Hello everyone,
I often grow endothelial cells in 3D gels, but one thing I would like to be certain of is whether or not they are developing a lumen. I have read several papers which show images of endothelial networks where the authors show images of endothelial cells labeled fluorescently to show what appear to be luminal voids where the cells are, but is there a more directly method that proves it, such as an antibody which is specific for markers expressed on the luminal surface? Thank you.
Hi !
I think that electron microscopy is the best approach to conclusively demonstrate the presence of a lumen and you don't even have to buy a new antibody! Apparently, in Binghamton, your electron microscopy facility is more oriented towards "material science" (http://www.binghamton.edu/adl). But the sample preparation in your case should be quite simple so they could either do it themselves or at least orient you towards a "life science" EMF which could do this for you in few days.
If you're willing to ship your samples to France, then we can also start a collaboration ;P
Nico
I recommend staining your endothelial cells with a robust cytoplasmic stain (F-actin or CellMask), and visualize the endothelial networks using confocal microscopy. You'll get the best imaging resolution if you plate your constructs on a thin glass coverslip as opposed to a standard glass slide or well-plate (unless you're using a plate specifically designed for imaging, such as the Ibidi u-Plate (http://ibidi.com/xtproducts/en/ibidi-Labware/m-Plates/m-Plate-96-Well) or Greiner Bio-One (http://nic.ucsf.edu/dokuwiki/doku.php?id=imaging_dishes). Nonetheless, you should use a small z-step (1-2um) and a 10x, 20x, or 40x objective, though depending on the thickness of your construct, you might only be able to use a 10X high working distance objective to capture enough thickness in the image.
An alternative to this approach is to use a microfluidic device to seed your endothelial cell-containing hydrogels. If your network is able to 'plumb' the inlet and outlet ports of the microfluidic chip, you should be able to flow through fluorescent microbeads and capture the position of the microbeads using real-time epifluorescence microscopy. I published on this approach (http://www.ncbi.nlm.nih.gov/pubmed/25190668), but this particular experiment was performed by my collaborator, so I do not have a lot of details other than what was written in the methods section.
I think electron microscopy would be difficult with hydrogels since the typical procedure for sample prep for scanning EM calls for a dehydration and gold sputtering, which would destroy the architecture of your gel. You may consider cryo-EM, but with this approach, you would need to fracture the hydrogel to image a cross section wherein you would be able to see endothelial lumens.
Best of luck to you!
What I meant was a standard Transmission Electron Microscopy procedure. It still involves dehydration yes but only after fixation, so it should not perturb the gel and even less the morphology of the cells. It is done routinely for decades now...
I have no doubt that TEM would work to visualize an endothelial lumen (http://www.ncbi.nlm.nih.gov/pubmed/16170819) if the hydrogel construct is cut into thin slices, but this is a far more difficult sample prep than confocal microscopy (http://onlinelibrary.wiley.com/doi/10.1002/app.38034/abstract).
From my own experience, I can recommend the TEM-approach with a slightly adapted processing method including agarose pre-embedding as described in my paper "Electron microscopy of cultured angiogenic endothelial cells". This approach also offers the advantage that other cellular features of lumen formation such as typical sealing cellular junctions etc. can be observed. Even the modus of lumen formation is detectable via TEM.
http://www.ncbi.nlm.nih.gov/pubmed/16170819
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