Hndfb

Human umbilical vein endothelial cells (HUVEC), human aortic smooth muscle cells (HASMC) and human normal dermal fibroblasts (HNDFB) were purchased from Lonza, Inc. (Walkersville, MD, USA). HUVEC, HASMC and HNDFB were cultured in an appropriate medium, i.e., endothelial cell medium (ECM), smooth muscle cell medium (SMCM), fibroblast medium (FM), respectively, with growth supplement (Lonza). The cells were passaged every 3 days and were used within the third to fifth passage in this study. The cells were cultured on 0.5% gelatin-coated dishes (Techno Plastic Products, Sigma-Aldrich) and were maintained at 37°C in a humidified atmosphere containing 5% CO₂. HUVEC were incubated with CellTracker Red (10μM) (Molecular Probes, Invitrogen Corp, Carlsbad, CA) for 30 minutes to allow visualization of the HUVEC in MCSs. Mixed cell suspensions (a total cell count of 2.5 x 10⁴) composed of HUVEC (40%), HASMC (10%), and HNDFB (50%) was plated into each well of ultra-low attachment round-shaped 96-U-well plates (Sumitomo Bakelite, Tokyo, Japan) filled with triple mixed media of ECM, SMCM, and FM with a ratio of 1:1:1. After 24 hours, the cells aggregated to form a round shaped MCS. According to the automated measurement function equipped on our Bio-3D printer, the size of the MCS was 615.0±51.3 μm (mean ±SD) for 500 MCSs.