Cell Lines from Tumor Tissue Samples - Advice/Protocols on best techniques (Aug/09/2006 )

I know that some of you out there get primary cell lines from tumor tissues. I was just wondering what sort of protocol you follow for that (I'm using frozen glioma tumor tissue samples, but I would like to find out what is done with samples from live donors as that is a more common practice). What sort of concentration of the sample do you put in the flask/plate? Do you pre-coat the flasks/plate with anything? How quickly does the tumor tissue shed cells and grow?

I just thawed a portion of a frozen tumor tissue sample (mouse glioma) and it is growing terribly. We used half of the sample for subcutaneous implantation into mice (so far no tumors or very small tumors have resulted) and the other half I plated. I may have plated them at too high of a concentration (I put them in small plates). We later split them and moved them to six well plates and from there to T25 flasks. Most likely my problems resulted either in how I plated them initially and/or the moving of the cells prematurely.

Therefore I am looking on advice on the best methods to do this and get better cell viability.

I will also need to know how to do this if we do get successful tumors from the mice implantations. So if you have advice or information about the growth of primary glioma cells and how to get the cells from tissue samples I would be very appreciative.

So nearly two weeks later I still am experiencing problems with these cells. I have been able to find some cells in one of my 25cm2 flasks, but only about 10 of them. I'm terrified to do anything to the flask for fear of losing the only viable cells I've been able to get from the frozen tumor sample. The flask is mostly full of what appears to be dead matter and debris. I am at a loss as to what to do. Do you think it is wise to just let the flask sit and see if I get more cells? What would you do in this situation?

Thanks so much!

If the cells are attached to the surface you can and should wash away the debris. These are quite harmful to your cells.