Oxygen plasma

Microfluidic devices with sidewall microgrooves were fabricated using the photolithography technique that has been previously developed [21 (link)–24 (link, link, link)] (Figure 1). The silicon master mould was made using a negative photoresist (SU-8 2050, Microchem, MA). To make sidewall microgroove patterns of 80 μm thickness, SU-8 2050 was spin-coated at 1,500 rpm for 60 sec, baked for 8 min and 25 min at 65 °C and 95 °C, respectively, and exposed to UV for 3 min. After UV exposure, the photoresist-patterned silicon master was post-baked for 1 min and 8 min at 65 °C and 95 °C, respectively. The negative replica of the microfluidic channel was moulded in poly (dimethylsiloxane) (PDMS) (Sylgard 184 Silicon elastomer, Dow Corning, MI). The PDMS prepolymer mixed with silicone elastomer and curing agent (10:1) was poured on the master and cured at 70 °C for two hours. PDMS moulds were removed from the photoresist-patterned master. An inlet and outlet of the microfluidic channel were punched by sharp punchers for cell seeding and medium perfusion. The sidewall microgroove-containing channel and the bottom PDMS substrate were irreversibly bonded using oxygen plasma (5 min at 30 W, Harrick Scientific, NY). Sidewall microgrooves in the microchannels were placed perpendicular to the fluidic flow direction in the microfluidic device.

Microfluidic channels in polydimethylsiloxane Sylgard 184 (PDMS) were manufactured using standard soft photolithographic techniques (Duffy et al., 2004 (link)) and sealed on glass via oxygen plasma treatment (Harrick Plasma, Ithaca, NY, United States). The geometry consisted in a 50 μm wide and 10 μm high channel, in which a succession of 15 tooth-like patterns have been implemented as illustrated in Figure 1. Each teeth-like pattern was composed of a 5 μm wide and 10 μm long constriction, associated with a 25 μm wide and 10 μm long enlargement. This width oscillation has been repeated over 290 μm and was chosen for its ability to significantly center the RBCs at the exit of the last constriction.

The microfluidic cell was fabricated using polydimethylsiloxane (PDMS) shaped by a 3D-printed mold. The mold was designed with Solidworks (Dassault Systèmes, Paris, France) and printed with a resolution of 25 µm using a laser stereolithographic (SLA) 3D printer (Form3, Formlabs Inc., Somerville, MA, USA) using their proprietary transparent resin (Clear V4, Formlabs Inc., Somerville, MA, USA).
The PDMS was mixed with a curing agent in 10:1 weight ratio, respectively. The mixture was sonicated for 10 min and then kept in a vacuum desiccator to remove all bubbles. A few mm thick layer of PDMS was poured into a flat petri dish, covered with the mold and subsequently heated at 80 °C for 2 h. The mold was then peeled, and holes punched for fitting PTFE tubing. The cell was completed by forming a glass-PDMS-glass assembly. A 150 µm thick standard 24 × 60 mm² glass slide was used to cover the open channel of the cell, and a standard 150− µm thick, 18 × 18 mm² glass cover slip was used to close the readout widow. Glass parts were glued to PDMS by placing the separated components in an oxygen plasma (45 s, 40 Pa, 1.2 L/min, 30 W; Harrick Plasma Inc., Ithaca, NY, USA) and subsequently heating the assembly at 80 °C overnight. The glass-PDMS-glass assembly was finally mounted on 3D-printed cartridge support, whose dimensions fit the Insplorion instrument.

The deployed microfluidic device architecture has been reported by Warkiani et al. [20 (link),24 (link),25 (link),26 (link)], and was adopted for the isolation of CTCs from the SF. A microfluidic chip was structured as two polydimethylsiloxane (PDMS) layers (Sylgard 184 from Dow Corning, Midland, MI, USA) made by mixing liquid PDMS and curing agent in a 10:1 ratio (w/w). The top layer of the device fabricated by employing the previously reported micromolding process was bonded to a flat layer of PDMS following oxygen plasma treatment for 2 min (Harrick Plasma, Ithaca, NY, USA); 1.5 mm holes were punched at the beginning and ends of the microfluidic channel and 1.5 mm tygon tubes were inserted into each inlet/outlet.

The framework of the microphysiological system (100 × 40 × 8 mm) was polydimethylsiloxane (PDMS) carved from polymethyl methacrylate (PMMA) moulds. The PMMA moulds were engraved utilising a computer numerical control (CNC) machine (Jingyan Technology). Then, the PDMS was cast at a ratio of 10:1 (w/w) monomer to curing agent in PMMA moulds and crosslinked at 70°C for 2 h. There were two layers of PDMS in the microdevice, with the top layer containing a medium channel (70 × 2 × 2 mm) and the bottom layer containing a gas channel (70 × 2 × 2 mm). After the construction of the two layers, the bottom layer was attached to the commercialised PDMS membrane using oxygen plasma (Harrick Plasma). Then, the top layer was attached to the bottom membrane layer with oxygen plasma. Finally, the medium channel and the gas channel were connected to different pumps, providing a medium flow and cyclic negative pressure.