A Microfluidic Chamber for Analysis of Neuron-to-Cell Spread and Axonal Transport of an Alpha-Herpesvirus

Alpha-herpesviruses, including herpes simplex virus and pseudorabies virus (PRV), infect the peripheral nervous system (PNS) of their hosts. Here, we describe an in vitro method for studying neuron-to-cell spread of infection as well as viral transport in axons. The method centers on a novel microfluidic chamber system that directs growth of axons into a fluidically isolated environment. The system uses substantially smaller amounts of virus inoculum and media than previous chamber systems and yet offers the flexibility of applying multiple virology and cell biology assays including live-cell optical imaging. Using PRV infection of cultured PNS neurons, we demonstrate that the microfluidic chamber recapitulates all known facets of neuron-to-cell spread demonstrated in animals and other compartmented cell systems

Robust Spatial Sensing of Mating Pheromone Gradients by Yeast Cells

Projecting or moving up a chemical gradient is a universal behavior of living organisms. We tested the ability of S. cerevisiae a-cells to sense and respond to spatial gradients of the mating pheromone α-factor produced in a microfluidics chamber; the focus was on bar1Δ strains, which do not degrade the pheromone input. The yeast cells exhibited good accuracy with the mating projection typically pointing in the correct direction up the gradient (∼80% under certain conditions), excellent sensitivity to shallow gradients, and broad dynamic range so that gradient-sensing was relatively robust over a 1000-fold range of average α-factor concentrations. Optimal directional sensing occurred at lower concentrations (5 nM) close to the Kd of the receptor and with steeper gradient slopes. Pheromone supersensitive mutations (sst2Δ and ste2300Δ) that disrupt the down-regulation of heterotrimeric G-protein signaling caused defects in both sensing and response. Interestingly, yeast cells employed adaptive mechanisms to increase the robustness of the process including filamentous growth (i.e. directional distal budding) up the gradient at low pheromone concentrations, bending of the projection to be more aligned with the gradient, and forming a more accurate second projection when the first projection was in the wrong direction. Finally, the cells were able to amplify a shallow external gradient signal of α-factor to produce a dramatic polarization of signaling proteins at the front of the cell. Mathematical modeling revealed insights into the mechanism of this amplification and how the supersensitive mutants can disrupt accurate polarization. Together, these data help to specify and elucidate the abilities of yeast cells to sense and respond to spatial gradients of pheromone

Two Distinct Filopodia Populations at the Growth Cone Allow to Sense Nanotopographical Extracellular Matrix Cues to Guide Neurite Outgrowth

The process of neurite outgrowth is the initial step in producing the neuronal processes that wire the brain. Current models about neurite outgrowth have been derived from classic two-dimensional (2D) cell culture systems, which do not recapitulate the topographical cues that are present in the extracellular matrix (ECM) in vivo. Here, we explore how ECM nanotopography influences neurite outgrowth

A hybrid microfluidic-vacuum device for direct interfacing with conventional cell culture methods

<p>Abstract</p> <p>Background</p> <p>Microfluidics is an enabling technology with a number of advantages over traditional tissue culture methods when precise control of cellular microenvironment is required. However, there are a number of practical and technical limitations that impede wider implementation in routine biomedical research. Specialized equipment and protocols required for fabrication and setting up microfluidic experiments present hurdles for routine use by most biology laboratories.</p> <p>Results</p> <p>We have developed and validated a novel microfluidic device that can directly interface with conventional tissue culture methods to generate and maintain controlled soluble environments in a Petri dish. It incorporates separate sets of fluidic channels and vacuum networks on a single device that allows reversible application of microfluidic gradients onto wet cell culture surfaces. Stable, precise concentration gradients of soluble factors were generated using simple microfluidic channels that were attached to a perfusion system. We successfully demonstrated real-time optical live/dead cell imaging of neural stem cells exposed to a hydrogen peroxide gradient and chemotaxis of metastatic breast cancer cells in a growth factor gradient.</p> <p>Conclusion</p> <p>This paper describes the design and application of a versatile microfluidic device that can directly interface with conventional cell culture methods. This platform provides a simple yet versatile tool for incorporating the advantages of a microfluidic approach to biological assays without changing established tissue culture protocols.</p

Fluorescent and photo-oxidizing TimeSTAMP tags track protein fates in light and electron microscopy.

Protein synthesis is highly regulated throughout nervous system development, plasticity and regeneration. However, tracking the distributions of specific new protein species has not been possible in living neurons or at the ultrastructural level. Previously we created TimeSTAMP epitope tags, drug-controlled tags for immunohistochemical detection of specific new proteins synthesized at defined times. Here we extend TimeSTAMP to label new protein copies by fluorescence or photo-oxidation. Live microscopy of a fluorescent TimeSTAMP tag reveals that copies of the synaptic protein PSD95 are synthesized in response to local activation of growth factor and neurotransmitter receptors, and preferentially localize to stimulated synapses in rat neurons. Electron microscopy of a photo-oxidizing TimeSTAMP tag reveals new PSD95 at developing dendritic structures of immature neurons and at synapses in differentiated neurons. These results demonstrate the versatility of the TimeSTAMP approach for visualizing newly synthesized proteins in neurons

Microvascularized tumor organoids-on-chips: advancing preclinical drug screening with pathophysiological relevance

Recent developments of organoids engineering and organ-on-a-chip microfluidic technologies have enabled the recapitulation of the major functions and architectures of microscale human tissue, including tumor pathophysiology. Nevertheless, there remain challenges in recapitulating the complexity and heterogeneity of tumor microenvironment. The integration of these engineering technologies suggests a potential strategy to overcome the limitations in reconstituting the perfusable microvascular system of large-scale tumors conserving their key functional features. Here, we review the recent progress of in vitro tumor-on-a-chip microfluidic technologies, focusing on the reconstruction of microvascularized organoid models to suggest a better platform for personalized cancer medicine.This work was supported by the National Institutes of Health Directors New Innovator Award 1DP2HL142050 (Y.K.) and the National Science Foundation under CAREER CMMI 1653006 (Y.K.)

Reconstituting ring-rafts in bud-mimicking topography of model membranes.

During vesicular trafficking and release of enveloped viruses, the budding and fission processes dynamically remodel the donor cell membrane in a protein- or a lipid-mediated manner. In all cases, in addition to the generation or relief of the curvature stress, the buds recruit specific lipids and proteins from the donor membrane through restricted diffusion for the development of a ring-type raft domain of closed topology. Here, by reconstituting the bud topography in a model membrane, we demonstrate the preferential localization of cholesterol- and sphingomyelin-enriched microdomains in the collar band of the bud-neck interfaced with the donor membrane. The geometrical approach to the recapitulation of the dynamic membrane reorganization, resulting from the local radii of curvatures from nanometre-to-micrometre scales, offers important clues for understanding the active roles of the bud topography in the sorting and migration machinery of key signalling proteins involved in membrane budding

Generation of dynamic temporal and spatial concentration gradients using microfluidic devices

This paper describes a microfluidic approach to generate dynamic temporal and spatial concentration gradients using a single microfluidic device. Compared to a previously described method that produced a single fixed gradient shape for each device, this approach combines a simple mixer module with gradient generating network to control and manipulate a number of different gradient shapes. The gradient profile is determined by the configuration of fluidic inputs as well as the design of microchannel network. By controlling the relative flow rates of the fluidic inputs using separate syringe pumps, the resulting composition of the inlets that feed the gradient generator can be dynamically controlled to generate temporal and spatial gradients. To demonstrate the concept and illustrate this approach, examples of devices that generate (1) temporal gradients of homogeneous concentrations, (2) linear gradients with dynamically controlled slope, baseline, and direction, and (3) nonlinear gradients with controlled nonlinearity are shown and their limitations are described.NSF (Grant No. DBI-0138055) U.S. Public Health Service-National Institutes of Health General Medical Science Grant (GM-6605

Optimal diameter reduction ratio of acinar airways in human lungs

In the airway network of a human lung, the airway diameter gradually decreases through multiple branching. The diameter reduction ratio of the conducting airways that transport gases without gas exchange is 0.79, but this reduction ratio changes to 0.94 in acinar airways beyond transitional bronchioles. While the reduction in the conducting airways was previously rationalized on the basis of Murray's law, our understanding of the design principle behind the acinar airways has been far from clear. Here we elucidate that the change in gas transfer mode is responsible for the transition in the diameter reduction ratio. The oxygen transfer rate per unit surface area is maximized at the observed geometry of acinar airways, which suggests the minimum cost for the construction and maintenance of the acinar airways. The results revitalize and extend the framework of Murray's law over an entire human lung

Patterned cell culture inside microfluidic devices

This paper describes a simple plasma-based dry etching method that enables patterned cell culture inside microfluidic devices by allowing patterning, fluidic bonding and sterilization steps to be carried out in a single step. This plasma-based dry etching method was used to pattern celladhesive and non-adhesive areas on the glass and polystyrene substrates. The patterned substrate was used for selective attachment and growth of human umbilical vein endothelial cells, MDAMB- 231 human breast cancer cells, NIH 3T3 mouse fibroblasts, and primary rat cortical neurons. Finally, we have successfully combined the dry-patterned substrate with a microfluidic device. Patterned primary rat neurons were maintained for up to 6 days inside the microfluidic devices and the neurons somas and processes were confined to the cell-adhesive region. The method developed in this work offers a convenient way of micropatterning biomaterials for selective attachment of cells on the substrates, and enables culturing of patterned cells inside microfluidic devices for a number of biological research applications where cells need to be exposed to wellcontrolled fluidic microenvironment.The Institute for Brain Aging and Dementia thanks NIA (AG17765