Engineered Microvascular Networks in Hydrogels

Microvessels were fabricated in collagen and hylauronan (HA) composite hydrogels polymerized inside polydimethylsiloxane (PDMS)-based microfluidic devices fabricated using soft lithography^{43 (link)}. p20–p23 human cerebral microvascular endothelial cells (hCMEC/D3) and p7–p15 GFP-labeled human brain vascular pericytes (hBVP) (Neuromics) were seeded in the hydrogels at densities of 2 M/mL and 0.4 M/mL, respectively. These ratios were obtained from a previous study that used co-cultures of human blood outgrowth endothelial cells and human pericytes to form microvasculature networks^{13 (link)}. hCMEC/D3 were cultured in Endothelial Cell Basal Medium (PromoCell) supplemented with 5 µg/mL ascorbic acid (Sigma), 1 ng/mL hBFGF (Sigma), 1/100 chemically defined lipid concentrate (Thermo Fisher), 5% fetal bovine serum (VWR Life Science), 10 mM HEPES (Quality Biological), 1.4 µM hydrocortisone (Sigma), and 1% penicillin-streptomycin (Corning). hBVP were cultured in DMEM (Corning) supplemented with 10% FBS, 1% penicillin-steptomycin, and 1X MEM Amino Acid Solution (Thermo Fisher). Final hydrogel formulation concentrations consisted of 3 mg/mL HA (Sigma), 5 mg/mL collagen type I (MP Biomedical), and 0.85–1 mg/mL Matrigel (Corning). These reagents were combined with 0.1 M sodium hydroxide (NaOH) and 10x phosphate buffer solution (PBS) to facilitate polymerization and maintain physiological pH.