Endothelial cells on different width membrane windows

Along with differently sized square membranes, I also designed a wafer with different sizes of slits.  On each chip, there is a 4000, 2000, 1000 and 500um long window.  The thicknesses of these windows are 200, 100, 50 and 25um.  I’m interested to see if there is an aspect ratio that causes endothelial cell alignment/tubulogenesis.  For this experiment, I seeded bEnd3 (P?) at 50000 cells/cm2 and allowed them to grow for 2 weeks.  These are SC604 and 605.  A couple of the membranes broke over this timeframe but I was able to get images from each chip.

Here are 20X images arranged from smallest to largest width.  There are vacuoles on all membranes.  All sizes of membranes have cells with interesting 2D, almost capillary-like features and spindly-looking cells.  These tubes are on the membranes but also extend onto supported pnc-Si.  Recently, I haven’t seen structures like these, but I’ve typically been staining cells before 2 weeks – the bEnd3 cells might progress to this state with longer culture periods.

I also took some pictures at 4X to get an idea of what the entire monolayer looks like.  Unfortunately, the larger width membranes broke (bottom 2 images).  You can clearly see that these tube-like features extend across supported pnc-Si, as well.  This isn’t the best news for us if cells can form tubes on nonporous pnc-Si.  However, you might be able to argue that these tubes originate around the membranes.  It looks like most (all?) of these tubes are connected to the membrane area somewhere.  Could these tubes start on pnc-Si and grow outward across the culture surface.  Or could cells forming vacuoles on free-standing membranes be releasing pro-angiogenic signaling factors to induce other cells in the monolayer to initiate vasculogenesis?

Also, it’s hard to say (since this is only n=1) but there might be more tubes on the smallest 3 slit widths (the top row).  It also seems like the tubes that are present are aligned with the skinnier slits better than wide slits, as well.  Pretty interesting and I’m repeating this now.