Omnicure s1500 uv spot curing system

MONHAs (2.5 × 10⁶ cells/ml or 5.0 × 10⁶ cells/ml) in media were mixed with 3.1 mg/ml methacrylate-conjugated bovine collagen type I, 1 mg/ml thiol-conjugated hyaluronic acid, and 0.025% (w/v) riboflavin (Sigma, R7774) (photoinitiator) on ice (Fig. 1). The chilled MONHA hydrogel precursor solution was pipetted as (1) 10 μl droplets onto PDMS-coated (Sylgard 184; Dow Corning) 24-well culture plates, (2) 30 μl droplets onto 12 mm round glass coverslips sandwiched with Surfasil (Fisher Scientific) coated coverslips on top, or 80 μl into custom 10 × 1 mm PDMS molds. Constructs were then crosslinked by exposure to UV light (OmniCure S1500 UV Spot Curing System; Excelitas Technologies, Mississauga, Ontario, Canada) at 405–500 nm, 10.3 mW/cm² for 5 min. MONHA growth medium was added to each well and replenished every 2–3 days, and constructs were cultured for 1–4 weeks.

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).