Endothelial basal medium 2 ebm 2

ECFCs were isolated from participants following previously published methods,[18 (link)] with modifications. Briefly, approximately 48 mL of venous blood was collected into BD Vacutainer® Cell Preparation Tubes^™ (BD Biosciences), centrifuged for 30 mins at 1600 RCF and the mononuclear cells resuspended in phosphate buffered-saline (PBS) supplemented with 10% fetal bovine serum (FBS). Centrifugation and resuspension was repeated two more times and finally cells were resuspended in endothelial growth media (Endothelial Basal Medium 2 [EBM-2]; Lonza) supplemented with 10% FBS, 1% Antibiotic-Antimycotic (Invitrogen), and the EGM-2 BulletKit^™ (Lonza) and seeded at a density of 4 × 10⁷ on 6-well collagen-coated plates. The following day, non-adherent cells were removed, adherent cells were washed once with media, and then media was added to each well. Media was changed daily for seven days, followed by every other day. All experiments used ECFCs from a single isolation and between passages 3 and 9. Established ECFCs were characterized using flow cytometry for the presence of endothelial cell surface markers CD31 (PECAM-1), CD144 (VE-cadherin), and/or CD146 (MCAM/cell surface glycoprotein MUC18), and the absence of leukocyte markers CD14 and CD45. All antibodies and isotypes were purchased from eBiosciences Inc. (San Diego, CA, USA).

Blood samples were obtained from VWD patients and healthy controls for the isolation of mononuclear cells (MNC), following previously published methods.[17 (link)] Briefly, blood was collected into BD Vacutainer® Cell Preparation Tubes™ (BD Biosciences) and MNC were resuspended in endothelial growth media (Endothelial Basal Medium 2 [EBM-2]; Lonza) supplemented with 10% fetal bovine serum, 1% Antibiotic-Antimycotic (Invitrogen), and the EGM-2 BulletKit™ (Lonza). Cells were seeded at a density of 4 x 10⁷ on 6-well collagen-coated plates. After 24 hours, non-adherent cells and debris were removed, adherent cells were washed once with media, and then media was added to each well. Culture media was refreshed daily for 7 days, followed by every other day. For all experiments BOECs were from a single isolation and used at passages 3–9.
The endothelial cell phenotype of isolated cells was confirmed by: 1) the formation of confluent monolayers of cells with characteristic endothelial cobblestone morphology, and 2) staining of endothelial cell surface markers CD31 (PECAM-1), CD144 (VE-cadherin), and leukocyte markers CD14 and CD45, followed by flow cytometry.

Methacrylamide-functionalized gelatin (GelMA) was synthesized as previously described, and degree of functionalization (DOF) was determined using ¹HNMR [33 (link)]. GelMA with a DOF of ~60% was used for this study. Hydrogels containing endothelial networks were formed by dissolving GelMA (5 wt%) in PBS at 65°C. Lithium acylphosphinate (LAP) was subsequently added (0.1% w/v) as a photoinitiator. A mixture of HUVECs and NHLFs (2:1 NHLF:HUVEC, 2 × 10⁶ NHLF/ml) was resuspended in the GelMA solution, and the resulting cell suspension was pipetted into Teflon molds (5 mm diameter, 1 mm thick). Hydrogels were formed after photopolymerization for 30 s using a UV lamp (λ = 365 nm, 5.69 mW/cm²). Hydrogels were deposited into 48-well plates and cultured for 6 days using EGM-2 media, with daily media changes. After 6 days, the media was switched to Endothelial Basal Medium 2 (EBM-2) (Lonza, Walkersville, MD) with 2% FBS. EBM-2 is EGM-2 without the added SingleQuot supplements. Conditioned media was collected after 24 hours, filtered using a 0.2 μm syringe filter, and stored at −20°C until further use (Fig. 1).