Directed endothelial cell migration is a key feature of vascular development, but the roles and interactions of soluble and extracellular matrix-bound (ECM) cues are poorly understood. The aim of this project is to develop solid-phase gradients to study VEGF-directed endothelial cell responses by live cell imaging, particularly focusing on developing an experimental platform to accurately quantify these gradients.
Surface bound gradients were created by physico- or chemisorption of proteins onto a range of surface chemistries generated by deposition of radio-frequency plasma polymers onto glass substrates. The amount and distribution of protein bound to the surface was controlled by a continuous-flow microfluidic device that generated steady linear soluble protein gradients over the surface. Surface chemistries included allylamine, propionaldehyde and ethanol plasmas deposited onto glass slides or coverslips . Controls were tissue culture polystyrene. This system was capable of generating a solid phase albumin gradient as visualised by fluorescence microscopy of Alexa-Fluoro®-488 labelled-albumin after washout of unadsorbed albumin.
Total Internal Reflection Fluorescence Microscopy (TIRF-M) was used to count the number of albumin molecules bound to the surface. Individual Alexa-labelled protein molecules were identified by their characteristic ‘flickering’ fluorescence. A novel software algorithm was developed to segment single molecules from TIRF-M movies by their stochastic emission patterns. Unlabelled albumin solutions were ‘spiked’ with very low concentrations of Alexa-Fluoro®-594 albumin (0.01- 1 nM) to demonstrate linearity between the number of immobilised molecules and concentration of label at limiting dilution (less than 1,000 molecules per square mm). Positively charged allylamine surfaces enhanced albumin adsorption in comparison to other surfaces. The attachment and spreading of human umbilical endothelial cells (HUVECs) over the first 24 hours was influenced by both the surface chemistry and the nature of the immobilised biomolecule species (albumin, fibronectin, or heparin). This robust methodology will be extended to examine the influence of ECM and VEGF isoforms on directed HUVEC migration.