Huvecs

TIFs were seeded onto the e-transmembrane scaffolds. Scaffolds were suspended in 1 ml of TIF media (see below) and placed onto an orbital shaker (IKA KS 260 Basic, IKA; Staufen, Germany) rotating at 60 rpm. A total of 200,000 TIFs in 75 μl of medium were pipetted into the well containing the scaffold in the device and left on the orbital shaker. After 24 hours, the medium was removed and replaced with 2 ml of fresh TIF medium. The devices were cultured under static conditions for the following 3 days. HUVECs (Lonza Biosciences, Basel, Switzerland) were added into the system in the same manner, applying 50,000 HUVECs within 75 μl of HUVEC medium (EGM-2; Lonza Biosciences, Basel, Switzerland). After 24 hours, the medium was removed and replaced with fresh HUVEC medium. These samples were left to culture statically for 2 days, at which point the orbital shaker was reinitiated to simulate blood flow. Cultures were carried out for an additional 7 days before samples were fixed for imaging.

Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza or Life Technologies, authenticated for EC marker expression and cultured until the fourth passage. HUVECs were cultured in endothelial basal medium-2 (EBM-2, Lonza) supplemented with foetal bovine serum, hydrocortisone, human basic fibroblast growth factor, vascular endothelial growth factor, R3-insulin-like growth factor, ascorbic acid, human epidermal growth factor, GA-1000 and heparin. Human embryonic kidney (HEK) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal calf serum (Thermo), 100 units/ml penicillin and 100 μg/ml streptomycin. Both cell lines were cultured in 5% CO₂ at 37 °C.

Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (Cat# CC-2519). HUVECs were cultured in EBM-2 (Cat# CC-3156, Lonza) supplemented with EGM-2 (Cat# CC-3162, Lonza), 2 mM L-glutamine (Cat# 25030081, Gibco^TM), 100 U/ml penicillin, and 100 μg/ml streptomycin (Cat# 15140122, Gibco^TM) on gelatin (Cat# G1890, Sigma‒Aldrich; 0.1% in PBS)-precoated plates in 5% CO₂ in a humidified incubator. The dorsal root ganglia (DRG) were isolated from C57BL/6 mice and digested as described previously^{26 (link)}. DRGs were plated onto poly-D-lysine (Cat# A3890401, Thermo Fisher Scientific)-coated dishes in Minimum Essential Medium (Cat# 11095080, Thermo Fisher Scientific) containing N-2 Supplement (Cat# 17502048, Thermo Fisher Scientific), 2 mM L-glutamine (Cat# 25030081, Thermo Fisher Scientific), 100 U/ml penicillin, and 100 μg/ml streptomycin (Cat# 15140122, Gibco^TM). Cells were cultured in a humidified 37 °C incubator with 5% CO₂. For LPHN2 knockdown in HUVECs and DRG neurons, shLPHN2 lentivirus particles were added to the culture medium at 5 × 10⁴ TU/ml as described previously^{20 (link)}. The scrambled shRNA was used as a control.

Primary Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from Lonza (C2519AS) and used up to passage five. Early passage HUVECs were cultured in EGM™2-Bulletkit™ medium with growth supplements CC-3156 & CC-4176 purchased from Lonza. At 80% confluency, HUVECs were trypsinized, washed twice with phosphate-buffered saline (PBS), pelleted, and frozen at −80°C.

In this study, human adipose stem cells (hASCs) (Lonza, USA) and human umbilical vein endothelial cells (HUVECs; Lonza) were used. The cells were cultured at 37 °C and 5% CO₂ with different culture media, namely, growth medium (GM) for hASCs consisting of Dulbecco's Modified Eagle's Medium-low-glucose (DMEM-L; Sigma-Aldrich, USA), 10% fetal bovine serum (FBS; BioWest, USA), and 1% penicillin-streptomycin (PS; Thermo-Fisher Scientific, USA) and EBM^TM-2 (Lonza) for HUVECs supplemented with the EGM^TM-2 endothelial SingleQuots^TM kit (Lonza) and 1% PS (EBM). The culture medium was changed every 2 days.
Before formulating the stem cell-loaded bioink, a porcine bone-derived dECM (BdECM) sponge, prepared using a previously described demineralization/decellularization protocol (described in Supplementary materials) 29 (link), was dissolved in deionized water and neutralized by mixing with 10×DMEM (Sigma-Aldrich) at a ratio of 1:1. The BdECM hydrogel was then mixed with b-TCP and hASCs (1.2 × 10⁷ cells/mL). The final concentrations of dECM and- TCP were 50 and 200 mg/mL, respectively. To obtain cell spheroids, mineral oil (Sigma-Aldrich) containing HUVECs (2.0 × 10⁷ cells/mL) was used. The total cell density to fabricate the cell-constructs was fixed as 2.0 × 10⁷ cells/mL in which the ratio of the hASCs and ECs was 3:2.

HUVECs obtained from Lonza Group Ltd. (Basel, Switzerland) were cultured in endothelial basal medium supplemented with 2% fetal bovine serum, 0.4% bovine brain extracts, 10 ng/ml human epidermal growth factor and 1 μg/ml hydrocortisone according to the manufacturer’s recommendation. AGE or non-glycated BSA treatment was carried out in a medium lacking epidermal growth factor and hydrocortisone. Cells at passage 4–11 were used for the present experiments. According to the certificate of analysis by Lonza Group Ltd., HUVECs were pooled from Caucasian newborn babies. Isolated cells were identified as HUVECs because cells expressed CD31/105, von Williebrand Factor VIII, and were positive for acetyated low-density lipoprotein uptake. All cells were negative for mycoplasma.