I need to extract live cells from ovarian tumors. I have tried this only twice so far and have not been successful. Once I get the tumor sample, usually immersed in PBS, I remove it in a petri plate and mince the tissue into fine pieces, or at least as small as I can manage. Then I add 0.2% collagenase type 2 ( prepared in serum free DMEM) and let it digest for 2-3 hourse at 37deg C with intermittent mixing. Finally when it all looks like a goopy mess I try to apply gentle mechanical force by pipetting. Once it looks like most of the tissue has disintegrated I pass this mix through a cell filter to collect the cells. Then I plate these cells but I see just a lot of floating mess and very very few cells. I would be very grateful if you can help me with suggestions/ protocols to improve my method and increase the yield of cells.
bonjour, la première des choses il faut considerer que les cellules tumorales sont aussi des cellules vivantes, une durée de temps sans glucose ne sera pas begnifiquue,
la recuperations de la pièce tumorale doit se faire dans de l'HBSS(tampon physiologique glucose) , en plus la digestion par la collagenase est lente et vous devez la preparer dans de PBS pas le DMEM!!, une demie heure suffit largement, puis vous pouvez separer des cellules tumorales des cellules non tumorale sur un gradient de densité de saccharose ou autre produit,
d'autre part je vous conseil de consulter springer protocol pour plus de détaille
bon chance
Hi Zankruti,
As above, but try without collagenase at least once and see how many/what cells you get. 2 - 3 hours of collagenase incubation seems rather long to me, try 15 - 30 min perhaps. You might have to push harder to get the pieces through a cell strainer (is that what you use?) - is yours 70 um or 40 um? (start with 70 um)
The next problem will be the growing of tumour cells: You could try the gradient centrifugation suggested above, or wait for the tumour cells to outgrow the normal ones. Good luck!
I think it is better to collect the tissue sample in DMEM and make small pieces.
Iam working with breast cancer tissues. I usually collect in DMEM, i use the sample for Nucleic acid isolations.
I prepare single cell suspension with collagenase Type IV (0.3%), and i use that cell for apoptotic assay by using flowcytometry.
Hi Zankruti,
The protocol we use in the lab is pretty much the same as yours, except for the medium (we use RPMI instead of DMEM) and the cell filter. We actually don't use a cell filter, we filter the tissue/cell suspension obtained after digestion (containing about 10 mL) trough a cotton gauze (pre-wetted with RPMI) inside the barrel of a 20 mL syringe. We never use a cell filter because big particles clogged the filter and we were losing the majority of the cells in the filtration process. I suggest you to try this or a bigger filter as Thomas suggested and then, when having a more uniform cell suspension you can centrifuge your cells a couple of times (1200 rpm x 10 min) to remove debris before plate. Boa sorte!
Hi,
May be the following articles can help you:
Long-term primary human tumor cell cultures and mixed autologous tumor-lymphocyte cultures for adoptive specific antitumoral immunotherapy
S Rizzo
Division of Pneumology, IRCCS Policlinico S Matteo, Pavia, Italy
Anticancer Res 18:41-4. 1998.
Growth curves: a method of monitoring established long-term primary human tumor cell cultures for autologous immunotherapy
S Rizzo
Division of Pneumology, IRCCS Policlinico S Matteo, Pavia, Italy
Anticancer Res 18:433-9. 1998
Thank you so much everybody for your suggestions. I will try out a few of your suggestions and see what works. Will surely update about the results.
Before filtering cells through filter incubate cells with DNAase at 37C for 15 minutes. It will increase your cells yield definitely.
We didn´t work with ovarian tumours. We work with breast cancer and protocol is more or less the same: Some points that I have to suggest is to work immediatly after rejection; we employ Colagenase IV and make as much as incubations we need for about 30 min, depending on the nature of the tumour.
Thank to all your suggestions, I do manage to get cells and growing. it is very interesting to see the heterogenous mix of cells. i have started using a gauze which was a BIG help,Collagenase in PBS is also effective. Thank you.
- Badges
- Science topic
- Similar topics
- Chemistry
- Pharmacology
- Pharmaceuticals
- Drugs