I am trying to isolate tumor infiltrating lymphocytes from melanoma tumors (B16F10) injected into mice subq and am having three issues with what is the best way to go about it. In the end I need millions of CD4+/CD8+ tumor infiltrating lymphocytes (TILs) for my future experiments and am therefore torn between:
1. Should I use Collagenase/DNase I? The B16F10 tumors seem to break up fine without it, but some papers use it and some don't. Does it improve isolation? If it does, will it diminish the number of CD4+/CD8+ TILs I can isolate?
2. Is it better to use a discontinuous Ficoll (100%/75% - 1.077 g/ml/1.055 g/ml) or a discontinuous Percoll (80%/40% - 1.1 g/ml/1.05 g/ml) gradient? And are my percentages/densities correct? Do I need more fractions? How exactly should I add the tumor digestion to the discontinuous gradient? This step is the most important I feel which is why I need to get it perfect.
3. I eventually want to sort out CD4+ and CD8+ cells for miRNA analysis and am wondering if I should use MACS beads after the gradient or just go straight to sorting with a FACS Aria III. I don't think the MACS beads will provide me with a more pure CD4+/CD8+ TIL population after the sort and probably will cause me to loose more TILs before my sort. But I could use some other opinions on whether I am correct.
Any advice would be appreciated!
Hi Nathan,
I have more experience using human TIL from melanoma biopsies, but I can try answering some of your questions...
1. I prefer the "fragments" approach (see Tran et al, Minimally cultured tumor-infiltrating lymphocytes display optimal characteristics for adoptive cell therapy, J Immunother 2008, and other similar and more recent publications), so TILs remain "untouched" for the first part of your experiment and you don't disrupt the microenvironment. If you prefer digestion, I may suggest you to try different timing for collagenase I, because I don't like too long exposures and sometimes they are uneffective and may perturb the cells viability.
2. Ficoll works fine in my hand, even if I have really small amounts of cells, but no experience with Percoll.
3. I usually go straight to MACS beads, and I'm currently running parallel tests like:
TIL - MACS separation - analysis
TIL - Ficoll - MACS separation - analysis
Hope this can somehow help...
I´d go with Ficoll + MACS. For the in vitro/in vivo experiments the purity obtained with this protocol should be fine. However, for the microRNA analysis I´d rather use Facs Aria for sorting the population of interest.
@Riccardo
1. That fragmented method from Dr. Tran is very useful for in vitro generation! I am focused on exhausted T cells, and tried an in vitro generation method where I plated splenocytes with B16F10 tumor cells, but maybe instead I shall try out that method of plating a digested tumor fragment with 6000 U/ml IL-2. I used 50U/mL before and found my lymphocytes died after 5 days which may be due to my low IL-2 levels.
2. I will try the 100%/75% Ficoll than! I usually get around 3-5x10^8 tumor cells from each mouse so I have had issues of too many cells. But I will try the Ficoll gradient in a 15 ml tube and put only 1x10^8 cells in so that I don't overload the density gradient.
3. I am still skeptical about MACS beads since I am isolating CD4+/PD1+/TIM3+ cells. Even if I use the beads I still need to use the Aria so I may skip the beads for now to try to maintain as high a cell count as possible. If I can get 80%+ lymphocytes from the Ficoll then I feel a sort with the Aria will let me get my population of interest with >95% purity while maintaining the highest amount of recovered cells. Which parallel test do you find works better for maintaining a high purity with a high recovery rate?
@Leonardo
I agree, for miRNA analysis it may be best to go to FACSAria right away, but to make it so I don't have to use too many antibodies for FACS I will use the ficoll first to isolate lymphocytes. Thanks!
As well,
If anyone can share their Ficoll protocol with me for TIL isolation that would be fantastic! Do you mix the digested tumor cells in the 100% Ficoll, then layer on 75% and centrifuge at 400G for 25 min with a little bit of acceleration and no deceleration?
Or is there a better way to do it?
One thing I found - collagenase (type IV) stripped CD4 from cells (from the colon, in my case) if left too long - I started out with a 2hr digestion, am now down to 30mins with agitation. Works well and CD4 remains intact!
I was told to use Dispase and DNase too, but when optimising the experiment I found they weren't necessary to get a decent digestion in my case, so I've since dropped them.
Hope this helps!
https://www.ncbi.nlm.nih.gov/pubmed/6329947
Have a look at this paper. might be usefull..
I guess you already have enough help.
I work with melanoma in mice and would like to add I use Liberase for digestion but I keep incubation time minimum 20 or 25 min max. Melanoma breaks very easily. Also, Over digestion leads to lose of surface receptor specially CD4. Also, I use Percoll gradient. Again keeping the time limited as Percoll is toxic to lymphocytes. It worked fine for FACS as long as it is single cell suspension. I am still figuring out the sorting/bead isolation issue for isolation of specific population. I am only concerned about the change in phenotype of cells (activation/exhaustion markers) using any of this methods.
I work with B16F10 melanoma in mice and also use the TIL for experiment.
The protocol I used as following.
1. Remove the tumor and transfer the tissues to a cell strainer (40µm) in the 35mm × 10mm dish containing 2 mL of PBS w/ 0.1% BSA.
2. After cutting it to small pieces and using the back of a syringe plunger to macerate the cells through the filter, the single spleen cells were washed with 20mL PBS.
3. The pellets were re-suspending with 1 ml PBC followed adding 3ml Ficoll (1.084) (total volume is 4ml)
4. Add Ficoll 3ml to the centrifuge tube (15ml tube).
5. Carefully layer the resuspending tumor pellets to Ficoll in the centrifuge tube.
Then following the manual of Ficol.
Hopefully it works for you.
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