Generating perfusable 3D microvessels in vitro is an important goal for tissue engineering, as well as for reliable modelling of blood vessel function. To date, in vitro blood vessel models have not been able to accurately reproduce the dynamics and responses of endothelial cells to grow perfusable and functional 3D vascular networks. Here we describe a microfluidic-based platform whereby we model natural cellular programs found during normal development and angiogenesis to form perfusable networks of intact 3D microvessels as well as tumor vasculatures based on the spatially controlled co-culture of endothelial cells with stromal fibroblasts, pericytes or cancer cells. The microvessels possess the characteristic morphological and biochemical markers of in vivo blood vessels, and exhibit strong barrier function and long-term stability. An open, unobstructed microvasculature allows the delivery of nutrients, chemical compounds, biomolecules and cell suspensions, as well as flow-induced mechanical stimuli into the luminal space of the endothelium, and exhibits faithful responses to physiological shear stress as demonstrated by cytoskeleton rearrangement and increased nitric oxide synthesis. This simple and versatile platform provides a wide range of applications in vascular physiology studies as well as in developing vascularized organ-on-a-chip and human disease models for pharmaceutical screening.

Molecular gradients play an important role in diverse physiological and pathological phenomena such as immune response, wound healing, development and cancer metastasis. In the past 10 years, engineering tools have been increasingly used to develop experimental platforms that capture important aspects of cellular microenvironments to allow quantitative and reproducible characterization of cellular response to gradients. This review discusses the emergence of microfluidics-based gradient generators and their applications in enhancing our understanding of fundamental biological processes such as chemotaxis and morphogenesis. The principles and applications of microfluidic gradient generation in both 2D and 3D cellular microenvironments are discussed with emphasis on approaches to manipulate spatial and temporal distribution of signaling molecules.

A crucial yet ill-defined phenomenon involved in the remodeling of vascular networks, including angiogenic sprouting, is flow-mediated endothelial dynamics and phenotype changes. Despite interstitial flow (IF) being ubiquitously present in living tissues surrounding blood capillaries, it is rarely investigated and poorly understood how endothelial cells respond to this flow during morphogenesis. Here we develop a microfluidic 3D in vitro model to investigate the role of IF during vasculogenic formation and angiogenic remodeling of microvascular networks. In the presented model, human blood endothelial cells co-cultured with stromal fibroblasts spontaneously organize into an interconnected microvascular network and then further expand to adjacent avascular regions in a manner of neovessel sprouting. We found that in the presence of IF, vasculogenic organization of the microvascular network was significantly facilitated regardless of the flow direction, whereas angiogenic sprouting was promoted only when the directions of flow and sprouting were opposite while angiogenic activity was suppressed into the direction of flow. We also observed that the vasculatures switch between active angiogenic remodeling and quiescent/non-sprouting state in the contexts provided by IF. This regulatory effect can be utilized to examine the role of anti-angiogenic compounds, clearly distinguishing the differential influences of the compounds depending on their mechanisms of action. Collectively, these results suggest that IF may serve as a critical regulator in tissue vascularization and pathological angiogenesis.

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Pericytes enveloping the endothelium play an important role in the physiology and pathology of microvessels, especially in vessel maturation and stabilization. However, our understanding of fundamental pericyte biology is limited by the lack of a robust in vitro model system that allows researchers to evaluate the interactions among multiple cell types in perfusable blood vessels. The present work describes a microfluidic platform that can be used to investigate interactions between pericytes and endothelial cells (ECs) during the sprouting, growth, and maturation steps of neovessel formation. A mixture of ECs and pericytes was attached to the side of a pre-patterned three dimensional fibrin matrix and allowed to sprout across the matrix. The effects of intact coverage and EC maturation by the pericytes on the perfused EC network were confirmed using a confocal microscope. Compared with EC monoculture conditions, EC-pericyte co-cultured vessels showed a significant reduction in diameter, increased numbers of junctions and branches and decreased permeability. In response to biochemical factors, ECs and pericytes in the platform showed the similar features with previous reports from in vivo experiments, thus reflect various pathophysiological conditions of in vivo microvessels. Taken together, these results support the physiological relevancy of our three-dimensional microfluidic culture system but also that the system can be used to screen drug effect on EC-pericyte biology.

COMMUNICATION (1 of 7)© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Biomimetic Model of Tumor Microenvironment on Microfluidic PlatformMinhwan Chung, Jungho Ahn, Kyungmin Son, Sudong Kim, and Noo Li Jeon* DOI: 10.1002/adhm.201700196 "cure." [4,5] The TME is a complex, inter acting system including the tumor itself, other noncancerous cell types such as immune, stromal and endothelial cells, and the extracellular matrix surrounding these cells. [6,7] This complexity has largely prevented a comprehensive understanding of TME in conventional in vitro models, which have been too simple to recapitulate the intricate interactions ( Figure 1A). [8,9] During recent decades, microfabrication technologies have been shown to be valu able tools in the exploration of tumor pro gression. Recently, several studies using microfluidic platform have been reported that recreate the 3D context of each aspect of the TME and metastasis in vitro. [10][11][12][13] However, to our knowledge, no versatile in vitro experimental model that reconstitutes direct interplay between constituents of the TME during tumorigenesis has yet been reported. Here we describe a biomimetic TME model to mimic the complex inter actions of the tumor, stromal, and endothe lial cells, all of which are essential components of the TME that hinder the effective treatment of cancers. [14][15][16] We used a micro fluidic platform from our previous studies. [17,18] Various effects of extracellular matrix (ECM) and the stroma on the physiology of tumor cells and angiogenesis were tested. Finally, we could mimic simultaneous angiogenesis and lymphangiogenesis in the TME with interactions between the tumor cells.Although the cancer stroma is well known for its many roles in multiple cancer stages, direct interactions between cancer and stromal cells have remained largely unknown. [19] Addition ally, cancer cell interaction with the ECM is another TME factor that regulates the behavior of the cancer cells and their resist ance to drugs. [20] Recently, in vitro activation of tumor stoma and corresponding ECM remodeling have been reported using a microfluidic platform. [21] However, single cell responses of the cancer cells in response to the stroma coculture and ECM composition has not yet been described. To examine cancer stromal cell interaction with a 3D ECM, we used a micro fluidic 3D cell culture platform previously reported from our group. [17,18] The array of microposts in the platform enabled straightforward micropatterning of the hydrogel which allowed flexible experimental configurations. We first cocultured cancer and stromal cells within fibrin gel in different microchannels, which still allowed the cells to interact with each other in a par acrine manner ( Figure 1B). Twice as many fibroblasts as cancer cells were patterned to exclude an autocrine or indirect effectThe "Tumor microenvironment" (TME) is a complex, interacting system of the tumor and its surrounding environment. The TME has drawn more attention recently in attempts to overcome current drug...

The mammalian lymphatic system consists of strategically located lymph nodes (LNs) embedded into a lymphatic vascular network. Mechanisms underlying development of this highly organized system are not fully understood. Using high-resolution imaging, we show that lymphoid tissue inducer (LTi) cells initially transmigrate from veins at LN development sites using gaps in venous mural coverage. This process is independent of lymphatic vasculature, but lymphatic vessels are indispensable for the transport of LTi cells that egress from blood capillaries elsewhere and serve as an essential LN expansion reservoir. At later stages, lymphatic collecting vessels ensure efficient LTi cell transport and formation of the LN capsule and subcapsular sinus. Perinodal lymphatics also promote local interstitial flow, which cooperates with lymphotoxin-β signaling to amplify stromal CXCL13 production and thereby promote LTi cell retention. Our data unify previous models of LN development by showing that lymphatics intervene at multiple points to assist LN expansion and identify a new role for mechanical forces in LN development.

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