Fibroblasts on pnc-Si transwells
One of the hidden secrets of endothelial angiogenesis assays on Matrigel is that non-endothelial cell types also form 2D capillary-like structures (tubes) on those gels. Motivated by this observation, I wanted to look at fibroblast behavior on pnc-Si transwells, specifically with respect to whether or not they formed vacuoles. Just to clarify – although Anant did many experiments with fibroblasts on pnc-Si, his experiments were done in 2D (flat chips) and he did not investigate cell behavior on free-standing pnc-Si membranes.
I got some 3t3-l1 fibroblasts from Nakul recently and plated them on SC604 (P35) at 50000 cells/cm2. I then stained the cells with Live/Dead after 2 days (I wanted to give them enough time to form vacuoles, if they could).
Here are the Live/Dead stained cells – the morphology is obviously different than endothelial cells. In the first image, the focal plane is at the supported pnc-Si surface and the cells over the free-standing membrane are out of focus. In the second image, I changed the focus to capture the cells over the membrane. There are several really interesting things to note here. First, the cell density on the free-standing membrane is ridiculously higher than elsewhere. It is so high that the cells are growing as multi-layers, which is why they are out of focus in the 1st image (I don’t think the membrane is sagging). Therefore, something seems to be recruiting cells to move over the free-standing membrane. Is it permeability, or are the cells already on the membrane releasing signaling factors to recruit more cells. Second, the cells are aligned almost perfectly with the membrane window even though there is a more random morphology on supported pnc-Si. Even endothelial cells don’t do this. Third, there are no vacuoles over the membrane. Unfortunately, there are so many cells that they may have choked off the membrane permeability and prevented vacuole formation. I need to repeat this experiment with a lower density of cells.
Image 1:
Image 2:
