Microfluidic technology in vascular research : the endothelial response to shear stress

Vascular endothelial cells form the inner lining of all blood vessels. These cells are highly responsive to the shear stress that is caused by blood flowing over their surface. In this thesis, several aspects of the endothelial response to shear stress are studied. The experiments focus on signal transduction, cytoskeletal remodeling, migration, micromechanical changes and uptake of low density lipoprotein. Most of these experiments were performed by using microfluidic set-ups. In these set-ups, cells are cultured and subjected to experimental conditions in micrometer-sized channels. Several advantages and challenges associated with applying this new technology in vascular research are discussed

Flow Cytometric Analysis of the Uptake of Low-Density Lipoprotein by Endothelial Cells in Microfluidic Channels

Acceptance of microfluidic technology in everyday laboratory practice by biologists is still low. One of the reasons for this is that the technology combines poorly with standard cell biological and biochemical analysis tools. Flow cytometry is an example of a conventional analytical tool that is considered to be incompatible with microfluidic technology and its inherent small sample sizes. In this study, it is shown that properly designed microfluidic devices contain cell populations that are large enough to be analyzed by flow cytometry. To illustrate this, the uptake of fluorescent human low-density lipoprotein (LDL) by human endothelial cells that were cultured in a microfluidic channel was analyzed. It was found that the uptake of LDL by the cells increased linearly over time. Moreover, the uptake decreased when cells were pretreated with fluid shear stress inside the microfluidic devices. This study shows that microfluidic technology can be combined with conventional flow cytometry, while retaining the advantages of working with microfluidics such as low reagent use and dynamic cell culture conditions. This approach of combining microfluidic technology with conventional laboratory tools may contribute to greater acceptance of microfluidic devices in biological research

A microfluidic wound-healing assay for quantifying endothelial cell migration

A microfluidic wound-healing assay for quantifying endothelial cell migration. Am J Physiol Heart Circ Physiol 298: H719–H725, 2010. First published November 20, 2009; doi:10.1152/ajpheart.00933.2009.—Endothelial migration is an important process in the formation of blood vessels and the repair of damaged tissue. To study this process in the laboratory, versatile and reliable migration assays are essential. The purpose of this study was to investigate whether the microfluidic version of the conventional wound-healing assay is a useful research tool for vascular science. Endothelial cells were seeded in a 500-mwide microfluidic channel. After overnight incubation, cells had formed a viable and confluent monolayer. Then, a wound was generated in this monolayer by flushing the channel with three parallel fluid streams, of which the middle one contained the protease trypsin. By analyzing the closing of the wound over time, endothelial cell migration could be measured. Although the migration rate was two times lower in the microfluidic assay than in the conventional assay, an identical 1.5-times increase in migration rate was found in both assays when vascular endothelial growth factor (VEGF165) was added. In the microfluidic wound-healing assay, a stable gradient of VEGF165 could be generated at the wound edge. This led to a two-times increase in migration rate compared with the untreated control. Finally, when a shear stress of 1.3 Pa was applied to the wound, the migration rate increased 1.8 times. In conclusion, the microfluidic assay is a solid alternative for the conventional wound-healing assay when endothelial cell migration is measured. Moreover, it offers unique advantages, such as gradient generation and application of shear stress

Microfluidic organ-on-chip technology for blood-brain barrier research

Organs-on-chips are a new class of microengineered laboratory models that combine several of the advantages of current in vivo and in vitro models. In this review, we summarize the advances that have been made in the development of organ-on-chip models of the blood-brain barrier (BBBs-on-chips) and the challenges that are still ahead. The BBB is formed by specialized e3ndothelial cells and separates blood from brain tissue. It protects the brain from harmful compounds from the blood and provides homeostasis for optimal neuronal function. Studying BBB function and dysfunction is important for drug development and biomedical research. Microfluidic BBBs-on-chips enable real-time study of (human) cells in an engineered physiological microenvironment, for example incorporating small geometries and fluid flow as well as sensors. Examples of BBBs-on-chips in literature already show the potential of more realistic microenvironments and the study of organ-level functions. A key challenge in the field of BBB-on-chip development is the current lack of standardized quantification of parameters such as barrier permeability and shear stress. This limits the potential for direct comparison of the performance of different BBB-on-chip models to each other and existing models. We give recommendations for further standardization in model characterization and conclude that the rapidly emerging field of BBB-on-chip models holds great promise for further studies in BBB biology and drug development

A microfluidic device for monitoring siRNA delivery under fluid flow

When studying particle uptake in vitro, it is favorable to mimic the in vivo situation as much as possible. In this study we present a microfluidic device to mimic the mechanical stress caused by the flow of blood while studying particle uptake in vitro. Human endothelial cells were treated with liposomes containing fluorescent siRNA. It was found that applying physiologically relevant mechanical stress during transfection diminishes the uptake of liposomes in the cells

Blood-brain barrier (BBB): an overview of the research of the blood-brain barrier using microfluidic devices

The blood-brain barrier (BBB) is a unique feature of the human body, preserving brain homeostasis and preventing toxic substances to enter the brain. However, in various neurodegenerative diseases, the function of the BBB is disturbed. Mechanisms of the breakdown of the BBB are incompletely understood and therefore a realistic model of the BBB is essential. This chapter highlights the anatomy and physiology of the BBB and gives an overview of the current available in vitro models to study the BBB in detail. Proof-of-concept work of BBB-on-Chips are described. Additionally, examples are given to optimize the present devices by engineering the microenvironment to better mimic the in vivo situation. This combination of biomedical science and micro-engineering will generate exciting new results in the field of neurovascular biology