Mp468

MDA-MB-231 cells (ATCC, Manassas, USA) were cultured in
Dulbecco’s modified eagle medium (Gibco, Grand Island, USA) with 10%
fetal bovine serum (Gibco, Grand Island, USA) at 37°C with 5%
CO₂. Cells were used between passages 5 and 7. The media was
changed every 48 hours. Human Umbilical Vein Endothelial Cells (HUVEC) (Thermo
Fisher Scientific, USA) were cultured in medium M200 (Thermo Fisher Scientific,
USA) supplemented with a low serum growth supplement kit (Thermo Fisher
Scientific, USA). HUVECs were used between passage 1–3. Cells were
enzymatically dissociated using TrypLE (ThermoFisher Scientific, MA, USA) for
5–7 minutes and centrifuged at 250G for 5 minutes, and resuspended.
MDA-MB-231 cells were seeded at a density of 30,000 cm⁻², and
HUVECs were seeded at 20,000 cm⁻². Before seeding, PDMS
channels were lifted off from the POMA functionalized coverslips to enable
direct access to the collagen gel. A 2mm × 20mm² laser-cut
poly(methyl methacrylate) (PMMA) well was cleaned with ethanol and attached to
the region surrounding the gel using a pressure-sensitive adhesive (MP468, 3M,
USA).

The UPP membrane was detached from the silicon wafer by immersing the
wafer in DI water until the UPP floated away from the substrate (1 minute). A
membrane holding frame was fabricated using 1.5mm × 18mm PMMA
(McMaster-Carr, California, USA) with a laser cut 2 mm × 2 mm region
central was attached to the UPP using pressure-sensitive adhesive (MP468, MP467,
3M Company, Minnesota, USA). The outer edges of the PMMA frame were attached to
the glass coverslip surrounding the exposed collagen gel using
pressure-sensitive adhesive such that the UPP membrane made direct contact with
the collagen.