Figure 5.
Cell culture in microfluidic assay. (a) After aspiration of medium in the reservoir, 50~60 μl of the hMVEC suspension is added into one reservoir connected to the center channel. (b) Hydrostatic pressure difference in the reservoirs pushes the suspended hMVECs onto the collagen gel to facilitate attachment. hMVECs right after seeding (right top) and tight monolayer at 1 day (right bottom). Scale bar indicates 250 microns. (c) To add cell suspension or media into the reservoir, use a pipette and gently inject medium into one reservoir after aspiration. The filled liquid automatically fills the channel and replaces the contained medium. (d) Cell seeded assays can be stored in incubator or 6 well plate with various experimental conditions. (e) In 4 days of culture, hMVECs form intact monolayer in channel and on type 1 collagen wall. Actin filaments and nuclei were then stained with rhodamine-phalloidin (yellow; Sigma-Aldrich) and 4',6-diamidino-2-phenylindole (DAPI; blue; Sigma-Aldrich), respectively. Scale bar indicates 50 microns. EC monolayer was imaged by confocal microscope13 and its diffusion coefficient was measured by fluorescent dextran diffusion13,14,18.