1.5. Microfluidic endothelial disease models for precision medicine Research
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The endothelium is a thin layer of cells lining all blood and lymphatic vessels in the body. This dynamic layer controls to a large extent which compounds cross over from the blood in the circulatory system into tissue. The barrier function is dictated largely by tight junctions formed between cells. However, in many regions the endothelium is fenestrated – that is, contains tiny holes with diameters on the order of 60 to 80 nm – in order to accommodate the passage of water and proteins. Blood vessels in the glomerulus, for example, are characterized by a high degree of fenestration. A polysaccharide gel layer known as the glycocalyx covers the luminal surface of the endothelium and acts as a filtration barrier in fenestrated regions to control transport of proteins such as albumin out of blood vessels.

  • The role of the endothelium extends beyond acting just as a barrier, and vascular endothelial cells are actively involved in almost all disease pathophysiologies [Aird, 2007]. The subject of this proposal is related to endothelial damage brought on as a result of persistent activation of the endothelium by cardiovascular risk factors such as diabetes mellitus [Rabelink 2015]. Glycocalyx function in patients with diabetes, obesity, or chronic kidney disease is diminished, leading to enhanced albumin excretion from blood vessels, which in turn drives vascular inflammation and progressive organ function loss. Experimental studies have suggested that glycocalyx dysfunction is reversible, rendering the glycocalyx a promising therapeutic target.

    We propose to develop new in vitro models for the glycocalyx to better be able to investigate this component of the vascular system as a target for potential therapeutic intervention in renal and cardiovascular disease. These models will be based on microfluidics, as this technology enables ultra-small-volume liquid handling in the femtoliter to microliter range in tiny channels having effective diameters from 1 to 1000 micrometers [Whitesides 2006]. Micofluidics has been applied to a multitude of life-science research questions, including those involving biochemical reactions and bioanalysis in the absence of cells (cell-free) [Mross 2015], as well as microperfusion cell culture (e.g. organs-on-a-chip). Organs-on-a-chip are microfabricated devices incorporating microchambers in which cells or tissue are cultured under continuous perfusion and other biochemical and physical stimuli to simulate tissue or organ physiology [Bhatia 2014]. These devices, currently under development for many different organs, enable the engineering of in vivo-like cellular microenvironments. It is also possible to implement dynamic experimental conditions to mimic the temporal changes to these microenvironments experienced by tissue in vivo. Microfluidic devices designed for both cell-free and cell-culture applications can provide real-time insight into physiological processes under different conditions, and the opportunity to test the efficacy of pharmacological interventions under varying circumstances.

  • In this project, we propose to develop microfluidic models for the glycocalyx with or without endothelial cells present to investigate the effects of glycocalyx degradation on the development of albuminuria and subsequent progression of renal and cardiovascular disease. We envision first the realization of a robust, cell-free model of the gel layer making up the glycocalyx. The degradation of this layer under different circumstances and resulting changes to glycocalyx permeability will be studied using this model. A second endothelium-on-a-chip model will combine cells with the glycocalyx layer under controlled flow and biochemical conditions. It will incorporate a means to directly probe the effect of albumin leakage (albuminuria) on vascular cell health to improve our understanding of the downstream consequences of glycocalyx dysfunction in diabetes.

  • Microfluidic devices will first be implemented for the development of a robust glyocalyx layer. A second generation of devices will combine the glycocalyx with perfusion culture of endothelial cells (primary human umbilical vein endothelial cells (HUVEC) will be used). Devices will be fabricated in the Verpoorte rapid prototyping lab, which will allow design-to-test cycles of less than a week. A protocol will be developed to introduce hydrogel solutions to microchambers. HUVEC will be cultured in microchannels as per protocols established in the Verpoorte lab for previous HUVEC work. The use of microfluidic devices will enable experiments in which the concentrations of various compounds are changed continuously in controlled spatiotemporal gradients. Cell cultures can be monitored in real time using fluorescence microscopy using specially adapted microscope stages designed in the Verpoorte group. An assay for albumin leakage in an epithelium organ-chip device will most likely be based on fluorescence.

    Technical objectives:

    1. a) robust, microperfusion glycocalyx model in-chip b) combined endothelial cell-glycocalyx model
    2. investigate effect of various parameters on induction of inflammation in combined model to gain more insight into pathophysiology of albuminuria in diabetic patients
    3. test potential drugs targeting the glycocalyx
    4. identify the circumstances under which these drugs offer the most benefit
    1. Establishment and characterization of a microfluidic, cell-free glycocalyx model
    2. Establishment of a glycocalyx-endthelium organ-on-a-chip (GEOoaC) model
    3. Design of an albumin leakage assay in the GEOoaC
    4. Characteristics of endogenous stimulation of albumin leakage effects. Consideration of glucose concentrations and profiles; inflammation by LPS; influence of cholesterol; etc.
    5. Investigation of the pharmacological inhibition of albumin leakage (inhibition of heparinase, restoration of glycocalyx layer, etc.)
  • Though in vitro drug screening is well established, it is often difficult to accurately extrapolate test results back to possible therapeutic or pathological effects in the human being. This is often due to species differences in drug response (when animal cells or models are used) or human cell models being too simplistic. As a result, a large majority of drug candidates fail in clinical trials. Using microfluidics, nanoliter cellular environments can be created that better mimic the in vivo situation with respect to both chemical and physical (e.g. flow) cues, allowing the behaviour of organs to be better reproduced ex vivo (Verpoorte, Pharmaceutical Analysis), irrespective of whether they are animal- or human-based. These more accurate “organ-on-a-chip” models will enable much earlier preclinical detection of the pharmacological efficacy of new chemical entities. Moreover, microfluidics lends itself very well to the culture and analysis of very small numbers of cells (few thousand) or small amounts (mg) of tissue, which allows the efficient use of often-scarce patient material and opens a route to precision medicine.

  • This project in Disease Mechanisms links well to collaborators working in Drug Development, as the model, once developed, can be used for testing potential pharmaceutical compounds. Colleagues Dömling and Poelarends in GRIP are foreseen as collaborators with respect to the drug testing aspects of this project.

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