| Summary: | In addition to the blood vasculature, the majority of tissues contain a secondary vascular system known as the lymphatics that supports tissue homeostasis and immune cell trafficking. As such, impairment of the lymphatic capillaries can result in diverse diseases including abnormal tissue swelling (edema), compromised immunity and cancer metastasis. Current in vitro methods to study the lymphatic vasculature, in health and disease, mostly rely on monolayer and transwell culture systems which only lend themselves to reductionist studies with a considerable lack of physiological relevance. In comparison, animal models provide the full spectrum of biological complexities; however, they offer limited control over biological events in the cellular microenvironment, thus making it increasingly difficult for mechanistic studies. To address these limitations, we developed a 3D lymphatic microvasculature model, that physiologically emulates the lymphatic structure and function, within a microfluidic system that allows for high spatial-temporal control over the biological transport phenomena to study cellular events.
For the first part in this thesis, we implemented a microfluidic-based cell culture system to screen for the optimal balance of growth factors, extracellular matrix compositing and interstitial fluid flow that would induce controlled-levels of angiogenic sprouting by the lymphatic endothelial cells. From this study, we developed two distinct approaches to generate 3D lymphatic microvasculature on-chip were lymphangiogenic-induction is achieved by diffusive exposure to growth factors or via mechanotransduction response to high levels of interstitial fluid flow. After validating the in vivo-like morphology of our engineered lymphatics, we quantified their solute drainage functionality using fluorescent tracers of varying molecular weights, resembling interstitial soluble proteins. Results validated that the lymphatic microvasculature exhibited solute drainage rates approaching in vivo lymphoscintigraphy standards. Computational and scaling analyses were performed to understand the underlying transport phenomena which elucidated the importance of a 3D geometry and the lymphatic endothelium to recapitulate physiological drainage. We then examined the capability of our on-chip lymphatics to elicit an immune response under a pathological-inflammatory condition by locally recruiting immune cells. Experimental and computational results demonstrate an increased infiltration of immune cells into the lymphatics guided by chemotactic gradients that trigger the CCR7-CCL21/19 and CXCR4-CXCL12 inflammatory axes. Finally, we demonstrate the utility of our microphysiological system for pre-clinical studies, specifically by screening the vascular absorption rate of therapeutic monoclonal antibodies developed by Amgen Inc. We coupled our experimental measurements with a physiological-based framework to describe their systemic transport which allowed us to quantitatively predict their corresponding pharmacokinetics.
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