Fabrication of Vessel-on-a-Chip Microfluidics

The vessels-on-a chip were made using the devices developed by the lab of Beebe, as previously described (8 (link)). Minor alterations to the protocol were made. Briefly, the devices were coated with 1% PEI (Polysciences Inc., Warrington, PA, USA, #23966) and incubated for 10 minutes at room temperature (RT). Sequentially chambers were coated with 0.1% glutaraldehyde (Merck, Darmstadt, Germany, #104239), washed 5x with water for injection (WFI; Gibco, #A12873-01) and air-dried. Collagen-1 was prepared according to the protocol above. 10 µl Collagen-1 was added to each chamber and polymerized for 30 minutes at 37°C, 5% CO₂. PBS-drenched cotton balls were added to the device to control humidity of the device. Rods were removed using tweezers and EGM-2 was added to the lumen. HUVECs were washed twice with PBS, trypsinized for 5 minutes, treated with trypsin neutralizing solution (TNS; Lonza, cat CC-5002) and centrifuged for 5 minutes at 200xg. HUVECs were then stained using CellTracker™ Green CMFDA Dye (1 µM; Molecular Probes, cat C7025) according to manufactures protocol, washed twice with PBS and pelleted. HUVECs were then resuspended to a concentration of 15*10⁶ cells/mL. 5µl of cell suspension was added to each lumen and placed in head-over-head at 37°C, 5% CO₂ for 2 hours at 1 RPM. Vessels were matured for 2 days with medium replacement twice daily.