* I would try to avoid FACS at first because it is quite violent for primary tumor cells beeing pressed throug a tiny little tube and then attach and grow well...

* Have a look on FibrOut mixes from ChiScientific to get rid of fibroblasts.

http://www.chiscientific.com/product.aspx?ID=93

they work very well in my hands. Take in account tthe source of your cells. If the tumor comes from Xenografts stroma and vasculature is entirely mouse. So you should use mouse specific Fibrout.

* Mantaininng cells in sphere culture (ultra low attachment plates Cultek) for a few days also help to reduce fibroblasts. here I use a serum free "stem cell medium" (Puig et al. DOI:10.1158/1078-0432.CCR-12-1740)

* Then you may clean life cells on a Ficoll gradient to get rid of debris.

* Try different coatings of your dishes. I plate in parallel to collagen and matrigel coated plates (1:100 in PBS coating for 1-2h). Some tumors attach better to one than to the other.

* The first weeks I feed the cells with stem cell medium and 20% inactivated FBS).

* Then you may use partial trypsinization when fibroblasts start to grow out. The usually detatch much quicker then tumor cells and can be washed away.

* Another point is right disgregation. We use mincing followed by Colagenase/DNase digestion and filtering through BD cell strainers (100um).

* Finally, you need patience... it takes weeks to ocasionally establish a line.

The final step is to confirm that what you established has to do with the original tumor. You may genotype some specific mutations. TRA85 antibody may be used to identify human cells in facs.

Good luck!!!

Hi Xiaoxiao,

I work with neural stem cells and glioblastoma cell lines and I think a can give you a little bit of advice. The most important part to separate your tumor cells from the fibroblasts is to know with surface protein is expressed in your tumor cells, and not in the fibroblasts. There are markers such as CD133 that are highly expressed in certain types of tumor cells, but not in other types of cells.

Once you know the surface marker expressed in this type of tumor cells, you can FACS sort your cells by that marker. FACS means Fluorescence-activated cell sorting. Basically, it is a method to separate cells by fluorescence labeling. You will have to incubate your cells with an antibody against your surface protein (e.g. CD133) linked to a fluorescent dye (e.g. GFP) and run your cells on a FACS machine. You will be able to separate your cells and grow them in vitro without fibroblasts in your culture.

Hope this information helped to your research.

Best,

Ale

Hi Ale,

Thank you very much for your advise. FACS is a good choice. However, it is not easy to select the surface marker which expresses in all or most tumor cells due to the heterogeneity. That would cause artificial selection of certain population of tumor cells. Maybe FACS sorting out of fibroblasts, and collecting the remaining as tumor cells would be a wise method?

Hello. I applied a device called Dako cytomation/ Denmark medimachin previously.

Instead of using FACS you can also go for magnetic separation using e.g. Miltenyi system - you're basically also working with surface markers and antibodies against them which are apparently magnetically labelled. Thereby, you can go for positive as well as negative selection, so either collect tumor cells (if you have an appropiate marker) or deplete the fibroblast with a specific marker. After cell isolation there is no problem to take cells in culture...

@venkata, I have found the Mitenyi products per your information. Many thanks : )

* I would try to avoid FACS at first because it is quite violent for primary tumor cells beeing pressed throug a tiny little tube and then attach and grow well...

* Have a look on FibrOut mixes from ChiScientific to get rid of fibroblasts.

http://www.chiscientific.com/product.aspx?ID=93

they work very well in my hands. Take in account tthe source of your cells. If the tumor comes from Xenografts stroma and vasculature is entirely mouse. So you should use mouse specific Fibrout.

* Mantaininng cells in sphere culture (ultra low attachment plates Cultek) for a few days also help to reduce fibroblasts. here I use a serum free "stem cell medium" (Puig et al. DOI:10.1158/1078-0432.CCR-12-1740)

* Then you may clean life cells on a Ficoll gradient to get rid of debris.

* Try different coatings of your dishes. I plate in parallel to collagen and matrigel coated plates (1:100 in PBS coating for 1-2h). Some tumors attach better to one than to the other.

* The first weeks I feed the cells with stem cell medium and 20% inactivated FBS).

* Then you may use partial trypsinization when fibroblasts start to grow out. The usually detatch much quicker then tumor cells and can be washed away.

* Another point is right disgregation. We use mincing followed by Colagenase/DNase digestion and filtering through BD cell strainers (100um).

* Finally, you need patience... it takes weeks to ocasionally establish a line.

The final step is to confirm that what you established has to do with the original tumor. You may genotype some specific mutations. TRA85 antibody may be used to identify human cells in facs.

Good luck!!!

I agree that FACS sorting or magnetic beads sorting depending on 1-2 markers does not cover the heterogeneity of cancer cells in fresh sample, whether in the same sample or from sample to sample. Attachment to plastic surface and serum support fibroblast growth, so I think using sphere culture condition+Ultra low attachment surface is good suggestion to reduce fibroblast in your culture.

Our group recently published a protocol in which we use FACS to negatively select highly tumorogenic cells from human prostate samples. We then use a xenograft model to study the biology but you could just as well perform serial passages and generate a cell line. Thought it might help illustrate the negative selection idea many above have proposed, good luck!

http://www.jove.com/video/51332/isolation-of-cancer-stem-cells-from-human-prostate-cancer-samples

You can do single cell cloning in soft agar. It takes patience.

Before utilising Facs, you can use Percol in order to enrich your tumor cells.

The protocol for this procedure was published by our group.

Georges et al.

Sequential biphasic changes in claudin1 and claudin4 expression are correlated to colorectal cancer progression and liver metastasis.

Enrich with ficoll / percol, then facs sorting with low pressure

I agree that Ficoll/Percol gradient is mandatory to get rid of dead cells and debris, but in my hands there is plenty of fibroblasts left in the top layer fraction. Before the gradient, I treat the tumor cell suspension with an ammonium cloride solution to lyse red blood cells from the mouse.

We use feeders (NIH 3T3 cells treated with mitomycin C) for establishing tumour keratinocyte line from human samples. Feeders have the double advantage to help tumour cells growing faster and they also limit fibroblasts growth by competing for the space. If you have never used feeders before, make sure first that the mitomycin C treatment actually stop 3T3 proliferation. The 3T3 should all be dead after a week. We have recently tried to use 3T3 J2 and never managed to kill them with mitomycin C, meaning that we have contaminated our primary tumour cells with very fast growing mouse fibroblasts!

FACS, magnetic selection, Ficol/Percol gradients, feeder cells, specfic serum free media, and a great deal of patience and diligence, are all useful in removing fibroblasts depending on the nature of the tumor cell culture. I could also suggest to add repeated rapid EDTA treatments with brief side tapping of the flask, instead of trypsinization, to detach fibroblasts. This would be safer to the neighbouring target tumor cells trying to grow in the same flask. Later, you may assess the percentage of contaminating fibroblasts using surface marker analyses.

I would look up the following:

Transforming Growth Factor β Signaling Is Disabled Early in Human Endometrial Carcinogenesis Concomitant with Loss of Growth Inhibition 1

Trilok V. Parekh, Patricia Gama2, Xie Wen, Rita Demopoulos, John S. Munger, Maria-Luisa Carcangiu3, Michael Reiss, and Leslie I. Gold4

Cancer Res May 15, 2002 62; 2778

The authors describe isolation of epithelial cells and stromal cells from endometrial tissue samples from patients following hysterectomy. The isolation protocol works really well and the cells look amazing and I have done this several times in the corresponding author's lab. This protocol is however for endometrial tissue samples and isolation of epithelial cells from these samples. It depends on your primary tumor sampleas to what would work best for you.

You can use single cell colony formation in multiwell plates after digestion of the tumor sample, count and diluent the cell to make sure one cell in one well of the plate.

We have recently published our protocol of developing primary cultures from both ascites and solid ovarian tumour samples. Hope this helps.

Article The Use of Ovarian Cancer Cells from Patients Undergoing Sur...

I have used differential trypsinization (use trypsin for approx 2 minutes; the cells left behind should be epithelial tumor cells). However I am currently working with an immortalized prostate tumor line and this is not working for me.

Hello all, just came across this discussion here, as I aslo would like to try to isolate tumor cells from a xenograft mass and reculture them .....

Can anyone tell me how important it is that I start with a "fresh" tumor ? 

Do I need to start isolating the cells immediately when taken the tumor from the mouse or could I snap freeze it and do it another day ? 

Big thanks 

What do you want to do with your tumor cells?

I would always isolate cells immediately.

I want to reisolate my breast cancer cells from the xenograft masses. 

I've injected cells, treated the mice when masses were 100mm3,  and now want to reculture the cells in vitro. I've already heard that I will need to isolate them and could freeze the single cell solution but not the mass. 

Now I search for a good protocol, as some people digest only with collagenase, others collagenase/hyaluronase , others add DNAse ..... but I couldn't figure out yet on what this depends.... if anyone can help I'd appreciate tips.