Oxygen plasma cleaner

To bond the hydrogels onto the glass surface, the following procedure was carried out. The microscopic glass slide was soaked in 0.4% v/v 3-(trimethoxysilyl) propyl methacrylate (TMS-PMA) (Sigma Aldrich, Singapore) for 12 h to provide bonding sites on the glass surface [54 (link)]. The glass side was then rinsed with water and dried at 70 °C for 2 h. Two coverslips were stacked up as spacers on the glass slide. Then the PDMS stamp was placed on top of the two coverslips (Figure 7B). To make the PDMS surface wettable, the PDMS stamp was treated using an oxygen plasma cleaner (Harrick Plasma, Ithaca, NY, USA) for 3 min. Afterwards, 50 μL of 5.0% w/v MeHA prepolymer solution was carefully added into the gap between the PDMS stamp and glass slide using a micropipette. The prepolymer solution was then exposed to ultraviolet (UV) radiation of 4.3 W/cm² for 40 s at 4 cm to the light source OmniCure s2000 (Excelitas Technologies, Waltham, MA, USA). After exposure, the PDMS stamp was peeled off from the surface, coverslip spacers were removed, and the formed hydrogel microwell array was placed in 10 mL of PBS solution.
The fabrication of hydrogel microwell arrays containing DPSCs is presented separately in Section 2.6.

We micro-fabricated millifluidic channels 30 mm in length, 1 mm in width and with heights ranging from 250 μm to 1 mm. A polydimethylsiloxane (PDMS) mixture (RTV615A+B from Momentive) was poured at ambient temperature in a polyvinyl chloride home-micromachined mold and left to cure at least 3 hours in an oven set at 65°C. Then, the recovered templates were drilled for further plugging of adapted connectors and tubings. PDMS templates and glass coverslips were then cleaned using an oxygen plasma cleaner (Harrick) and immediately bound together to seal the channels. The last step consisted in adapting connections: we used stainless steel connectors (0.013" ID and 0.025" OD) and microbore Tygon tubing (0.020" ID and 0.06" OD) supplied by Phymep (France). The thin metallic connectors accommodate on the flow circuit a bottleneck which prevented upstream colonization. Next the plugged device—usually a set of five channels 250 μm; 350 μm; 500 μm; 750 μm and 1 mm in height, respectively—was fixed on the microscope stage using a customized holder. The medium was pushed into the channels at a controlled rate using syringe pumps.

Before seeding cells in the microfluidic channels, the inner surface of PDMS was modified to covalently bond extracellular matrix molecules^{33 (link)}. Briefly, PDMS chips and standard glass slides were cleaned, activated in an oxygen plasma cleaner (Harrick Plasma, New York, USA) at 650 mTorr for 3 min, and bonded together. Immediately after bonding, the hydrophobic PDMS surface in the microchannels was silanized to make it hydrophilic by filling the channels with 5% 3-triethoxysilylpropylamine (APTES, Sigma-Aldrich, Buchs, Switzerland) and incubation for 20 min at room temperature. The channels were then washed with ultrapure water and treated with 0.1% glutaraldehyde (Sigma-Aldrich) for 30 min to provide a crosslinking substrate for the immobilization of extracellular matrix proteins. Microchannels were incubated with 50 µg/ml human fibronectin (Millipore, Schaffhausen, Switzerland) in PBS for 1 h at 37 °C or at room temperature overnight under UV light, followed by 100 μg/ml bovine collagen I in 0.2 mol/l acetic acid (Gibco, Thermo Fisher Scientific) at room temperature for 1.5 h. Cell culture medium containing 10% FBS was then rinsed through the microfluidic channels to block unspecific protein binding sites as well as to wash out unbound collagen I before cell loading.