Microfluidic-Based Reconstitution of Functional Lymphatic Microvasculature: Elucidating the Role of Lymphatics in Health and Disease.

The knowledge of the blood microvasculature and its functional role in health and disease has grown significantly attributable to decades of research and numerous advances in cell biology and tissue engineering; however, the lymphatics (the secondary vascular system) has not garnered similar attention, in part due to a lack of relevant in vitro models that mimic its pathophysiological functions. Here, a microfluidic-based approach is adopted to achieve precise control over the biological transport of growth factors and interstitial flow that drive the in vivo growth of lymphatic capillaries (lymphangiogenesis). The engineered on-chip lymphatics with in vivo-like morphology exhibit tissue-scale functionality with drainage rates of interstitial proteins and molecules comparable to in vivo standards. Computational and scaling analyses of the underlying transport phenomena elucidate the critical role of the three-dimensional geometry and lymphatic endothelium in recapitulating physiological drainage. Finally, the engineered on-chip lymphatics enabled studies of lymphatic-immune interactions that revealed inflammation-driven responses by the lymphatics to recruit immune cells via chemotactic signals similar to in vivo, pathological events. This on-chip lymphatics platform permits the interrogation of various lymphatic biological functions, as well as screening of lymphatic-based therapies such as interstitial absorption of protein therapeutics and lymphatic immunomodulation for cancer therapy.

Microfluidic platform for three-dimensional cell culture under spatiotemporal heterogeneity of oxygen tension.

Cells in a tumor microenvironment are exposed to spatial and temporal variations in oxygen tension due to hyperproliferation and immature vascularization. Such spatiotemporal oxygen heterogeneity affects the behavior of cancer cells, leading to cancer growth and metastasis, and thus, it is essential to clarify the cellular responses of cancer cells to oxygen tension. Herein, we describe a new double-layer microfluidic device allowing the control of oxygen tension and the behavior of cancer cells under spatiotemporal oxygen heterogeneity. Two parallel gas channels were located above the media and gel channels to enhance gas exchange, and a gas-impermeable polycarbonate film was embedded in the device to prevent the diffusion of atmospheric oxygen. Variations in oxygen tension in the device with the experimental parameters and design variables were investigated computationally and validated by using oxygen-sensitive nanoparticles. The present device can generate a uniform hypoxic condition at oxygen levels down to 0.3% O2, as well as a linear oxygen gradient from 3% O2 to 17% O2 across the gel channel within 15 min. Moreover, human breast cancer cells suspended in type I collagen gel were introduced in the gel channel to observe their response under controlled oxygen tension. Hypoxic exposure activated the proliferation and motility of the cells, which showed a local maximum increase at 5% O2. Under the oxygen gradient condition, the increase in the cell number was relatively high in the central mild hypoxia region. These findings demonstrate the utility of the present device to study cellular responses in an oxygen-controlled microenvironment.

Cooperative Effects of Vascular Angiogenesis and Lymphangiogenesis.

In this study, we modeled lymphangiogenesis and vascular angiogenesis in a microdevice using a tissue engineering approach. Lymphatic vessels (LV) and blood vessels (BV) were fabricated by sacrificial molding with seeding human lymphatic endothelial cells and human umbilical vein endothelial cells into molded microchannels (600 µm diameter). During subsequent perfusion culture, lymphangiogenesis and vascular angiogenesis were induced by addition of phorbol 12-myristate 13-acetate (PMA) and VEGF-C or VEGF-A characterized by podoplanin and Prox-1 expression. The lymphatic capillaries formed button-like junctions treated with dexamethasone. To test the potential for screening anti-angiogenic (vascular and lymphatic) factors, antagonists of VEGF were introduced. We found that an inhibitor of VEGF-R3 did not completely suppress lymphatic angiogenesis with BVs present, although lymphatic angiogenesis was selectively prevented by addition of a VEGF-R3 inhibitor without BVs. To probe the mechanism of action, we focus on matrix metalloproteinase (MMP) secretion by vascular endothelial cells and lymphatic endothelial cells under monoculture or co-culture conditions. We found that vascular angiogenesis facilitated lymphangiogenesis via remodeling of the local microenvironment by the increased secretion of MMP, mainly by endothelial cells. Applications of this model include a drug screening assay for corneal disease and models for tumorigenesis including lymphatic angiogenesis and vascular angiogenesis.

Adaptive responses of murine osteoblasts subjected to coupled mechanical stimuli.

Restitution of the natural organization and orientation of cells is imperative for the construction of functional tissue scaffolds. While numerous studies have exploited mechanical methods to engineer tissues with the desired cellular architecture, fundamental knowledge is still lacking in understanding the manner in which morphological features can be modulated through coupled mechanical cues. To address this knowledge gap, the adhesion and alignment response of murine osteoblast cells under the synergistic effects of matrix rigidity and cyclic mechanical loading was investigated. This was accomplished by applying cyclic mechanical strain (1% at 0.05Hz) to MC3T3-E1 cells seeded on PDMS substrates of different elastic moduli (1.22, 1.70 and 2.04MPa). Results demonstrate that the overall cell density and expression of inactive vinculin increased on substrates subjected to cyclic stimulus in comparison to substrates under static loading. Conversely, in terms of the adhesion response, osteoblasts exhibited an increased growth of focal adhesion complexes under static substrates. Interestingly, results also elucidate that substrates of a stiffer matrix exposed to cyclic stimulus, had a significantly higher percentage of osteoblasts aligned parallel to the direction of the applied strain, as well as a higher degree of internal order with respect to the strain axis, in comparison to both cells seeded on substrates of lower stiffness under cyclic loading or under static conditions. These findings suggest the role of cyclic mechanical strain coupled with matrix rigidity in eliciting mechanosensitive adaptations in cell functions that allow for the reconstitution of the spatial and orientational assembly of cells in vivo for tissue engineering.