Microfluidic Cell Assay

Photolithography (a, b, c) and Soft Lithography (d, e, f):

[1]   SU-8 is spin-coated and pre-baked on a bare wafer;

[2]   Use a transparency photo mask (black), ultraviolet (UV) light is exposed on the SU-8;

[3]   Exposed SU-8 is then post-exposure baked and developed to define channel patterns;

[4]   Polydimethylsiloxane (PDMS) mixed solution is poured on the wafer and cured;

[5]   Cured PDMS is then peeled from the wafer;

[6]   Device is trimmed, punched and autoclaved ready for assembly.

Hydrogel IncorporatingMicrofluidic Assay:

[1]   Autoclaved PDMS device and coverslip are assembled with plasma treatment;

[2]   To close the microfluidic channels;

[3]   Surface coating solution (PDL) solution is filled and device is placed in an incubator;

[4]   After washing and aspiration, coated device is stored in dry oven for 24 hours to render the microfluidic channel surface hydrophobic;

[5]   Hydrogel is filled into the hydrogel region;

[6]   Medium is added into the microfluidic channel.

Device is ready for cell seeding in incubator.

Cell Culturein Microfluidic Assay:

[1]  After aspiration of medium in the reservoir, 50~60 μL of the human microvascular endothelial cell (hMVEC) suspension is added into one reservoir connected to the center channel.

[2]   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.

[3]   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.

[4]   Cell seeded assays can be stored in incubator or 6-well plate with various experimental conditions.

[5]   In 4 days of culture, hMVECs form intact monolayer in channel and on Type-1 collagen wall. Actin filaments and nuclei were then stained withrhodamine-phalloidin (yellow, Sigma-Aldrich) and 4’,6-diamidino-2-phenylindole (DAPI, blue, Sigma-Aldrich), respectively. Scale bar indicates 50 microns. Endothelialcell monolayer was imaged by confocal microscopeand its diffusion coefficient was measured by fluorescent dextran diffusion.