A 3‐D Biomicrofluidic Lymphatic Vessel Analogue for Studying Lymphangiogenesis and Lymphatic Vessel Function

Abstract

Lymphatic vessels are critical in physiology by maintaining tissue fluid homeostasis and coordinating immune responses. Lymphatic vessels possess the unique function of draining the excess fluids that accumulates in the tissue spaces to alleviate elevated interstitial fluid pressure that can occur in tumors and during wound healing. The lymphatic vessel system is also believed to play a critical role in facilitating tumor cell invasion and dissemination. Nevertheless, while the structure and functions of blood endothelial cell biology have been extensively studied, comparatively much less is known about lymphatic endothelial cell biology. This outcome can be attributed to in part to the challenges of recapitulating fluid dynamic environment of lymphatic vessels in vitro and manipulating this environment in vivo. Thus, there is an important need to develop a new experimental platform that reconstitutes the tissue‐level function of lymphatic vessels, such as interstitial flow dynamics, biomolecular signaling, and the 3‐D ECM conditions. To this end, we developed a 3‐D microfluidic lymphatic vessel analogue to investigate lymphangiogenesis and lymphatic vessel functions. This system features three parallel microchannels where the lymphatic endothelial cells (LECs) fully line the two side microchannels that are separated a single microchannel that contains type I collagen gel (Figure 1). To mimic the physiochemical environment of lymphatic pathology, such as what is observed in tumor, our lymphatic vessel analogues were stimulated with tumor‐derived cytokines (IL‐6 and VEGF‐C, both applied at the concentration of 50 ng/ml) and interstitial flow (applied at ~ 7 μm/sec, which is in line with pathological levels of interstitial flow). A sprouting ratio, defined as the change in lymphatic sprouting area over time and normalized to the area of collagen gel region, was employed to quantify lymphangiogenesis under both biochemical and mechanical stimulations. Moreover, vessel permeability was also used as a measurement in the presence of physiochemical stimulations. We found that lymphangiogenesis was promoted by treating IL‐6 and VEGF‐C individually in the static condition while no significant sprouting ratio observed over the experimental periods, suggesting tumor‐derived biomolecules are potent to trigger lymphangiogenesis (Figure 2). Interestingly, upon introducing interstitial flow to microfluidic device, we observed that interstitial flow on its own was able to significantly increase lymphatic vessel sprouting by ~ 5‐fold compared to static conditions, suggesting that such mechanical stimuli can potently promote lymphatic vessel remodeling. Furthermore, interstitial flow also upregulated the sprouting ratio for both IL‐6 and VEFC‐C treated conditions, demonstrating that interstitial flow augment biochemically stimulated lymphangiogenesis. In addition, our vessel permeability measurements showed that both IL‐6 and VEGF‐C potently induce heighted lymphatic permeability, which are coincident with the sprouting measurements for these same conditions. Immunofluorescent staining of LECs was also performed to characterize the changes in cell morphology during vessel sprout formation. Collectively, our results highlight the importance of incorporating interstitial flow for studying lymphangiogenesis and vessel permeability and its importance in pathophysiological conditions, such as elevated levels of IL‐6 and VEGF‐C.Support or Funding InformationFunding was provided by the American Heart Association (15SDG25480000) and the Pelotonia Junior Investigator Award.C.‐W. C. acknowledge the support from the Pelotonia fellowship.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.