Rather a nuisance if you are trying to study apoptosis...

The mismatch repair deficiency by the way is the reason why it is possible to make knock-outs for specific genes in this cell line.

The are tend to change their behaviour if kept continously in culture for a longer time, so I fear the only solution indeed is to make a large stock of frozen aliquots at the beginning of a project, and thaw fresh cells frequently.

Thanks everyone for your suggestions. To David Flavell: Thanks for your valuable suggestions regarding master and working stock. Can I keep working stock in -70.C for a while (as you know LN2 has limited space)?

One more thing, actually I was preparing resistant cell lines using repeated drug shocks. While doing this I also maintained untreated SKOV3 cell line with it as a control. Most of the cases to develop resistant strain it takes 4 to 6 months. What to do?

To Attila Lehotzky, Its an established cell line. However, I am getting higher IC50 in resistant cells (developed by shock treatment with drug) of same cell line. Maintained exactly same time period in culture.

Hm. Do you shock SCOV3 by the same procedure as primary cells... why do you think that an established cell line would not response such type of shock? You did the selection pressure on it, it responsed. In this case you must stricktly go for banking at certain level at least. If i understood well.

you need an unshocked stock of SCOV3 line in every control experiment. because your selection procedure long, you should do as Prof Flavell suggested.

If you need consistency only for say one 3-year project, you can scale down a bit from what David F suggested - say 20 vials of master stock and make 20 vials from each of those, giving a potential 400 vials for thawing (and plenty of margin). If you need more but are short of LN2 space, you could have (for example) 10 master vials, 10 secondary from each of those, and 10 tertiary from each of those, giving a potential 1000 similar vials.

We freeze many types of cells, and we typically use the culture medium the cells normally grow in, with 20% serum and 7.5% DMSO. We expect at least 90%, often 99% viability, so long as the cells were healthy and growing (not yet confluent) when frozen.

Cells are ok at -80 at least for a few weeks, but may lose viability after that.