Retina-on-a-chip: a microfluidic platform for point access signaling studies.

We report on a microfluidic platform for culture of whole organs or tissue slices with the capability of point access reagent delivery to probe the transport of signaling events. Whole mice retina were maintained for multiple days with negative pressure applied to tightly but gently bind the bottom of the retina to a thin poly-(dimethylsiloxane) membrane, through which twelve 100 μm diameter through-holes served as fluidic access points. Staining with toluidine blue, transport of locally applied cholera toxin beta, and transient response to lipopolysaccharide in the retina demonstrated the capability of the microfluidic platform.

Three-Dimensional Blood-Brain Barrier Model for in vitro Studies of Neurovascular Pathology.

Blood-brain barrier (BBB) pathology leads to neurovascular disorders and is an important target for therapies. However, the study of BBB pathology is difficult in the absence of models that are simple and relevant. In vivo animal models are highly relevant, however they are hampered by complex, multi-cellular interactions that are difficult to decouple.

NF2/Merlin mediates contact-dependent inhibition of EGFR mobility and internalization via cortical actomyosin.

The proliferation of normal cells is inhibited at confluence, but the molecular basis of this phenomenon, known as contact-dependent inhibition of proliferation, is unclear. We previously identified the neurofibromatosis type 2 (NF2) tumor suppressor Merlin as a critical mediator of contact-dependent inhibition of proliferation and specifically found that Merlin inhibits the internalization of, and signaling from, the epidermal growth factor receptor (EGFR) in response to cell contact. Merlin is closely related to the membrane-cytoskeleton linking proteins Ezrin, Radixin, and Moesin, and localization of Merlin to the cortical cytoskeleton is required for contact-dependent regulation of EGFR.

Neuronal uptake and propagation of a rare phosphorylated high-molecular-weight tau derived from Alzheimer's disease brain.

Tau pathology is known to spread in a hierarchical pattern in Alzheimer's disease (AD) brain during disease progression, likely by trans-synaptic tau transfer between neurons. However, the tau species involved in inter-neuron propagation remains unclear. To identify tau species responsible for propagation, we examined uptake and propagation properties of different tau species derived from postmortem cortical extracts and brain interstitial fluid of tau-transgenic mice, as well as human AD cortices.

Detection of T790M, the Acquired Resistance EGFR Mutation, by Tumor Biopsy versus Noninvasive Blood-Based Analyses.

Purpose: The T790M gatekeeper mutation in the EGFR is acquired by some EGFR-mutant non-small cell lung cancers (NSCLC) as they become resistant to selective tyrosine kinase inhibitors (TKI). As third-generation EGFR TKIs that overcome T790M-associated resistance become available, noninvasive approaches to T790M detection will become critical to guide management.

Experimental Design: As part of a multi-institutional Stand-Up-To-Cancer collaboration, we performed an exploratory analysis of 40 patients with EGFR-mutant tumors progressing on EGFR TKI therapy.

Microfluidic mazes to characterize T-cell exploration patterns following activation in vitro.

The migration of T-cell subsets within peripheral tissues is characteristic of inflammation and immunoregulation. In general, the lymphocyte migratory response is assumed directional and guided by local gradients of chemoattractants and/or chemorepellents. However, little is known about how cells explore their tissue environment, and whether lymphocyte activation may influence speed and exploratory patterns of migration.

Human Neutrophils Are Primed by Chemoattractant Gradients for Blocking the Growth of Aspergillus fumigatus.

The contribution of human neutrophils to the protection against fungal infections by Aspergillus fumigatus is essential but not fully understood. Whereas healthy people can inhale spores of A. fumigatus without developing disease, neutropenic patients and those receiving immunosuppressive drugs have a higher incidence of invasive fungal infections.

Fast sorting of CD4+ T cells from whole blood using glass microbubbles.

The isolation of CD4 positive T lymphocyte (CD4+) from peripheral blood is important for monitoring patients after HIV infection. Here, we demonstrate a fast isolation strategy for CD4+ cells that involves mixing blood and glass microbubbles. After the specific binding of target cells to the microbubbles carrying specific antibodies on their surface, target cells will spontaneously float to the top of the blood vial and can be quickly separated.

Continuous Flow Microfluidic Bioparticle Concentrator.

Innovative microfluidic technology has enabled massively parallelized and extremely efficient biological and clinical assays. Many biological applications developed and executed with traditional bulk processing techniques have been translated and streamlined through microfluidic processing with the notable exception of sample volume reduction or centrifugation, one of the most widely utilized processes in the biological sciences. We utilize the high-speed phenomenon known as inertial focusing combined with hydraulic resistance controlled multiplexed micro-siphoning allowing for the continuous concentration of suspended cells into pre-determined volumes up to more than 400 times smaller than the input with a yield routinely above 95% at a throughput of 240 ml/hour.

Whole blood human neutrophil trafficking in a microfluidic model of infection and inflammation.

Appropriate inflammatory responses to wounds and infections require adequate numbers of neutrophils arriving at injury sites. Both insufficient and excessive neutrophil recruitment can be detrimental, favouring systemic spread of microbes or triggering severe tissue damage. Despite its importance in health and disease, the trafficking of neutrophils through tissues remains difficult to control and the mechanisms regulating it are insufficiently understood.

"Universal" vitrification of cells by ultra-fast cooling.

Long-term preservation of live cells is critical for a broad range of clinical and research applications. With the increasing diversity of cells that need to be preserved (e.g.

Translational implications of endothelial cell dysfunction in association with chronic allograft rejection.

Advances in therapeutics have dramatically improved short-term graft survival, but the incidence of chronic rejection has not changed in the past 20 years. New insights into mechanism are sorely needed at this time and it is hoped that the development of predictive biomarkers will pave the way for the emergence of preventative therapeutics. In this review, we discuss a paradigm suggesting that sequential changes within graft endothelial cells (EC) lead to an intragraft microenvironment that favors the development of chronic rejection.

Spontaneous neutrophil migration patterns during sepsis after major burns.

Finely tuned to respond quickly to infections, neutrophils have amazing abilities to migrate fast and efficiently towards sites of infection and inflammation. Although neutrophils ability to migrate is perturbed in patients after major burns, no correlations have yet been demonstrated between altered migration and higher rate of infections and sepsis in these patients when compared to healthy individuals. To probe if such correlations exist, we designed microfluidic devices to quantify the neutrophil migration phenotype with high precision.

A Food and Drug Administration-approved asthma therapeutic agent impacts amyloid β in the brain in a transgenic model of Alzheimer disease.

Interfering with the assembly of Amyloid β (Aβ) peptides from monomer to oligomeric species and fibrils or promoting their clearance from the brain are targets of anti-Aβ-directed therapies in Alzheimer disease. Here we demonstrate that cromolyn sodium (disodium cromoglycate), a Food and Drug Administration-approved drug already in use for the treatment of asthma, efficiently inhibits the aggregation of Aβ monomers into higher-order oligomers and fibrils in vitro without affecting Aβ production. In vivo, the levels of soluble Aβ are decreased by over 50% after only 1 week of daily intraperitoneally administered cromolyn sodium.

Stochastic variations of migration speed between cells in clonal populations.

We combined microfluidic tools and molecular probes to monitor the migration speed of successive generations of cancer cells. We found that the migratory speed of individual cells changes stochastically from parent cells to their descendants, while the average speed of successive generations of cells remains constant. Further studies of the interrelations between cell migration and division processes may help identify the molecular determinants of cell speed and lead to new therapies to slow the invasion of cancer cells and delay metastases.

Three-Dimensional Holographic Refractive-Index Measurement of Continuously Flowing Cells in a Microfluidic Channel.

Refractive index of biological specimens is a source of intrinsic contrast that can be explored without any concerns of photobleaching or harmful effects caused by extra contrast agents. In addition, RI contains rich information related to the metabolism of cells at the cellular and subcellular levels. Here, we report a no-moving parts approach that provides three-dimensional refractive index maps of biological samples continuously flowing in a microfluidic channel.

A neutrophil treadmill to decouple spatial and temporal signals during chemotaxis.

After more than 50 years of debates, the role of spatial and temporal gradients during cell chemotaxis is still a contentious matter. One major challenge is that when cells move in response to a heterogeneous chemical environment they are exposed to both spatial and temporal concentration changes. Even in the presence of perfectly stable chemical gradients, moving cells experience temporal changes of concentration simply by moving between locations with different chemical concentrations in a heterogeneous environment.

New methods for investigation of neuronal migration in embryonic brain explants.

Background: Proper migration of neurons is essential for the formation and normal functioning of the nervous system. Defects in neuronal migration underlie a number of neurologic diseases in humans. Although cell migration is crucial for neural development, molecular mechanisms guiding neuronal migration remain to be elucidated fully.

Microfluidic platform for the quantitative analysis of leukocyte migration signatures.

Leukocyte migration into tissues is characteristic of inflammation. It is usually measured in vitro as the average displacement of populations of cells towards a chemokine gradient, not acknowledging other patterns of cell migration. Here, we designed and validated a microfluidic migration platform to simultaneously analyse four qualitative migration patterns: chemoattraction, -repulsion, -kinesis and -inhibition, using single-cell quantitative metrics of direction, speed, persistence and fraction of cells responding.

Collective and individual migration following the epithelial-mesenchymal transition.

During cancer progression, malignant cells in the tumour invade surrounding tissues. This transformation of adherent cells to a motile phenotype has been associated with the epithelial-mesenchymal transition (EMT). Here, we show that EMT-activated cells migrate through micropillar arrays as a collectively advancing front that scatters individual cells.

Microfluidic platform for measuring neutrophil chemotaxis from unprocessed whole blood.

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur.

Inertial focusing in microfluidics.

When Segré and Silberberg in 1961 witnessed particles in a laminar pipe flow congregating at an annulus in the pipe, scientists were perplexed and spent decades learning why such behavior occurred, finally understanding that it was caused by previously unknown forces on particles in an inertial flow. The advent of microfluidics opened a new realm of possibilities for inertial focusing in the processing of biological fluids and cellular suspensions and created a field that is now rapidly expanding. Over the past five years, inertial focusing has enabled high-throughput, simple, and precise manipulation of bodily fluids for a myriad of applications in point-of-care and clinical diagnostics.

Migration of neutrophils targeting amyloid plaques in Alzheimer's disease mouse model.

Immune responses in the brain are thought to play a role in disorders of the central nervous system, but an understanding of the process underlying how immune cells get into the brain and their fate there remains unclear. In this study, we used a 2-photon microscopy to reveal that neutrophils infiltrate brain and migrate toward amyloid plaques in a mouse model of Alzheimer's disease. These findings suggest a new molecular process underlying the pathophysiology of Alzheimer's disease.

On-demand, competing gradient arrays for neutrophil chemotaxis.

Neutrophils are the most abundant type of white blood cells in the circulation, protecting the body against pathogens and responding early to inflammation. Although we understand how neutrophils respond to individual stimuli, we know less about how they prioritize between competing signals or respond to combinational signals. This situation is due in part to the lack of adequate experimental systems to provide signals in controlled spatial and temporal fashion.