Microporous MgF2 Cell Seeding Density over 3 days
One of the issues with my microporous MgF2 cell culture experiments is the apparent multilayer of cells that form on the freestanding area; ideally we would have a nice cell monolayer for imaging. It has been unclear if this was an effect of overseeding or if cells were migrating to the freestanding area.
To this end, I have cultured 4 devices with P6 ARPE-19 cells in a set of open well devices. Here the active well of each device is a 4 mm circle (12.56 
). The active windows of these devices are 0.1 mm squares, so the area is .01 
.
I counted 1.56e6 cells/mL from my suspension, and seeded 1 uL of cells into the device C well (1,560 cells). Each of the subsequent devices had a higher seeding volume as follows.
- A = 5x
- B = 10x
- C =1x
- D = 50x
The first picture is taken 10 minutes after seeding. Device D looks very similar to the types of cell layers I had previously been getting. Devices A-C become confluent at different timepoints, in order of cell seeding magnitude (B, A, C). We shall observe if more cells migrate into the freestanding region.
have you run this data via Emilia?
Is it possible that the medium is wicking through the nanomembrane and thus accumulating extra cells on the active area as time goes on?
Further, is the initial stack a projection from confocal images or is this all wide field? If the latter then i suggest you look with confocal and count the number of layers you have in the stack – each cell has only one nucleus so looking at a DAPI channel would make this unambiguous if the stack was re-projected in XZ/YZ
The wicking possibility exists, though I have tried to make sure that the fluid height in all wells is the same height post feeding. We have observed previously that the MgF2 membrane sags with larger area windows in some of the confocal work we did in Nottingham, though these membrane also had many cell layers on top of them. Using a smaller window and low levels of cells should help.