Co-Culture Cross Sections
To supplement our upcoming manuscript, we decided that we needed to provide a visual of how thin our ultrathin SiO2 membranes actually are. To demonstrate the thinness of the SiO2 membranes, in comparison to the commercially available Track-Etched hanging membrane inserts, we performed a co-culture using 0.5µm high porosity SiO2 membranes (thickness ~0.1-0.3µm) and 0.5µm high porosity Track-Etched hanging membrane inserts (thickness ~10µm).
The 0.5µm SiO2 high porosity membrane was built into the standard CytoVu device. The CytoVu devices and 0.5µm Track-Etched hanging membrane inserts were placed into a 24-well TCP plate. HUVEC and ADSC were seeded on opposite sides of each membrane type. After several days, the cells were stained for nuclei (DAPI, blue), F-actin cytoskeleton (phalloidin, green), and VE-cadherin (anti-cd144, red) which is a cell-cell junctional molecule only present in HUVEC monolayers.
The SiO2 membranes were removed from their CytoVu devices and enclosed between two glass coverslips that were adhered to a metal slide using vacuum grease. The Track-Etched membranes were cut out of their hanging inserts and sandwiched between a glass slide and a glass coverslip, sealed with clear nail polish. Planar images were captured using the three common fluorescence channels, DAPI, FITC, and TRITC using the 20x objective on our wide-field microscope.
The samples were then imaged using the COS confocal microscope. Z-stack images (315µm x 315µm, step size z=1 µm) were acquired to image the entire co-culture, including both cell layers and the membranes. Z-stack images were cropped to approximately 315µm x 10µm. From these crops, 3D maximum projections were created and then rotated 90deg in the x-axis, synthesizing a cross-sectional view of the partial z-stack.
The cross-sectional images show the thickness of the SiO2 membrane to be less than 1 µm. The thickness of the Track-Etched membrane is approximately 10 µm, in agreement with the dimensional values provided by the manufacturer.