Genipin

Type A gelatin (300 Bloom) from porcine skin was supplied by Sigma-Aldrich, genipin was provided by Wako Chemicals USA and was used without further purification. Acetic acid (AcAc) was supplied by Sigma-Aldrich.

Gelatin supplied from Nitta-Gelatin Ltd. (Japan headquarter) is a high-grade quality, low-endotoxin unit essential for diminishing immune rejection post-implantation. It is currently manufactured at Nitta-Gelatin Ltd. (India branch) and is certified halal, originating from a buffalo’s raw bone. Quercetin (QC) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and utilized without further purification. Elastin was procured from the Faculty of Science and Technology, Universiti Kebangsaan Malaysia (FST, UKM) and Genipin was purchased from FUJIFILM Wako, Osaka, Japan. Pharmaceutical-grade solvents and reagents were used in this study and were used as received.
The location for this study was the Centre for Tissue Engineering and Regenerative Medicine (CTERM), Faculty of Medicine, and several tests were run at FKAB, UKM, and iCRIM UKM. The study design was approved by the Universiti Kebangsaan Malaysia Research Ethics Committee (UKM PPI/111/8/JEP-2021-301).

Gelatin (250 Bloom; bovine bone origin) was supplied by Nitta Gelatin India Limited, Cochin, India. Genipin was purchased from FUJIFILM Wako Pure Chemical Inc., Osaka, Japan, while EDC and N-hydroxysuccinimide (NHS) were obtained from Sigma-Aldrich, St. Louis, MO, USA. Various gelatin concentrations ranging from 6% (w/v) to 10% (w/v) were utilized to create hydrogel. The gelatin was dissolved in DPBS at 50 °C. Upon obtaining a homogeneous solution, different concentrations of crosslinkers (Genipin or EDC; both 0.1% (w/v)–0.5% (w/v)) were then added to achieve hydrogel crosslinking. The gelatin solution was mixed with Genipin powder at 50 °C for three minutes to ensure homogenous polymerization until a yellow mixture was obtained. The fabrication method was demonstrated in Supplementary Vid. 1. EDC:NHS at a ratio of 4:1 was dissolved in ultrapure water and added to the gelatin solution. In calculations, all concentrations of hydrogel (%) represented as calculation of (w/v). Gelatin as "G" and Genipin as "gn" were also used as abbreviations.

PMECs (Lonza) were used to generate 3D pulmonary microvessels. PMECs were cultured in EC medium from passages 6 to 8 using routine procedures. Three-dimensional, 150 μm diameter microvessels were fabricated based on published protocols (45 (link)). Microchannels were patterned in 7 mg/mL type I collagen (Corning) using nitinol wire, then cross-linked with 20 mM genipin (Wako). PMECs were suspended at 1 × 10⁶ cells/mL in EC medium and seeded into microchannels under static conditions for 30 minutes to promote cell adhesion. Microchannels were then perfused at 3 dyne/cm² for 48 hours in EC medium to form confluent pulmonary endothelium. To replicate the inflammatory state, microvessels were perfused with EC medium supplemented with 1000 U/mL recombinant human IFN-α2 (BioLegend) and 50 ng/mL recombinant human IFN-γ (R&D Systems) for 24 hours.

Gelatin (Gel) with mesh
4 and bloom 280 was extracted from pig skin from Italgelatine (Cuneo,
Italy). Chitosan (Chit) of low molecular weight (75,774.77 g/mol)^{21 (link)} and deacetylation degree of 79 ± 1%^{21 (link)} was purchased by Sigma Aldrich (Saint Louis,
Missouri). Genipin (Gen, purity 98%) of natural source was purchased
by Wako Chemicals (Wako Pure Chemical) and obtained by extraction
from Gardenia Jasminoides Ellis.