Prior to seeding into the microfluidic device, three different cell types were thawed and expanded. Normal human astrocytes (NHA) (Lonza, catalog CC-2565) were thawed at passage 5 and cultured for 10 days in astrocyte growth medium (AGM) (Lonza, catalog CC-3186). Human Coronary Arterial Smooth Muscle Cells (HCASMC) (Sigma, catalog 350-05A) were also thawed at passage 5 and cultured in smooth muscle growth medium (Sigma, catalog 311-500) until confluency, then passaged and cultured in 6 well plates using smooth muscle cell differentiation medium (Sigma, catalog 311D-500) for 10 days prior to vessel fabrication. Additionally, human cerebral microvascular endothelial cells (HCMEC/D3) received at passage 19 (gifted from Dr. Robert Nagele’s laboratory) were cultured and expanded on tissue culture plates coated with 1% gelatin (Sigma, catalog 1288485) in EBM-2 (Lonza, catalog CC-3156) modified with CD lipid concentrate (Life Technologies, catalog 11905031), HEPES buffer (Sigma, catalog 83264), 10% fetal bovine serum (VWR, catalog 97068-085), 5 μg/mL ascorbic acid (Sigma, catalog A92902), 1 ng/mL of bFGF (Peprotech, catalog 100-18B), 1 μM hydrocortisone (Sigma, catalog H0888), and 1% penicillin/streptomycin (VWR, catalog 97062-806), consistent with a previously described protocol [
23]. The vessel seeding protocol consists of three different steps. First, NHA were seeded at a density of 1 million cells per mL in a 10 mg/mL collagen, 1.33 mg/mL hyaluronan (HA) (Sigma, catalog H388) and 1 mg/mL Matrigel (Corning, catalog 354234) hydrogel formulation used in a previous study [
24]. After injecting 100 μL of the astrocyte-seeded hydrogel directly into the reservoir, an 18-g needle coated with 0.1% Bovine Serum Album (BSA) (Sigma, catalog 05470) was inserted into the device through a needle guide. After waiting 20 min to allow for gel polymerization, AGM was added on the top of the device to maintain cell viability. After an additional 20 min, the needle was pulled leaving a cylindrical void. HCASMC were then seeded into a 10 mg/mL collagen at a density of 1 million cells per mL. In lumican add-in studies, these hydrogels were supplemented with 10 μg/mL lumican (VWR, catalog PRSI96-526). A 20-g needle coated in 0.1% BSA was inserted inside the void left by the 18-g needle. After polymerization, the 20-g needle was removed leaving a final cylindrical void and surrounding annulus with a 944 μm diameter. In the final step, HCMEC/D3 (passages 22–24) were resuspended in EGM-2 at a density of 5 million per mL, then 15 μL of the HCMEC/D3-containing media was injected into the cylindrical void and incubated for 10 min, yielding a seeding density of approximately 150,000 cells per cm
2. This process was repeated for an additional 10 min after inverting the device to assure uniform lumen coverage throughout the vessel. Devices were then submerged in EGM-2 media and left in static conditions for 2 days to ensure cell spreading and viability prior to exposure to flow or static conditions. For the flow experiments, silicone grease (Sigma, catalog Z273554) was used to close the needle guides to prevent flow leakage.