Irgacure 2959

Poly(ethylene glycol) diacrylate (M_n 575) was obtained commercially (Sigma Aldrich, St. Louis, MO) along with reduced glutathione (Chem-Impex International, Wood Dale, IL) and Irgacure 2959 (Ciba, Basel, Switzerland). Analytical grade meropenem (Supplementary Figure 1A) and vancomycin hydrochloride (Supplementary Figure 1B) were obtained from Sigma Aldrich (St. Louis, MO) along with pravastatin sodium hydrochloride (Supplementary Figure 1C) from Cayman Chemical (Ann Arbor, MI). Dulbecco’s modified phosphate buffered saline (PBS) without calcium and magnesium was obtained from Corning (Manassas, VA) and used for all loading and release experiments unless otherwise noted. Cation-adjusted Mueller-Hinton broth (CAMHB) and agar along with tryptic soy agar with 5% sheep’s blood plates were obtained commercially (Becton Dickinson, Franklin Lakes, NJ). Recombinant glutathione-S-transferase proteins were expressed and purified from Escherichia coli as previously described (27 (link)). Pseudomonas aeruginosa isolates (ATCC 27853 and AR 0440) were obtained from our internal biorepository and the Centers for Disease Control and Prevention (CDC) Antibiotic Resistance (AR) isolate bank (28 ).

Fabrication: PEG-acrylate (4 arm, 20,000 Da; Laysan biomaterials; 10% w/v) was dissolved into PBS with photoinitiator (Irgacure 2959, Ciba; 0.5% w/v). To form porous hydrogels, the solution was frozen for 16 hours at −20 °C and then exposed while frozen to UV light (365 nm, 50 mW/cm²; 2 min). To form non-porous hydrogels, UV exposure was employed to crosslink the liquid-phase PEG solution. The formed hydrogel was then immersed in PBS or distilled water until use.
Mesh Size and Swelling Ratio: PEG hydrogels were formed as before (n = 4 per condition), weighed, and placed in distilled water. After 24 hours, the hydrogels were weighed, frozen in liquid nitrogen, lyophilized for 24 hours, and weighed again. Swelling ratio and mesh size was approximated using a modified Flory-Rehner model [20 (link)-22 ].

20 uL hydrogels were fabricated in a one-pot synthesis reaction within a 1 mL syringe from which the tip was removed. Between 3 and 5 wt% PEG8a-NB was mixed with appropriate concentration of adhesion peptide (0 – 3mM nominal) and crosslinked at stoichiometric ratios between 0.35 and 0.65, referred to as 35 or 65% crosslinking, respectively using LW. In experiments were adhesion peptide concentration was variable, a scrambled adhesion peptide (RDG) was added to keep the total concentration of adhesion peptide in the system consistent between conditions. Cells were added to the precursor solution in serum free medium at a final concentration of 12 million cells/mL. Gels were polymerized using 0.05 wt% IRGACURE 2959 (Ciba, Prod. No. 0298913AB) UV-irradiated for 45 seconds at ~100mW/cm².

Hydrogel arrays were formed in two steps on subsequent days as previously described[34 ]. Induced pluripotent stem cell-derived endothelial cells (iPSC-ECs; Cellular Dynamics International, Inc.) were expanded before use in Growth Medium containing 10 vol.% serum supplement (Cellular Dynamics International, Inc.), VEGF LifeFactors kit (LifeLine), penicillin/streptomycin, and VascuLife (LifeLine) and were used at passage 5 for experiments. On day 0 of experiments, iPSC-ECs were encapsulated in eight-arm PEG-norbornene hydrogels containing cell-adhesion peptide (CRGDS; GenScript) and matrix metalloproteinase-degradable crosslinker (KCGGPQGIWGQGCK; GenScript). iPSC-ECs were suspended at 8 × 10⁷ cells/mL in 0.1 wt.% Irgacure 2959 (Ciba) in PBS and immediately diluted 1:1 in a 2X hydrogel precursor solution to make a final solution containing 4 wt.% eight-arm PEG-NB, 2 mM CRGDS, 3.6 mM KCGGPQGIWGQGCK. Cells were then encapsulated by exposing 0.5 μL cell-dense “spheres”, formed at the end of a 10 μL pipet tip, to UV at 0.18 J/cm². iPSC-EC cell-dense spheres were cultured overnight in 16 well ProPlate (Grace Bio) slide chambers (with 64-well PDMS stencils installed in place of the 16-well silicone stencil) in Growth Medium at 37°C, 95% relative humidity, and 5% CO₂.