What tissue are you trying to culture as success varies widely. The fresher the tumor sample the better. a rapidly proliferating part of the tumor offers better chance of success.

Proper dissociation of cells prior to plating of the cells in the culture dish is vital to the success of cell line isolation from tumor tissue. Depending on the tumor type, dissociation times can vary from 1 to 24 hrs. In the first days of culturing make sure there is not an overgrowth of faster growing, non tumorigenic cells in the plate, if this is the case, replate your cells.

Try different culturing and isolation conditions !

We always had good success with mechanical dissociation instead of enzyme digestion. Start with high serum concentration - optimally autologous serum or at least homlogous - and try to reduce serum concentration later. Start without antibiotics - of course that means really clean working !

My experience suggest three fundamental variables:

1.The site of sampling, it is important to prefer the part of tumour tissue with the more concentration of cancer stem cells. Their presence induces and guarantees the expansion of seeded cancer cells

2. The time between from the tissue sampling and its preparation to culture. It must be very short.

3. Finally, for the procedure used to dissociate the tumour tissue, I recommend to choose the procedure more brief and non-invasive procedure to preserve the original characteristics.

We are trying to cultivate tumor cells from ovarian cancer tissues. The aim is to get pure culture of tumor cells. At the beginning, you always get a mixture of tumor cells, fibroblasts and probably mesenchymal cells. There are two strategies to process the mixture: to selectively trypsinize the culture (fibroblasts detach earlier) or simply split culture until fibroblasts die. Using the first strategy, you tent to loose tumor cells as well, so that you’ll probably end up with a couple of areas with tumor cells cluster, which will not grow out. With the second one, you’ll reach a period that tumor cells are left alone and concentrated in the culture flask because of the death of fibroblasts. In both cases, you’d better bring the cells back to smaller culture areas, so that they may get contact and can proliferate again. This is the case with ovarian cancer.

One of the problems with making tumour cell lines is that tumours are heterogeneous and you will potentially get a number of different cell lines from each tumour sample. Try and disaggregate the tumour, physically and/or mechanically as quickly as possible in order to get the cells into culture before they start to loose viability. You could try using Primaria tissue culture dishes/flasks from BD which have positively and negatively charged groups, as well as some other functional groups on them, and are designed to culture primary cells and tumour cells. I have used these and pre-incubated them with a bit of serum before I plated the tumour cells down for the first time, and they work.

If they grow well then you can either keep passaging them as Dan Cacsire Castillo Tong suggested and get a nice pool of tumour cells from which you can then clone different lines by limiting dilution or ring cloning. The former will give you a population which has different cell types in it, and may behave more like the source material, the latter will give you a clone, but it might not be representative of the source and it takes longer. It depends what you want to do with the cell line once you have made it.

My question was rather the genetic background. 10% of the samples show outgrow. There are two possibilities: it is totally coincidental? Or is it due to tumor heterogenity (e.g. a certain type of mutation is needed)?

Collectin of fresh material into a transport medium is essential to keep the cells in condition prior to disaggregation and plating. SOme lines prefer to establsih out of small clumps rather than single cells so try both ways. When I was establishing Merkel cell carcinomas, I found that putitng them in the incubator and leaving them alone for a few days was beneficial. Too much movement in and out of the incubator to see whether anything is growing stops cells settling and disturbs any microenvironments being established. These are often beneficial if you are not sure what added supplements are required. Good quality medium and serum is required. Use of irradiated feeder layers of fibroblasts or endothelial cells can help depending on the type of cells you need to establish.

Also if you are not having much luck try out some of the different plastics for culture dishes. Just changing brands can work, but there are also ones now with specific coatings on the surface which assist various cell types to establish.

Sorry i cannot answer to your question cause i work only with primary cultures of cancer cells

It's probably only the tumor stem cells growing out - but I don't know any proof for that...

Perhaps facs sorting after digestion of the tissue.

Try changing culture medium to Opti-MEM + Glutamax (Invitrogen) containing 20%heat inactivated serum (Atlas Biologicals). The combination worked quite well with our adult-derrived stem cells, where other medias gave less than optimal results.