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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.

Inhibition of the vascular endothelial growth factor (VEGF) pathway has failed to improve overall survival of patients with glioblastoma (GBM). We previously showed that angiopoietin-2 (Ang-2) overexpression compromised the benefit from anti-VEGF therapy in a preclinical GBM model. Here we investigated whether dual Ang-2/VEGF inhibition could overcome resistance to anti-VEGF treatment. We treated mice bearing orthotopic syngeneic (Gl261) GBMs or human (MGG8) GBM xenografts with antibodies inhibiting VEGF (B20), or Ang-2/VEGF (CrossMab, A2V). We examined the effects of treatment on the tumor vasculature, immune cell populations, tumor growth, and survival in both the Gl261 and MGG8 tumor models. We found that in the Gl261 model, which displays a highly abnormal tumor vasculature, A2V decreased vessel density, delayed tumor growth, and prolonged survival compared with B20. In the MGG8 model, which displays a low degree of vessel abnormality, A2V induced no significant changes in the tumor vasculature but still prolonged survival. In both the Gl261 and MGG8 models A2V reprogrammed protumor M2 macrophages toward the antitumor M1 phenotype. Our findings indicate that A2V may prolong survival in mice with GBM by reprogramming the tumor immune microenvironment and delaying tumor growth.

Evaporation is a critical problem when handling submicroliter volumes of fluids. This paper characterizes this problem as it applies to microfluidic cell culture in poly(dimethylsiloxane) (PDMS) devices and provides a practical solution. Evaporation-mediated osmolality shifts through PDMS membranes with varying thicknesses (10, 1, 0.2, or 0.1 mm) were measured over 96 h. Even in humidified cell culture incubators, evaporation through PDMS and associated shifts in the osmolality of culture media was significant and prevented mouse embryo and human endothelial cell growth and development. A simple diffusion model, where the measured diffusion coefficient for PDMS matches reported values of ~[10.sup.-9] [m.sup.2]/s, accounts for these evaporation and osmolality shifts. To overcome this problem, a PDMS-parylene-PDMS hybrid membrane was developed that greatly suppresses evaporation and osmolality shifts, yet possesses thinness and the flexibility necessary to interface with deformation-based microfluidic actuation systems, maintains the clarity for optical microscopy, and enables the successful development of single-cell mouse embryos into blastocysts under static conditions and culture of human endothelial cells under dynamic recirculation of submicroliter volumes of media. These insights and methods demonstrated specifically for embryo and endothelial cell studies will be generally useful for understanding and overcoming evaporation-associated effects in microfluidic cell cultures.

Endothelial cells (ECs) lining the inner lumen of blood vessels are continuously subjected to hemodynamic shear stress, which is known to modify EC morphology and biological activity. This paper describes a self-contained microcirculatory EC culture system that efficiently studies such effects of shear stress on EC alignment and elongation in vitro. The culture system is composed of elastomeric microfluidic cell sheafing chambers interfaced with computer-controlled movement of piezoelectric pins on a refreshable Braille display. The flow rate is varied by design of channels that allow for movement of different volumes of fluid per variable-speed pump stroke. The integrated microfluidic valving and pumping system allowed primary EC seeding and differential sheafing in multiple compartments to be performed on a single chip. The microfluidic flows caused ECs to align and elongate significantly in the direction of flow according to their exposed levels of shear stress. This microfluidic system overcomes the small flow rates and the inefficiencies of previously described microfluidic and macroscopic systems respectively to conveniently perform parallel studies of EC response to shear stress.