G2500

Gelatin channels were fabricated using the molding technique. A mold was prepared using a silicon container and a cylindrical plastic tube (outer diameter: 1 mm) in which 10 wt% molten gelatin in PBS was crosslinked into a tubular channel structure. To prepare 10 wt% gelatin solution, 10 g of gelatin powder (G2500, Sigma-Aldrich) was dissolved in 100 mL of PBS buffer heated to ~60 °C. Then, the gelatin solution was poured into the mold and allowed for crosslinking at 4 °C for ~30 min. After crosslinking, the plastic tube was gently slipped out and the gelatin channel block was removed from the mold. The gelatin block was then placed in a Petri dish at room temperature (~24 °C) for ~15 min before one end was connected to a syringe pump (GenieTouch Syringe Pump, Kent Scientific) via a polymer tubing (Masterflex, Cole-Parmer) for in vitro cell delivery experiments.

The electrospinning solutions were prepared in 1,1,1,3,3,3-hexafluoroisopropanol using PLGA 50:50 (Lactel, B6010-1, Birmingham, AL, USA), pure PCL (Aldrich, 440744, St Louis, MO, USA) or PCL blended with Gl (PCL-Gl) from porcine skin (Sigma, G-2500, Switzeland). 3D matrices with a thickness of 120–150 µm were produced from 15% (w/v) PLGA, 7% (w/v) PCL or 5% (w/v) PCL-Gl (9:1 w/w) solutions on an NF-103 (MECC CO., LTD., Ogori-shi, Fukuoka, Japan) electrospinning device using a blunt-end surgical needle 27G (0.4 mm inner diameter). Fibers were electrospun onto a rotating drum collector (diameter 60 mm, length 35 mm) under the following conditions: feed rate 1–1.15 mL/h, capillary-collector distance 19–20 cm, voltage 18.5–24 kV, drum collector rotation speed 300 rpm, humidity 25–35%. After fabrication, 3D matrices were removed from the collector, incubated under vacuum for 12 h and stored in sealed zip-lock polyethylene containers at 4 °C.

LLC Scaffold production: Polycaprolactone (PCL) pellets (Sigma, 440744) and type A porcine-derived gelatin (Sigma, G2500) solution were prepared at 10% (w/v) concentration, by dissolving in HFP at a ratio of 90:10. The solution was left to homogenise overnight in a rotator at room temperature. PCL-gelatin grafts (2-mm inner diameter) were fabricated using standard electrospinning parameters of +20/−1 kV electrode voltages, 500 rpm mandrel rotation and a flow rate of 4 mL/h [25 (link)].
Cell culture: PCL-gelatin grafts were secured inside a resin printed bioreactor chamber, filled with EBM-2 medium. ECFCs at a cell density of 1 × 10⁶ were seeded into the graft lumen and allowed to attach for 1 h. Pulsatile flow was generated using a Welco WP10-P1/8 peristaltic roller pump. Pressure characterisation was performed using a PendoTech PREPS-N-012 in-line strain gauge pressure sensor. After 24 h, the graft was harvested from the bioreactor, and fixed with 10% formalin (Sigma, HT501128). For CD31 staining, the sample was first permeabilised in 0.1%Triton-X diluted in PBS and blocked with 5% BSA. The cells were then stained using AlexaFluor 488 conjugated anti-CD31 antibody (Abcam, ab215911) at 1:500 dilution with PBST and counter-stained with DAPI (ThermoFisher, R37605). The cells were then visualised under a fluorescent microscope at Ex/Em: 495/519 nm.