Rapid Tissue Perfusion Using Sacrificial Percolation of Anisotropic Networks.

Tissue engineering has long sought to rapidly generate perfusable vascularized tissues with vessel sizes spanning those seen in humans. Current techniques such as biological 3D printing (top-down) and cellular self-assembly (bottom-up) are resource intensive and have not overcome the inherent tradeoff between vessel resolution and assembly time, limiting their utility and scalability for engineering tissues. We present a flexible and scalable technique termed SPAN - Sacrificial Percolation of Anisotropic Networks, where a network of perfusable channels is created throughout a tissue in minutes, irrespective of its size. Conduits with length scales spanning arterioles to capillaries are generated using pipettable alginate fibers that interconnect above a percolation density threshold and are then degraded within constructs of arbitrary size and shape. SPAN is readily used within common tissue engineering processes, can be used to generate endothelial cell-lined vasculature in a multi-cell type construct, and paves the way for rapid assembly of perfusable tissues.

Rapid time-lapse 3D oxygen tension measurements within hydrogels using widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) and image segmentation.

Understanding the influence of oxygen tension on cellular functions and behaviors is crucial for investigating various physiological and pathological conditions. In vitro cell culture models, particularly those based on hydrogel extracellular matrices, have been developed to study cellular responses in specific oxygen microenvironments. However, accurately characterizing oxygen tension variations with great spatiotemporal resolutions, especially in three dimensions, remains challenging. This paper presents an approach for rapid time-lapse 3D oxygen tension measurements in hydrogels using a widely available inverted fluorescence microscope. Oxygen-sensitive fluorescent microbeads and widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) are utilized for oxygen tension estimation. To incorporate the third dimension, a motorized sample stage is implanted that enables automated image acquisition in the vertical direction. A machine learning algorithm based on K-means clustering is employed for microbead position identification. Using an upside-down microfluidic device, 3D oxygen gradients are generated within a hydrogel sample, and z-stack images are acquired using the FD-FLIM system. Analyses of the acquired images, involving microbead position identification, lifetime calculation, and oxygen tension conversion, are then performed offline. The results demonstrate the functionality of the developed approach for rapid time-lapse 3D oxygen tension measurements in hydrogels. Furthermore, the 3D oxygen tension adjacent to a tumor spheroid within a hydrogel during media exchange is characterized. The results further confirm that the 3D spatiotemporal oxygen tension profiles can be successfully measured quantitatively using the established setup and analysis process and that the approach may have great potential for investigating cellular activities within oxygen microenvironments.

Studying sprouting angiogenesis under combination of oxygen gradients and co-culture of fibroblasts using microfluidic cell culture model.

Sprouting angiogenesis is an essential process for expanding vascular systems under various physiological and pathological conditions. In this paper, a microfluidic device capable of integrating a hydrogel matrix for cell culture and generating stable oxygen gradients is developed to study the sprouting angiogenesis of endothelial cells under combinations of oxygen gradients and co-culture of fibroblast cells. The endothelial cells can be cultured as a monolayer endothelium inside the device to mimic an existing blood vessel, and the hydrogel without or with fibroblast cells cultured in it provides a matrix next to the formed endothelium for three-dimensional sprouting of the endothelial cells. Oxygen gradients can be stably established inside the device for cell culture using the spatially-confined chemical reaction method. Using the device, the sprouting angiogenesis under combinations of oxygen gradients and co-culture of fibroblast cells is systematically studied. The results show that the oxygen gradient and the co-culture of fibroblast cells in the hydrogel can promote sprouting of the endothelial cells into the hydrogel matrix by altering cytokines in the culture medium and the physical properties of the hydrogel. The developed device provides a powerful in vitro model to investigate sprouting angiogenesis under various in vivo-like microenvironments.

A 3D culture system for evaluating the combined effects of cisplatin and anti-fibrotic drugs on the growth and invasion of lung cancer cells co-cultured with fibroblasts.

Fibrosis and fibroblast activation usually occur in the tissues surrounding a malignant tumor; therefore, anti-fibrotic drugs are used in addition to chemotherapy. A reliable technique for evaluating the combined effects of anti-fibrotic drugs and anticancer drugs would be beneficial for the development of an appropriate treatment strategy. In this study, we manufactured a three-dimensional (3D) co-culture system of fibroblasts and lung cancer cell spheroids in Matrigel supplemented with fibrin (fibrin/Matrigel) that simulated the tissue microenvironment around a solid tumor. We compared the efficacy of an anticancer drug (cisplatin) with or without pretreatments of two anti-fibrotic drugs, nintedanib and pirfenidone, on the growth and invasion of cancer cells co-cultured with fibroblasts. The results showed that the addition of nintedanib improved cisplatin's effects on suppressing the growth of cancer cell spheroids and the invasion of cancer cells. In contrast, pirfenidone did not enhance the anticancer activity of cisplatin. Nintedanib also showed higher efficacy than pirfenidone in reducing the expression of four genes in fibroblasts associated with cell adhesion, invasion, and extracellular matrix degradation. This study demonstrated that the 3D co-cultures in fibrin/Matrigel would be useful for assessing the effects of drug combinations on tumor growth and invasion.

Revealing anisotropic elasticity of endothelium under fluid shear stress.

Endothelium lining interior surface of blood vessels experiences various physical stimulations in vivo. Its physical properties, especially elasticity, play important roles in regulating the physiological functions of vascular systems. In this paper, an integrated approach is developed to characterize the anisotropic elasticity of the endothelium under physiological-level fluid shear stress. A pressure sensor-embedded microfluidic device is developed to provide fluid shear stress on the perfusion-cultured endothelium and to measure transverse in-plane elasticities in the directions parallel and perpendicular to the flow direction. Biological atomic force microscopy (Bio-AFM) is further exploited to measure the vertical elasticity of the endothelium in its out-of-plane direction. The results show that the transverse elasticity of the endothelium in the direction parallel to the perfusion culture flow direction is about 70% higher than that in the direction perpendicular to the flow direction. Moreover, the transverse elasticities of the endothelium are estimated to be approximately 120 times larger than the vertical one. The results indicate the effects of fluid shear stress on the transverse elasticity anisotropy of the endothelium, and the difference between the elasticities in transverse and vertical directions. The quantitative measurement of the endothelium anisotropic elasticity in different directions at the tissue level under the fluid shear stress provides biologists insightful information for the advanced vascular system studies from biophysical and biomaterial viewpoints. STATEMENT OF SIGNIFICANCE: In this paper, we take advantage an integrated approach combining microfluidic devices and biological atomic force microscopy (Bio-AFM) to characterize anisotropic elasticities of endothelia with and without fluidic shear stress application. The microfluidic devices are exploited to conduct perfusion cell culture of the endothelial cells, and to estimate the in-plane elasticities of the endothelium in the direction parallel and perpendicular to the shear stress. In addition, the Bio-AFM is utilized for characterization of the endothelium morphology and vertical elasticity. The measurement results demonstrate the very first anisotropic elasticity quantification of the endothelia. Furthermore, the study provides insightful information bridging the microscopic sing cell and macroscopic organ level studies, which can greatly help to advance vascular system research from material perspective.

Study 3D Endothelial Cell Network Formation under Various Oxygen Microenvironment and Hydrogel Composition Combinations Using Upside-Down Microfluidic Devices.

Formation of 3D networks is a crucial process for endothelial cells during development of primary blood vessels under both normal and pathological conditions. In order to investigate effects of oxygen microenvironment and matrix composition on the 3D network formation, an upside-down microfluidic cell culture device capable of generating oxygen gradients is developed in this paper. In cell experiments, network formation of human umbilical vein endothelial cells (HUVECs) within fibrinogen-based hydrogels with different concentrations of hyaluronic acid (HA) is systematically studied. In addition, five different oxygen microenvironments (uniform normoxia, 5%, and 1% O2 ; oxygen gradients under normoxia and 5% O2 ) are also applied for the cell culture. The generated oxygen gradients are characterized based on fluorescence lifetime measurements. The experimental results show increased 3D cell network length when the cells are cultured under the oxygen gradients within the hydrogels with the HA addition suggesting their roles in promoting network formation. Furthermore, the formed networks tend to align along the direction of the oxygen gradients indicating the presence of gradient-driven cellular response. The results demonstrate that the developed upside-down microfluidic device can provide an advanced platform to investigate 3D cell culture under the controlled oxygen microenvironments for various biomedical studies in vitro.