Engineered 3D vessel-on-chip using hiPSC-derived endothelial- and vascular smooth muscle cells.

Full details are provided in supplemental experimental procedures . hiPSC lines Research on hiPSC was approved by the medical ethical committee at Leiden University Medical Center, the Netherlands. A detailed list of the hiPSC lines and batches used for each experiment is provided in Table S2 . Differentiation of hiPSC-ECs and hiPSC-VSMCs hiPSC differentiation to ECs was performed as described previously ( Orlova et al., 2014a , 2014b ). hiPSC differentiation to VSMC was performed as previously described ( Halaidych et al., 2019 ). Setting up VoCs hiPSC-ECs and mural cells were prepared prior to incorporation in VoCs as described in supplemental experimental procedures . Commercially available microfluidic chips with one gel channel and two media channels (AIM Biotech) were used. Cells were resuspended and combined to obtain 10 × 10 6 hiPSC-ECs/mL and 2 × 10 6 mural cells/mL (5:1 ratio). Three different mural cell suspensions were tested in combination with hiPSC-ECs: (1) hiPSC-VSMCs, (2) HBVSMCs, and (3) HBVPs. Cell were resuspended in EGM-2 supplemented with Thrombin (4 U/mL) and then gently mixed with fibrinogen (final concentration 3 mg/mL, Sigma) at 1:1 vol ratio. Cell/hydrogel mixture was quickly loaded into the middle gel-loading channel of the microfluidic chip. Chips were incubated at room temperature for 15 min before the addition of EGM-2 supplemented with VEGF (50 ng/mL) to both flanking media channels. The γ-secretase inhibitor DAPT (10 μM) was also added to the medium on day 1 for 24 h. Gravity-driven flow was induced by the addition of 100 μL medium to the right media ports and 50 μL media to left media ports. Medium was refreshed daily.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 9 software. Normality of the data was evaluated by the D'Agostino-Pearson test. One-way and two-way ANOVA with Tukey's multiple comparison test was used for the analysis of three groups. For paired or unpaired analysis of two groups, either Student's t test or Wilcoxon-Mann-Whitney test was used. Analyses are indicated in the figure legends. The data are reported as mean ± SD. Statistical significance was defined as p < 0.05.

Supplemental information Document S1. Supplemental experimental procedures, Figures S1–S4, and Tables S1 and S2 Document S2. Article plus supplemental information