Bovine skin

Human BM-MSCs were obtained from Lonza (Basel, Switzerland) and were maintained with Mesenchymal Stem Cell Growth Medium (Lonza). The cells between passages 4 and 7 were used for all experiments. MDA-MB-231 cells and MCF7 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle medium/high glucose (DMEM/HG; HyClone, Logan, UT, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) and 100 U/mL penicillin/streptomycin (P/S; Thermo Fisher Scientific). MCF7 cells were cultured in DMEM/F-12 (1:1) (Thermo Fisher Scientific) supplemented with 10% FBS, 2 mM L-glutamine (Thermo Fisher Scientific) and 100 U/mL P/S. Human umbilical vein endothelial cells (HUVECs; Lonza) were maintained on 0.2% gelatin from a bovine skin (Sigma-Aldrich, St. Louis, MO, USA)-coated dish using Endothelial Cell Growth Media-Plus (Lonza), and HUVECs between passages 4 and 8 were used for all experiments. All of the cells were maintained at 37 °C in a humidified incubator containing 5% CO₂.

Un-/cellularized scaffolds were fixed in 3.7% PFA for 15 min and washed with 1 × PBS. Samples were incubated in 5% gelatin (bovine skin; Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 °C followed by an incubation step in 5%, 10%, or 25% gelatin solution for 30 min at 37 °C. Afterwards, the samples were incubated for 15 min at 4 °C to harden the gelatin-scaffold block. The construct was transferred to a larger well, prewarmed gelatin solution (5%, 10%, or 25%) was added, and the samples were cooled for 15 min at 4 °C. The blocks were transferred into a 3.7% PFA solution [room temperature (RT), 24 h] and washed in 1 × PBS at RT. Finally, the PFA-fixed and gelatin-stabilized PCL/PLA scaffolds were embedded in standard paraffin with a Tissue-Tek^® VIP^® 5 Vacuum Infiltration Processor (Sakura^®, Alphen aan den Rijn, The Netherlands) and TES Valida (MEDITE Cancer Diagnostics, Orlando, USA) using the parameters, as listed in Table 1.
Formalin-fixed-paraffin-embedded (FFPE) blocks were cut into 7 µm sections using an automated rotary microtome (HM355 with STS & Cool-Cut, Thermo Fisher Scientific, Waltham, Massachusetts, USA), and the sections were then transferred to SuperFrost Plus™ slides and dried overnight at 45 °C. HE staining was performed on cellularized scaffolds.

Sacrificial ink was formulated by dissolving gelatin powder (bovine skin, Sigma‐Aldrich) in sterile water at 10% w/v concentration by constant mixing at 60 °C under sterile conditions. Before printing, gelatin solution was liquified by heating and loaded into a 1 mL glass syringe (Hamilton). Printing was performed through a 21 G (500 µm inner diameter) blunt metal needle with extrusion rate of 22 µL min⁻¹ and lateral speed of 0.6 mm s⁻¹. Printing was performed in custom containers mounted on a glass slide with defined inlets and outlets. The containers were manufactured from Dental LT clear resin using stereolithography Form 2 printer (FormLabs) according to a CAD design created in Fusion 360 software (Autodesk). During printing, the syringe was heated to 50 °C to liquify gelatin and allow smooth extrusion into SHAPE support. When deposited into the printing support, gelatin rapidly solidifies creating the sacrificial template for channel generation. After printing, the composite support containing the printed construct was incubated at 37 °C overnight to anneal the support and liquify the sacrificial ink. Liquified gelatin structure was evacuated by injecting warm cell culture media. Food dye and colored polystyrene microspheres (Sigma‐Aldrich) were added for better visualization of the perfused channels.