Hmecs

HMECs were purchased from Lonza (Walkersville, MD, USA) and cultured in MCDB170 (US Biological, Salem, MA, USA) supplemented with 0.1 mM ethanolamine (Sigma-Aldrich, St Louis, MO, USA), 0.1 mM ortho-phosphoethanolamine (Sigma), 0.25% bovine pituitary extract (Hammond Cell Tech, Windsor, CA, USA), 5 ng/ml EGF (Peprotech, Rocky Hill, NJ, USA), 0.5 μg/ml hydrocortisone (Sigma), 5 μg/ml insulin (Sigma), 5 μg/ml transferrin (Sigma), and 5 μM isoproterenol (Sigma).^{44 (link)} For immortalization, HMECs were transduced with hTERT-expressing retrovirus as described previously.^{17 (link)}For suspension culture, cells were detached by treatment with 0.025% trypsin, followed by resuspension in the growth medium supplemented with 0.5% methylcellulose and plated on polyHEMA-coated dishes. Anisomycin, pepstatin A, and polyHEMA were obtained from Sigma-Aldrich. E64d and Z-VAD-fmk (Z-VAD) were purchased from Peptide Institute, Inc. (Osaka, Japan), staurosporine (STS) and bafilomycin from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and necrostatin-1 from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). BD Matrigel Matrix Growth Factor Reduced (GFR) was obtained from BD Biosciences (Franklin Lakes, NJ, USA).

Primary human dermal blood microvascular endothelial cells (hMECs) and growth media (EGM-2-MV) were purchased from Lonza (MD, USA). hMECs (5x10⁵ cells) were resuspended in 25 μL of human fibrinogen (15 mg/mL in PBS, Sigma-Aldrich) and dripped onto the upper surface of collagen scaffolds sitting flat on a petri dish. Following passive uptake of the cell/fibrinogen mixture, 25 μL of human thrombin (25 U/mL in PBS, Sigma-Aldrich) was added to form the fibrin gel. Scaffolds were then transferred to a 24-well plate containing 2 mL growth media and cultured for the specified time. Media was replaced after 2 days. At day 3, scaffolds were harvested and fixed in 4% paraformaldehyde for 24 hours before subjection to tissue processing and embedding in paraffin.