Vascular Transcriptomics: Investigating Endothelial Activation and Vascular Dysfunction Using Blood Outgrowth Endothelial Cells, Organ-Chips, and RNA Sequencing.

Vascular organ-chip or vessel-chip technology has significantly impacted our ability to model microphysiological vasculature. These biomimetic platforms have garnered significant interest from scientists and pharmaceutical companies as drug screening models. However, these models still lack the inclusion of patient-specific vasculature in the form of patient-derived endothelial cells. Blood outgrowth endothelial cells are patient blood-derived endothelial progenitors that have gained interest from the vascular biology community as an autologous endothelial cell alternative and have also been incorporated with the vessel-chip model. Next-generation sequencing techniques like RNA sequencing can further unlock the potential of personalized vessel-chips in discerning patient-specific hallmarks of endothelial dysfunction. Here we present a detailed protocol for (1) isolating blood outgrowth endothelial cells from patient blood samples, (2) culturing them in microfluidic vessel-chips, (3) isolating and preparing RNA from individual vessel-chips for sequencing, and (4) performing differential gene expression and bioinformatics analyses of vascular dysfunction and endothelial activation pathways. This method focuses specifically on identification of pathways and genes involved in vascular homeostasis and pathology, but can easily be adapted for the requirements of other systems. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Isolation of blood outgrowth endothelial cells from patient blood Basic Protocol 2: Culture of blood outgrowth endothelial cells in microfluidic vessel-chips Basic Protocol 3: Isolation of RNA from autologous vessel-chips Basic Protocol 4: Differential gene expression and bioinformatics analyses of endothelial activation pathways.