Hyperpermeable tumor vessels are responsible for elevated interstitial fluid pressure and altered flow patterns within the tumor microenvironment. These aberrant hydrodynamic stresses may enhance tumor development by stimulating the angiogenic activity of endothelial cells lining the tumor vasculature. However, it is currently not known to what extent shear forces affect endothelial organization or paracrine signaling during tumor angiogenesis. The objective of this study was to develop a three-dimensional (3D), in vitro microfluidic tumor vascular model for coculture of tumor and endothelial cells under varying flow shear stress conditions. A central microchannel embedded within a collagen hydrogel functions as a single neovessel through which tumor-relevant hydrodynamic stresses are introduced and quantified using microparticle image velocimetry (μ-PIV). This is the first use of μ-PIV in a tumor representative, 3D collagen matrix comprised of cylindrical microchannels, rather than planar geometries, to experimentally measure flow velocity and shear stress. Results demonstrate that endothelial cells develop a confluent endothelium on the microchannel lumen that maintains integrity under physiological flow shear stresses. Furthermore, this system provides downstream molecular analysis capability, as demonstrated by quantitative RT-PCR, in which, tumor cells significantly increase expression of proangiogenic genes in response to coculture with endothelial cells under low flow conditions. This work demonstrates that the microfluidic in vitro cell culture model can withstand a range of physiological flow rates and permit quantitative measurement of wall shear stress at the fluid-collagen interface using μ-PIV optical flow diagnostics, ultimately serving as a versatile platform for elucidating the role of fluid forces on tumor-endothelial cross talk.

In vitro arterial flow bioreactor systems are widely used in tissue engineering to investigate response of endothelial cells to shear. However, the assumption that such models reproduce physiological flow has not been experimentally tested. Furthermore, shear stresses experienced by the endothelium are generally calculated using a Poiseuille flow assumption. Understanding the performance of flow bioreactor systems is of great importance, since interpretation of biological responses hinges on the fidelity of such systems and the validity of underlying assumptions. Here we test the physiologic reliability of arterial flow bioreactors and the validity of the Poiseuille assumption for a typical system used in tissue engineering. A particle image velocimetry system was employed to experimentally measure the flow within the vessel with high spatial and temporal resolution. Two types of vessels were considered: first, fluorinated ethylene propylene (FEP) tubing representative of a human artery without cells; and second, FEP tubing with a confluent layer of endothelial cells on the vessel lumen. Instantaneous wall shear stress (WSS), time-averaged WSS, and oscillatory shear index were computed from velocity field measurements and compared between cases. The flow patterns and resulting wall shear were quantitatively determined to not accurately reproduce physiological flow, and that the Poiseuille flow assumption was found to be invalid. This work concludes that analysis of cell response to hemodynamic parameters using such bioreactors should be accompanied by corresponding flow measurements for accurate quantification of fluid stresses.

Physiological flow parameters such as pressure and stress inside the vascular system strongly influence the physiology and function of vascular endothelial cells [1]. Variations in the shear stress experienced by endothelial cells affect morphology, alignment with the flow, mechanical strength, rate of proliferation, and gene expression [2]. Although it is known that these factors are dependent on the hemodynamics of the flow, the relationship has not been accurately quantified. In vitro bioreactor flow loops have been developed to simulate vascular flow for tissue conditioning and measurement of the endothelial cell response to varying shear [3–5]; however, wall shear stresses (WSS) have been estimated from the bulk flow rate by assuming Poiseuille flow [2, 6]. Due to the pulsatility of the flow, biochemical interactions, and the typically short vessel length, this assumption is fundamentally incorrect; however, the level of inaccuracy has not been quantified.

Current in vitro studies of tumor angiogenesis and metastasis are limited by the use of static 2D culture systems or 3D models that poorly reflect the pathological tumor microenvironment. While these systems have provided insight into tumor-inherent mechanisms of neovascularization, they are unable to couple local cellular response with specific biochemical and mechanical cues [1]. Interstitial flow plays an important role in regulating tumor growth; however, there are currently no in vitro cell culture models specifically designed to investigate the effect of fluid shear on tumorigenesis. By integrating tissue-engineering strategies with microfluidics and particle image velocimetry, we have developed a 3D in vitro cell culture model that allows the relationship between shear stress and tumor-endothelial cell cross-talk to be monitored. This research strategy will greatly improve our understanding of shear-stress mediated angiogenesis.

As solid tumors develop, a variety of physical stresses arise including growth induced compressive force, matrix stiffening due to desmoplasia, and increased interstitial fluid pressure and altered flow patterns due to leaky vasculature and poor lymphatic drainage [1]. These microenvironmental stresses likely contribute to the abnormal cell behavior that drives tumor progression, and have become an increasingly significant area of cancer research. Of particular importance, is the role of flow shear stress on tumor-endothelial signaling, vascular function, and angiogenesis. Compared to normal vasculature, blood vessels in tumors are poorly functional due to dysregulated expression of angiogenic growth factors, such as vascular endothelial growth factor (VEGF) or the angiopoietins. Also, because of the abnormal vessel structure, blood velocities can be an order of magnitude lower than that of normal microvessels. Recently published work utilizing intravital microscopy to measure blood velocities in mouse mammary fat pad tumors, demonstrated for the first time that shear rate gradients in tumors may help guide branching and growth of new vessels [2]. However, much still remains unknown about how shear stress regulates endothelial organization, permeability, or expression of growth factors within the context of the tumor microenvironment.