An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Our protocol enables unique insights into the molecular and bulk tissue level mechanisms of vasculogenesis that may aid in the engineering functional vasculature for cardiovascular disease therapy in modeling. This protocol allows the creation of robust, three-dimensional vascular networks for evaluating the vasculogenic potential of various platforms, and also provides a free, open source computational pipeline for analyzing final network topology. Begin by adding 250 microlitres of cell detachment solution per well of the D5D6 differentiated cell culture for 10 minutes at 37 degrees Celsius.

At the end of the incubation, use a P1000 pipette tip to dissociate the cells into single cell suspensions. Pool the cell solutions in a single 15 milliliter conical tube for centrifugation. We suspend the pellet in 200 microliters of ice cold sorting buffer and label the cells with five microliters of concentrated fluorescence conjugated CD34 antibody for 10 minutes at four degrees Celsius.