Cells on squares and slits (2)
I few weeks ago, I had a couple wafers made with different size squares and slits. I posted my first results here and here. In that experiment, I showed pretty dramatic 2D structures over both supported and free-standing membranes with rectangle shapes. Also, I showed vacuole formation at (almost) the single cell level on square membranes. For this experiment, I wanted to try a repeat but this time with bEnd3 on flat TCPS as a non-permeable control culture surface.
Once again, these were RTP’ed SC604 and 605 with P13 bEnd3 cells seeded on the bottom of the transwells at 50000 cells/cm2. All images were taken after 2 weeks of growth.
Most of these transwells broke over the 2 week culture period, so I’m only including a couple of samples in this post. For the smallest square membranes, I found no 2D structures on the pnc-Si surface at 4 weeks – I didn’t include 4X images in my original post but there didn’t appear to be any tubes on those samples, either. At 20X, cells over the membranes express vacuoles. Also, cells right next to cells over the membranes are showing vacuoles. So, although these windows are bigger than 1 cell, vacuole formation is certainly a localized/few cells response.
In contrast with my previous experiment, cells on the rectangular membrane windows did not form 2D/tube structures (see 4X). The large patch of dead cells in the corner is where I accidently banged into the cells with the pipette tip. These slits are so thing that they are only a single cell body wide. The cells over this membrane are aligned with the window and they express vacuoles. Interestingly, vacuole formation seems to be confined to the cells over or touching the membranes only, unlike the small square membrane windows sample.
The flat control surface had cells with no vacuoles and no tubular structures:
Therefore, this was not a repeat of tube formation on these pnc-Si samples even though the wafers and growth conditions were the same. I’m not sure what to make of this. This was a repeat of localized vacuole formation over very small free-standing pnc-Si windows, though.