Bryson M. Brewer

Recreating blood-brain barrier physiology and structure on chip: A novel neurovascular microfluidic bioreactor.

The blood-brain barrier (BBB) is a critical structure that serves as the gatekeeper between the central nervous system and the rest of the body. It is the responsibility of the BBB to facilitate the entry of required nutrients into the brain and to exclude potentially harmful compounds; however, this complex structure has remained difficult to model faithfully in vitro. Accurate in vitro models are necessary for understanding how the BBB forms and functions, as well as for evaluating drug and toxin penetration across the barrier. Many previous models have failed to support all the cell types involved in the BBB formation and/or lacked the flow-created shear forces needed for mature tight junction formation. To address these issues and to help establish a more faithful in vitro model of the BBB, we have designed and fabricated a microfluidic device that is comprised of both a vascular chamber and a brain chamber separated by a porous membrane. This design allows for cell-to-cell communication between endothelial cells, astrocytes, and pericytes and independent perfusion of both compartments separated by the membrane. This NeuroVascular Unit (NVU) represents approximately one-millionth of the human brain, and hence, has sufficient cell mass to support a breadth of analytical measurements. The NVU has been validated with both fluorescein isothiocyanate (FITC)-dextran diffusion and transendothelial electrical resistance. The NVU has enabled in vitro modeling of the BBB using all human cell types and sampling effluent from both sides of the barrier.

TDP-43 is intercellularly transmitted across axon terminals

A protein complementation assay quantifying TDP-43 oligomerization in living neurons shows microvesicular and bidirectional synaptic transmission of TDP-43 and TDP-43 seeding activity in human ALS postmortem brain tissue.

Stretching fibroblasts remodels fibronectin and alters cancer cell migration.

Most investigations of cancer-stroma interactions have focused on biochemical signaling effects, with much less attention being paid to biophysical factors. In this study, we investigated the role of mechanical stimuli on human prostatic fibroblasts using a microfluidic platform that was adapted for our experiments and further developed for both repeatable performance among multiple assays and for compatibility with high-resolution confocal microscopy. Results show that mechanical stretching of normal tissue-associated fibroblasts (NAFs) alters the structure of secreted fibronectin. Specifically, unstretched NAFs deposit and assemble fibronectin in a random, mesh-like arrangement, while stretched NAFs produce matrix with a more organized, linearly aligned structure. Moreover, the stretched NAFs exhibited an enhanced capability for directing co-cultured cancer cell migration in a persistent manner. Furthermore, we show that stretching NAFs triggers complex biochemical signaling events through the observation of increased expression of platelet derived growth factor receptor α (PDGFRα). A comparison of these behaviors with those of cancer-associated fibroblasts (CAFs) indicates that the observed phenotypes of stretched NAFs are similar to those associated with CAFs, suggesting that mechanical stress is a critical factor in NAF activation and CAF genesis.

Metabolic consequences of interleukin-6 challenge in developing neurons and astroglia

BackgroundMaternal immune activation and subsequent interleukin-6 (IL-6) induction disrupt normal brain development and predispose the offspring to developing autism and schizophrenia. While several proteins have been identified as having some link to these developmental disorders, their prevalence is still small and their causative role, if any, is not well understood. However, understanding the metabolic consequences of environmental predisposing factors could shed light on disorders such as autism and schizophrenia.MethodsTo gain a better understanding of the metabolic consequences of IL-6 exposure on developing central nervous system (CNS) cells, we separately exposed developing neuron and astroglia cultures to IL-6 for 2 hours while collecting effluent from our gravity-fed microfluidic chambers. By coupling microfluidic technologies to ultra-performance liquid chromatography-ion mobility-mass spectrometry (UPLC-IM-MS), we were able to characterize the metabolic response of these CNS cells to a narrow window of IL-6 exposure.ResultsOur results revealed that 1) the use of this technology, due to its superb media volume:cell volume ratio, is ideally suited for analysis of cell-type-specific exometabolome signatures; 2) developing neurons have low secretory activity at baseline, while astroglia show strong metabolic activity; 3) both neurons and astroglia respond to IL-6 exposure in a cell type-specific fashion; 4) the astroglial response to IL-6 stimulation is predominantly characterized by increased levels of metabolites, while neurons mostly depress their metabolic activity; and 5) disturbances in glycerophospholipid metabolism and tryptophan/kynurenine metabolite secretion are two putative mechanisms by which IL-6 affects the developing nervous system.ConclusionsOur findings are potentially critical for understanding the mechanism by which IL-6 disrupts brain function, and they provide information about the molecular cascade that links maternal immune activation to developmental brain disorders.

Cancer-associated fibroblasts promote directional cancer cell migration by aligning fibronectin

Cancer-associated fibroblasts (CAFs) are major components of the carcinoma microenvironment that promote tumor progression. However, the mechanisms by which CAFs regulate cancer cell migration are poorly understood. In this study, we show that fibronectin (Fn) assembled by CAFs mediates CAF–cancer cell association and directional migration. Compared with normal fibroblasts, CAFs produce an Fn-rich extracellular matrix with anisotropic fiber orientation, which guides the cancer cells to migrate directionally. CAFs align the Fn matrix by increasing nonmuscle myosin II- and platelet-derived growth factor receptor &agr;–mediated contractility and traction forces, which are transduced to Fn through &agr;5&bgr;1 integrin. We further show that prostate cancer cells use &agr;v integrin to migrate efficiently and directionally on CAF-derived matrices. We demonstrate that aligned Fn is a prominent feature of invasion sites in human prostatic and pancreatic carcinoma samples. Collectively, we present a new mechanism by which CAFs organize the Fn matrix and promote directional cancer cell migration.

A microfluidic cell co-culture platform with a liquid fluorocarbon separator

A microfluidic cell co-culture platform that uses a liquid fluorocarbon oil barrier to separate cells into different culture chambers has been developed. Characterization indicates that the oil barrier could be effective for multiple days, and a maximum pressure difference between the oil barrier and aqueous media in the cell culture chamber could be as large as ~3.43 kPa before the oil barrier fails. Biological applications have been demonstrated with the separate transfection of two groups of primary hippocampal neurons with two different fluorescent proteins and subsequent observation of synaptic contacts between the neurons. In addition, the quality of the fluidic seal provided by the oil barrier is shown to be greater than that of an alternative solid-PDMS valve barrier design by testing the ability of each device to block low molecular weight CellTracker dyes used to stain cells in the culture chambers.

The Rho family GEF Asef2 regulates cell migration in three dimensional (3D) collagen matrices through myosin II

Cell migration is fundamental to a variety of physiological processes, including tissue development, homeostasis, and regeneration. Migration has been extensively studied with cells on 2-dimensional (2D) substrates, but much less is known about cell migration in 3D environments. Tissues and organs are 3D, which is the native environment of cells in vivo, pointing to a need to understand migration and the mechanisms that regulate it in 3D environments. To investigate cell migration in 3D environments, we developed microfluidic devices that afford a controlled, reproducible platform for generating 3D matrices. Using these devices, we show that the Rho family guanine nucleotide exchange factor (GEF) Asef2 inhibits cell migration in 3D type I collagen (collagen I) matrices. Treatment of cells with the myosin II (MyoII) inhibitor blebbistatin abolished the decrease in migration by Asef2. Moreover, Asef2 enhanced MyoII activity as shown by increased phosphorylation of serine 19 (S19). Furthermore, Asef2 increased activation of Rac, which is a Rho family small GTPase, in 3D collagen I matrices. Inhibition of Rac activity by treatment with the Rac-specific inhibitor NSC23766 abrogated the Asef2-promoted increase in S19 MyoII phosphorylation. Thus, our results indicate that Asef2 regulates cell migration in 3D collagen I matrices through a Rac-MyoII-dependent mechanism.

Biomechanics of cell reorientation in a three-dimensional matrix under compression

Abstract Although a number of studies have reported that cells cultured on a stretchable substrate align away from or perpendicular to the stretch direction, how cells sense and respond to compression in a three‐dimensional (3D) matrix remains an open question. We analyzed the reorientation of human prostatic normal tissue fibroblasts (NAFs) and cancer‐associated fibroblasts (CAFs) in response to 3D compression using a Fast Fourier Transform (FFT) method. Results show that NAFs align to specific angles upon compression while CAFs exhibit a random distribution. In addition, NAFs with enhanced contractile force induced by transforming growth factor &bgr; (TGF‐&bgr;) behave in a similar way as CAFs. Furthermore, a theoretical model based on the minimum energy principle has been developed to provide insights into these observations. The model prediction is in agreement with the observed cell orientation patterns in several different experimental conditions, disclosing the important role of stress fibers and inherent cell contractility in cell reorientation. HighlightsNAFs and CAFs show very different reorientation upon compression in 3D.NAFs treated with TGF‐&bgr; and of high contractility behave like CAFs.The difference between NAFs and CAFs is due to different cell contractility.A theoretical model based on minimum energy principle is established.The model results correctly predict the behavior of NAFs and CAFs.

A Study of Small Molecule Absorption in Polydimethylsiloxane

While polydimethylsiloxane (PDMS) has proven to be a popular material in the construction of microfluidic devices, the polymer network structure of PDMS makes it permeable to some molecules. This feature can be problematic for applications where thin PDMS walls are used to separate two different molecular streams or cell populations. Therefore, a better understanding of factors affecting small molecule absorption in PDMS is important for better design and operation of microfluidic devices.We report on studies of various factors in the absorption of fluorescent dyes in PDMS. Results show significant effects of thermal aging of PDMS on the absorption of the hydrophobic molecule Nile Red. In addition, the short-term hydrophobic recovery of the PDMS devices after being plasma bonded to a glass slide was investigated. Furthermore, we have tested the effects of aspiration and flushing, which show that the wetting property (hydrophilic or hydrophobic) of the flushing solvents can significantly affect the absorption of the Nile Red dye. Finally, parallel tests of PDMS absorption of fluorescein isothiocyanate (FITC) were conducted with no absorption observed.Copyright

Ultrasensitive Graphene Optoelectronic Probes for Recording Electrical Activities of Individual Synapses

The complex neuronal circuitry connected by submicron synapses in our brain calls for technologies that can map neural networks with ultrahigh spatiotemporal resolution to decipher the underlying mechanisms for multiple aspects of neuroscience. Here we show that, through combining graphene transistor arrays with scanning photocurrent microscopy, we can detect the electrical activities of individual synapses of primary hippocampal neurons. Through measuring the local conductance change of graphene optoelectronic probes directly underneath neuronal processes, we are able to estimate millivolt extracellular potential variations of individual synapses during depolarization. The ultrafast nature of graphene photocurrent response allows for decoding of activity patterns of individual synapses with a sub-millisecond temporal resolution. This new neurotechnology provides promising potentials for recording of electrophysiological outcomes of individual synapses in neural networks.