Cells in free space
Tom recently designed a wafer with 1cm2 chips containing a 2×2 array of 1mm2 square membrane windows that fit on 384-well plates. I wanted one of these chips to get a preliminary data image for my R21 application. Instead, I saw something much cooler.
I plated bEnd3 cells on the pnc-Si side of this chip on Friday and let them grow over the weekend. Since this is in 2D, the chip discolored. When I looked at the cells yesterday, I noticed a strange coloration:
The upper left corner of the membrane has a darker tan/light brown color, which is how intact membranes typically appear with cells growing on them in transmission/phase microscopy. I wasn’t sure what to make of the transition from that color to the brighter region on the bottom/right.
So, I looked at this sample in reflection mode:
at 5X:
at 10X:
There is no membrane reflecting light on the right side of this window and it seems like the membrane is bunched up underneath the cells. The membrane-less area of the window corresponds almost exactly with the discoloration pattern (see 5X). Even though the membrane only spans ~3/4 of the window area, the cells are still growing over the entire window. These cells are growing completely suspended in space (this matches the 10X image above):
This is the 1st time I’ve seen a partially degraded membrane window with cells spanning the entire window. This proves one of the things we’ve been claiming – that we can grow cells on a membrane and then elicit membrane degradation in order to have direct access to both sides of cell monolayers (either for manipulation or for plating a new cell type directly on the cells). This is basically a free-standing membrane composed only of cells.
Here are 10X images of an intact membrane window with cells as a comparison:
Transmission:
Reflection:
Several comments: First, what could be cooler than getting data for a R21? Second, moving the reflection microscope to the basement has paid dividends (glad I thought of it Nakul!). I never imagined we could image cells and the membranes so clearly with this scope, this is a fantastic ability I didn’t know that we had. Third, the cells are not really ‘growing’ in these images. Its likely that they are just hanging on for dear life as the membrane degrades beneath them. Its interesting to ask if they have the ability to recover to make a confluent layer by patching the holes that develop in the monolayer. I bet with smaller membranes, they can. It certainly demonstrates that they apply tension to each other to make this rather taught looking cell-based trampoline. Finally – yes – these images do help us make the case that we can do controlled degradation of the membrane to create freely suspended monolayers. It is cool.
Are these membranes wrinkled to start with, or do they wrinkle over time as the media attacks it? Had the membrane been tight and flat, it probably would have broken more violently when it got very thin, potentially disturbing the layer of cells. Here, it looks like the membrane wrinkle and gradually broke away from the support, apparently a gentle process. Nice images!
These membranes are pretty flat on the bench – they are the square 2×2 arrays that Tom designed.
I’m curious whether the wrinkles are created by the cells. The only other way to wrinkle would be by oxidizing the membrane, I think. I guess it would make sense that the cells are pulling and stretching the membrane…
True, both are possible.