Ensemble Analysis of Angiogenic Growth in Three-Dimensional Microfluidic Cell Cultures

We demonstrate ensemble three-dimensional cell cultures and quantitative analysis of angiogenic growth from uniform endothelial monolayers. Our approach combines two key elements: a micro-fluidic assay that enables parallelized angiogenic growth instances subject to common extracellular conditions, and an automated image acquisition and processing scheme enabling high-throughput, unbiased quantification of angiogenic growth. Because of the increased throughput of the assay in comparison to existing three-dimensional morphogenic assays, statistical properties of angiogenic growth can be reliably estimated. We used the assay to evaluate the combined effects of vascular endothelial growth factor (VEGF) and the signaling lipid sphingoshine-1-phosphate (S1P). Our results show the importance of S1P in amplifying the angiogenic response in the presence of VEGF gradients. Furthermore, the application of S1P with VEGF gradients resulted in angiogenic sprouts with higher aspect ratio than S1P with background levels of VEGF, despite reduced total migratory activity. This implies a synergistic effect between the growth factors in promoting angiogenic activity. Finally, the variance in the computed angiogenic metrics (as measured by ensemble standard deviation) was found to increase linearly with the ensemble mean. This finding is consistent with stochastic agent-based mathematical models of angiogenesis that represent angiogenic growth as a series of independent stochastic cell-level decisions

Validating Antimetastatic Effects of Natural Products in an Engineered Microfluidic Platform Mimicking Tumor Microenvironment

Development of new, antimetastatic drugs from natural products has been substantially constrained by the lack of a reliable in vitro screening system. Such a system should ideally mimic the native, three-dimensional (3D) tumor microenvironment involving different cell types and allow quantitative analysis of cell behavior critical for metastasis. These requirements are largely unmet in the current model systems, leading to poor predictability of the in vitro collected data for in vivo trials, as well as prevailing inconsistency among different in vitro tests. In the present study, we report application of a 3D, microfluidic device for validation of the antimetastatic effects of 12 natural compounds. This system supports co-culture of endothelial and cancer cells in their native 3D morphology as in the tumor microenvironment and provides real-time monitoring of the cells treated with each compound. We found that three compounds, namely sanguinarine, nitidine, and resveratrol, exhibited significant antimetastatic or antiangiogenic effects. Each compound was further examined for its respective activity with separate conventional biological assays, and the outcomes were in agreement with the findings collected from the microfluidic system. In summary, we recommend use of this biomimetic model system as a new engineering tool for high-throughput evaluation of more diverse natural compounds with varying anticancer potentials

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference

Design and development of a low-cost thermal response rig

A thermal response test (TRT) is a controlled insitu test during which a known quantity of heat energy is injected into a closed loop heat-exchanger pipe while the heat dissipation rate into the surrounding ground is monitored. Results from a test can be interpreted to determine a number of ground thermal parameters with are vital design requirements for any medium to large scale ground source energy system. This paper describes the design and construction of a low cost TRT rig and compares the results obtained from a test using the constructed rig and a commercially built rig in order to evaluate the accuracy of the constructed equipment. The TRT rig is designed in accordance with the following principles: keep construction costs low, improve the cost-efficiency of TRT testing by incorporating remote data transmission capability and ensure attainment of sufficient accuracy to satisfy the design requirements of ground source energy systems. Analysis of data collected by the TRT rigs result in a calculated thermal conductivity of 1.9 W/mK in both cases. This value falls within the range expected for the tested geological formation and confirms the accuracy of both test rigs.Irish Research Council for Science, Engineering and Technolog

Three-dimensional microfluidic model for tumor cell intravasation and endothelial barrier function

Entry of tumor cells into the blood stream is a critical step in cancer metastasis. Although significant progress has been made in visualizing tumor cell motility in vivo, the underlying mechanism of cancer cell intravasation remains largely unknown. We developed a microfluidic-based assay to recreate the tumor-vascular interface in three-dimensions, allowing for high resolution, real-time imaging, and precise quantification of endothelial barrier function. Studies are aimed at testing the hypothesis that carcinoma cell intravasation is regulated by biochemical factors from the interacting cells and cellular interactions with macrophages. We developed a method to measure spatially resolved endothelial permeability and show that signaling with macrophages via secretion of tumor necrosis factor alpha results in endothelial barrier impairment. Under these conditions intravasation rates were increased as validated with live imaging. To further investigate tumor-endothelial (TC-EC) signaling, we used highly invasive fibrosarcoma cells and quantified tumor cell migration dynamics and TC-EC interactions under control and perturbed (with tumor necrosis factor alpha) barrier conditions. We found that endothelial barrier impairment was associated with a higher number and faster dynamics of TC-EC interactions, in agreement with our carcinoma intravasation results. Taken together our results provide evidence that the endothelium poses a barrier to tumor cell intravasation that can be regulated by factors present in the tumor microenvironment.National Cancer Institute (U.S.) (R21CA140096)National Institutes of Health (U.S.) (Grant GM58801)Department of Defense Breast Cancer Research Program (Grant W81XWH-10-1-0040

In vivo

The capability of currently available echocardiography-based strain estimation techniques to fully map myocardial abnormality at early stages of myocardial ischemia is yet to be investigated. In this study, myocardial elastography (ME), a radio-frequency (RF)-based strain imaging technique that maps the full 2D transmural angle-independent strain tensor in standard echocardiographic views at both high spatial and temporal resolution is presented. The objectives were to (1) evaluate the performance of ME on mapping the onset, extent and progression of myocardial ischemia at graded coronary constriction levels (from partial to complete coronary flow reduction), and (2) validate the accuracy of the strain estimates against sonomicrometry (SM) measurements. A non-survival canine ischemic model (n = 5) was performed by gradually constricting the left anterior descending (LAD) coronary blood flow from 0% (baseline blood flow) to 100% (zero blood flow) at 20% increments. An open-architecture ultrasound system was used to acquire RF echocardiograms in a standard full short-axis view at the frame rate of 211 fps, at least twice higher than what is typically used in conventional echocardiographic systems, using a previously developed, fully automated composite technique. Myocardial deformation was estimated by ME and validated against sonomicrometry. ME estimates and maps transmural (1) 2D displacements using RF cross-correlation and recorrelation; and (2) 2D polar (radial and circumferential) strains, derived from 2D (i.e. both lateral and axial) displacement components, at high accuracy. Full-view strain images were shown and found to reliably depict decreased myocardial function in the region at risk at increased levels of coronary flow reduction. The ME radial strain was deemed to be a more sensitive, quantitative, regional measure of myocardial ischemia as a result of coronary flow reduction when compared to the conventional wall motion score index and ejection fraction. Good agreement (0.22% strain bias, 95% limits of agreement) using Bland-Altman analysis and good correlation (r = 0.84) were found between the ME and SM measurements. These findings demonstrate for the first time that ME could map angle-independent strains to non-invasively detect, localize and characterize the early onset of myocardial ischemia, i.e. at 40%, and possibly as low as 20%, LAD flow reduction, which could be further associated with the severity of coronary stenosis. © 2011 Institute of Physics and Engineering in Medicine.link_to_subscribed_fulltex

High-throughput microfluidic platform for 3D cultures of mesenchymal stem cells

The design of innovative tools for generating physiologically relevant three-dimensional (3D) in vitro models has been recently recognized as a fundamental step to study cell responses and long-term tissue functionalities thanks to its ability to recapitulate the complexity and the dimensional scale of the cellular microenvironment, while directly integrating high-throughput and automatic screening capabilities. This chapter addresses the development of a poly(dimethylsiloxane)-based microfluidic platform to (1) generate and culture 3D cellular microaggregates under continuous flow perfusion while (2) conditioning them with different combinations/concentrations of soluble factors (i.e., growth factors, morphogens or drug molecules), in a high-throughput fashion. The proposed microfluidic system thus represents a promising tool for establishing innovative high-throughput models for drug screening, investigation of tissues morphogenesis, and optimization of tissue engineering protocols