Glycosil

Hydrogel precursors methacrylate-conjugated bovine collagen type I (MA-COL, Advanced BioMatrix, Carlsbad, CA, USA; 3.6 mg/ml), thiol-conjugated hyaluronic acid (SH-HA, Glycosil^®, Advanced BioMatrix; 0.5 mg/ml, 0.025% (w/v) photoinitiator Irgacure^® 2959; Sigma-Aldrich, St. Louis, MO, USA) and in-house expressed elastin-like polypeptide (SH-ELP, thiol via cysteine in KCTS flanks ^{37 (link)}; 2.5 mg/ml) were thoroughly mixed. Thirty microliters of the hydrogel solution were pipetted onto a Surfasil (Fisher Scientific) coated 12-mm round glass coverslip followed by placing a regular 12-mm round glass coverslip onto the hydrogels. Constructs were crosslinked by exposure to UV light (OmniCure S1500 UV Spot Curing System; Excelitas Technologies, Mississauga, Ontario, Canada) at 320–500 nm, 2.2 W/cm² for 5 s, as previously described ^{37 (link)}. The hydrogel-adhered coverslips were removed with fine-tipped tweezers and placed in 24-well culture plates (Corning; Thermo Fisher Scientific). HTM cell-laden hydrogels were prepared by mixing HTM cells (1.0 × 10⁶ cells/ml) with MA-COL (3.6 mg/ml [all final concentrations]), SH-HA (0.5 mg/ml, 0.025% (w/v) photoinitiator) and ELP (2.5 mg/ml) on ice, followed by pipetting 10 μl droplets of the HTM cell-laden hydrogel precursor solution onto polydimethylsiloxane-coated (PDMS; Sylgard 184; Dow Corning) 24-well culture plates (Suppl. Fig. 2C).

FibriCol^® (10 mg/mL) and Glycosil^® were obtained from Advanced BioMatrix (San Diego, CA). pCMV3-VEGF-A plasmids were acquired from Sino Biological (Wayne, PA). Following the manufacturer’s protocols, pCMV3-VEGF-A plasmids were amplified in MAX Efficiency^™ DH5α competent Escherichia coli (Thermo Fisher, Waltham, MA) and purified using a Qiagen Maxiprep Kit (Germantown, MD). Murine recombinant VEGF-A was obtained from Pepro Tech. (Cranbury, NJ). CellTiter 96^® Aqueous One Solution Cell Proliferation Assay (MTS) was purchased from Promega (Madison, WI). Geltrex^™ and Calcein-AM were acquired from ThermoFisher (Waltham, MA). Alexa Fluor^® 555-conjugated Rabbit IgG-CD31 polyclonal antibody (PECAM-1) and Alexa Fluor^® 555-conjugated Rabbit IgG Isotype Control were purchased from Bioss Antibodies Inc. (Woburn, MA). Mouse IgG2a monoclonal α-SMA-FITC antibody and mouse IgG2a-FITC antibody were obtained from Sigma-Aldrich (St. Louis, MO).