Abstract
Cellular activation and inflammation leading to endothelial dysfunction is associated with cardiovascular disease (CVD). We investigated whether a single cell label-free multi parameter optical interrogation system can detect endothelial cell and endothelial progenitor cell (EPC) activation in vitro and ex vivo, respectively. Cultured human endothelial cells were exposed to increasing concentrations of tumour necrosis factor alpha (TNF-α) or lipopolysaccharide (LPS) before endothelial activation was validated using fluorescence-activated cell sorting (FACS) analysis of inflammatory marker expression (PECAM-1, E-selectin and ICAM-1). A centrifugal microfluidic system and V-cup array was used to capture individual cells before optical measurement of light scattering, immunocytofluorescence, auto-fluorescence (AF) and cell morphology was determined. In vitro, TNF-α promoted specific changes to the refractive index and cell morphology of individual cells concomitant with enhanced photon activity of fluorescently labelled inflammatory markers and increased auto-fluorescence (AF) intensity at three different wavelengths, an effect blocked by inhibition of downstream signalling with Iκβ. Ex vivo, there was a significant increase in EPC number and AF intensity of individual EPCs from CVD patients concomitant with enhanced PECAM-1 expression when compared to normal controls. This novel label-free ‘lab on a disc’ (LoaD) platform can successfully detect endothelial activation in response to inflammatory stimuli in vitro and ex vivo.
Highlights
Among the numerous inflammatory mediators that contribute to endothelial activation leading to dysfunction, tumour necrosis factor alpha (TNF-α) is central to the initiation and termination of long-term inflammatory responses[4]
There was a marked increase in the proportion of Human umbilical vein endothelial cells (HUVECs) expressing intercellular adhesion molecule-1 (ICAM-1) and PECAM-1 in cells treated with TNF-α and LPS [Fig. 2a–c] concomitant with a significant increase in the photonic fluorescent counts for PECAM-1, ICAM-1 and E-Selectin [Fig. 2d]
The main finding of the present study is that TNF-α induced endothelial cell activation can be successfully detected label-free using a novel “Load” platform by measuring single cell autofluorescence (AF) and cell morphology in vitro
Summary
Among the numerous inflammatory mediators that contribute to endothelial activation leading to dysfunction, tumour necrosis factor alpha (TNF-α) is central to the initiation and termination of long-term inflammatory responses[4]. The combination of forward and side scatter has been successfully used to identify specific cell types and subpopulations using flow cytometry[22] In this context, altered light scattering properties associated with disease-induced cellular and molecular events may provide discrete photonic signatures that can be detected using label-free spectroscopic platforms in vivo[23,24]. Label-free prediction of DNA content and quantification of the mitotic cell cycle phases has recently been reported by applying supervised machine learning to morphological features extracted from brightfield and the typically ignored darkfield images of cells from an imaging flow cytometer[25] This method facilitates non-destructive monitoring of cells avoiding potentially confounding effects of fluorescent stains while maximizing available fluorescence channels. A disc for holding three biochips and for mounting onto the centrifugal test stand was manufactured using 3D printing [Fig. 1c] before the V-cup array occupancy distribution for 20 μm polystyrene beads, mammalian cells (HUVECs and EA.hy[926] cells) was confirmed [Fig. 1d]
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