In-vivo Cell Tracking

[GabeSauce]

Hello fellow scientists!!

 

I am the lead researcher of a group that is trying to produce a functioning tissue-engineered ligament for anterior cruciate ligament regeneration.

Anyway... we are currently using a rat model and implanting "scaffolds" (basically a polymer construct) with cells seeded onto them into the knee capsule. I am trying to figure out how to "track" the cells to evaluate survival and proliferation after we sacrifice the animals. By "tracking" I mean differentiate between our implanted cells and the host cells that infiltrate our construct. There are two transplantation models I am trying to figure out:

 

1) Rat cells into rats. We have GFP positive fibroblast cells (harvested from a transgenic GFP rat) that we implant into normal (non-GFP) animals (for weeks to months), but there are a whole host of problems trying to actually TRACK these GFP+ cells in post-mortem histological sections. The biggest problem is that after a few passages in culture, these fibroblasts hardly transcribe the GFP gene anymore. If I take a flask of these GFP+ fibroblasts and put them under a fluorescent scope, I don't see any green fluorescence. So tracking the cells in-vivo using their endogenous fluorescence is a no-go. Next we took tissue-sections of our implants and tried anti-GFP immunohistochemistry (IHC) using a primary antibody against the GFP protein and a fluorescent secondary and once again saw basically no GFP signal (probably due to extremely low levels of GFP expression by the implanted cells). Now, we know we have cells in our constructs because we can do PCR and detect the GFP gene, but we want to visualize the distribution of cells.

 

Any ideas for alternative cell-tracking techniques (with or without relying on GFP) would be greatly appreciated. Do any of you have any experience with FISH? Could I use FISH to visualize the GFP genes in our construct? What about CM-Dil? I'v heard mixed reviews about CM-Dil and heard that it can photobleach during processing. Also, if I use CM-Dil (which is red, I believe), does that mean I cannot do IHC using a red fluorescent secondary (like AlexaFluor 594)? Our sections are formaldehyde fixed and paraffin embedded.

 

2) Human cells into athymic rats. We are working on a model of xenograft fibroblast transplantation, basically human fibroblasts onto a "scaffold" implanted into an athymic rat knee. How do I track the human cells and differentiate them from rat cells? I assume there are a whole host of proteins that are uniquely human that I can stain for using immunohistochemistry. I've heard human alkaline phosphatase is commonly used for tracking human xenograft implant survival but I'm not certain.

 

Once again, tracking rat cells implanted into other rats (lewis rat cells into lewis rats) and tracking human cells into rats. If anyone understands what I'm saying, PLEASE HELP!!!

Edited July 27, 2012 by GabeSauce

[ecoli]

Do you actually need vizualization of cells? If you're having problems visualizing GFP, than FISH wouldn't be much of an improvement. However, you could do Flow/ FACS / cell sorting with an anti-GFP antibody (no visualization or microscopy necessary) and you get quantitative composition of a mixed cell population.

[CharonY]

I am surprised that the transcription would go down. The question I would have is what the reasons are. For instance, under which promotor is the GFP? Technically, with a very good optical setup (say, confocal) one could detect GFP down to a single molecule, though this is obviously not always possible. However, if there is at least some expression but even IHC does not work, I am also not sure whether FISH would yield much more. FISH in tissue does not have terribly good sensitivity. And RNA Fish is not going to help if transcription is really low.

Maybe first check the transcription level (as opposed to only look for the gene?).