Lower density fibroblasts on pnc-Si transwells
Last time I cultured fibroblasts on pnc-Si transwells, the cells got super-confluent over the membrane and made the analysis difficult. Basically, I wanted to see if fibroblasts formed vacuoles/2D structures over free-standing membranes. I thought the multi-layer cells could have choked off permeability and prevented the higher layers of cells from even sensing that they were above a permeable floor. This time, I seeded the cells @ 50000 cells/cm2 and let them grow for just over 1 day instead of 2 days, as before. These were P37 cells on SC309 transwells.
Live(green)/Dead(red)/Phase(blue):
Here’s another field of view in just phase:
So, definitely no vacuoles on these lower density cells. It seems like there might be some 2D organization into tube-like structures but this is also happening away from the free-standing membrane. Also, there is no alignment of these organized cells with the free-standing membrane. So now I’ve shown that 2 endothelial tissue cell types but not connective tissue cells (these fibroblast-like 3t3-L1).
The lack of vacuoles is good news but its becoming unclear if the 3-D structures are being induced by the membranes since they are happening everywhere. It seems we need to routinely do sham (no windows and/or glass coverslips) controls in order to interpret our results. I hope there isn’t something happening to all our cultures to produce these network-like cell structures.