Probing of peripheral blood mononuclear cells anchoring on TNF-alpha challenged-vascular endothelia in an in vitro model of the retinal microvascular

Abstract

Retinopathy is a complication of diabetes that affects the eyes; it stems from damage to the microvasculature of the retina and eventually compromises vision. The diagnosis of retinopathy is difficult to make because there are no early symptoms or warning signs. Dysfunction of the retina's microvascular networks is believed to be associated with inflammatory cytokines and tumor necrosis factor alpha (TNF-α). To investigate the effect of these cytokines, such as TNF-α, a polydimethylsiloxane (PDMS)/glass hydride microfluidic device reflecting the physiological structure of the retina's microvasculature was developed. In this model, the bifurcations and tortuosity of branch vessels were based on photographs of the fundus and an endothelial cell layer (EA.hy926 cells) were reconstructed within the microfluidic network. The adhesion, spreading, and growth of cells was ensured by optimizing the conditions for cell seeding and perfusion. Fluorescent staining was used to visualize the cytoskeleton and measurement of the nitric oxide (NO) level proved that the endothelial EA.hy926 cells had spread in the direction of flow perfusion system, forming artificial vascular networks. The endothelial layer was further challenged by TNF-α perfusion. Cytokine treatment increased the anchoring of peripheral blood mononuclear cells (PBMCs) on the endothelial layer. The microfluidic device developed in this study provides a low-cost platform reflecting the physiological structures of the retina's microvasculature. It is anticipated that this device will be useful in evaluating the diseased retina as well as in drug screening.

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