Cell growth in micovasculature mimetic device
I am trying to grow HUVECs under flow in my device to establish a tight and confluent monolayer for TEER analysis. My device schematic looks like this
and the whole setup is as below
I tried to use the NPN chip in sepcon format so that I don’t have to be restricted by number of chips to be used. I first tried using the 5-slot membrane due to its availability. However I had a tough time in outgassing the system and getting rid of the bubbles beneath the trenches. Hence I decided to switch to 3-slot membrane hoping that that’ll solve my problem. I used the chip from wafer 1024; it has pnc-Si cap on it. I RTPed the chip with the cytovu non degradable recipe. I didn’t coat or prime the surface with any other material. I am using the P3 HUVECs from Vectec using their MCDB media for cell culture. I harvested the cells and seeded inside the device. Usually I start the flow on the back side after cells are seeded inside the device. (This is to promote gas exchange and fresh nutrient replenishment via the back side.) Instead I waited for the cells on the pncSi to adhere to the substrate. After 2 hours of incubation at 37C, I started the flow. The flow rate was abut 100 ul/min, which, as per my device geometry, yielded much lesser than 1 dyne/cm2 shear stress. As soon as I started my flow, I started seeing the cells are slowly changing from phase dark to phase bright, i.e. they are rounding up and getting washed away. So I stopped the flow and decided to continue with the static culture. The cell media was static but was connected to a media reservoir, which is in turn was exposed to 5% CO2 via a 0.22 micron filter. I performed the imaging after 12,24 and 46 hours. I wanted to wait till the cells get confluent and then start the flow.
Cells after 12 hours
After 24 hours
After ~2 days
As seen from the last image, there was a bubble formation occurring inside the device. I did start the flow, but there was a leakage inside the device causing the whole media to get washed away. Also I can’t see much difference in the confluency after 12 hours time point. Nonetheless, it is my first successful attempt in growing cells in a closed loop fashion, inside any device.