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1.  Engineering microscale cellular niches for three-dimensional multicellular co-cultures 
Lab on a chip  2009;9(12):1740-1748.
Modeling the in vivo microenvironment typically involves placing cells in a three-dimensional (3D) extracellular matrix (ECM) in physiologically relevant context with respect to other cells. The mechanical and chemical features of 3D microenvironments play important roles in tissue engineering, tumor growth and metastasis, and in defining stem cell niches, and it is increasingly recognized that cells behave much differently when surrounded by a 3D ECM than when anchored to a 2D substrate. To create microenvironments that more closely mimic in vivo settings, here we describe a novel microfluidic device that allows multiple discrete constructs of 3D cell-laden hydrogels to be patterned in a sequence of simple steps. The microfluidic platform allows for real time imaging of the interactions between multiple cell types exposed to both autocrine and paracrine signaling molecules, all within a 3D ECM environment. Detailed modeling determined that surface tension, hydrophobic interactions, and spatial geometry were important factors in containing the gels within distinct separate channels during the filling process. This allowed us to pattern multiple gel types side-by-side and pattern 3D gels spatially with tight dimensional control. Cells embedded in gels could be patterned by culturing MDA-MB-231 metastatic breast cancer cells and RAW 264.1 macrophage cells within distinct collagen type I and Matrigel ECM environments, respectively. Over a 7 day culture experiment, RAW cells invaded into neighboring gels containing MDA-MB-231 cells, but not into gels lacking cells. These studies demonstrate the versatility and potential of this new microfluidic platform to engineer 3D microscale architectures to investigate cell-cell and cell-matrix interactions.
PMCID: PMC3758562  PMID: 19495458
2.  Monitoring the Differentiation and Migration Patterns of Neural Cells Derived from Human Embryonic Stem Cells Using a Microfluidic Culture System 
Molecules and Cells  2014;37(6):497-502.
Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells.
PMCID: PMC4086344  PMID: 24938227
axons; human embryonic stem cells; microfluidics; migration; neural differentiation
3.  Adipocyte Induction of Preadipocyte Differentiation in a Gradient Chamber 
Tissue Engineering. Part C, Methods  2012;18(12):958-967.
Adipose tissue expansion involves enlargement of mature adipocytes and the formation of new adipocytes through the differentiation of locally resident preadipocytes. Factors released by the enlarged adipocytes are potential cues that induce the differentiation of the preadipocytes. Currently, there are limited options to investigate these cues in isolation from confounding systemic influences. A gradient generating microfluidic channel-based cell culture system was designed to enable solution patterning, while supporting long-term culture and differentiation of preadipocytes. Solution patterning was confirmed by selectively staining a fraction of uniformly seeded preadipocytes. An adipogenic cocktail gradient was used to induce the differentiation of a fraction of uniformly seeded preadipocytes and establish a spatially defined coculture of adipocytes and preadipocytes. Varying the adipogenic cocktail gradient generated cocultures of preadipocytes and adipocytes with different compositions. Transient application of the cocktail gradient, followed by basal medium treatment showed a biphasic induction of differentiation. The two phases of differentiation correlated with a spatial gradient in adipocyte size. Our results provide in vitro data supporting the size-dependent release of preadipocyte differentiation factors by enlarged adipocytes. Prospectively, the coculture system developed in this study could facilitate controlled, yet physiologically meaningful studies on paracrine interactions between adipocytes and preadipocytes during adipose tissue development.
PMCID: PMC3491637  PMID: 22651694
4.  Quantitative Analysis of Axonal Transport by Using Compartmentalized and Surface Micropatterned Culture of Neurons 
ACS Chemical Neuroscience  2012;3(6):433-438.
Mitochondria, synaptic vesicles, and other cytoplasmic constituents have to travel long distance along the axons from cell bodies to nerve terminals. Interruption of this axonal transport may contribute to many neurodegenerative diseases including Alzheimer’s disease (AD). It has been recently shown that exposure of cultured neurons to β-amyloid (Aβ) resulted in severe impairment of mitochondrial transport. This Letter describes an integrated microfluidic platform that establishes surface patterned and compartmentalized culture of neurons for studying the effect of Aβ on mitochondria trafficking in full length of axons. We have successfully quantified the trafficking of fluorescently labeled mitochondria in distal and proximal axons using image processing. Selective treatment of Aβ in the somal or axonal compartments resulted in considerable decrease in mitochondria movement in a location dependent manner such that mitochondria trafficking slowed down more significantly proximal to the location of Aβ exposure. Furthermore, this result suggests a promising application of microfluidic technology for investigating the dysfunction of axonal transport related to neurodegenerative diseases.
PMCID: PMC3643179  PMID: 24358503
β-Amyloid; axonal transport; mitochondrial trafficking; microfludics; surface micropatterning; image processing
5.  Fluorescent and photo-oxidizing TimeSTAMP tags track protein fates in light and electron microscopy 
Nature neuroscience  2012;15(12):1742-1751.
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.
PMCID: PMC3509268  PMID: 23103964
6.  Yeast G-proteins mediate directional sensing and polarization behaviors in response to changes in pheromone gradient direction 
Molecular Biology of the Cell  2013;24(4):521-534.
G-proteins, heterotrimeric and Cdc42, modulate in a ligand-dependent fashion two fundamental cell polarity behaviors (projection bending growth and second projection formation) in response to gradient directional change.
Yeast cells polarize by projecting up mating pheromone gradients, a classic cell polarity behavior. However, these chemical gradients may shift direction. We examine how yeast cells sense and respond to a 180o switch in the direction of microfluidically generated pheromone gradients. We identify two behaviors: at low concentrations of α-factor, the initial projection grows by bending, whereas at high concentrations, cells form a second projection toward the new source. Mutations that increase heterotrimeric G-protein activity expand the bending-growth morphology to high concentrations; mutations that increase Cdc42 activity result in second projections at low concentrations. Gradient-sensing projection bending requires interaction between Gβγ and Cdc24, whereas gradient-nonsensing projection extension is stimulated by Bem1 and hyperactivated Cdc42. Of interest, a mutation in Gα affects both bending and extension. Finally, we find a genetic perturbation that exhibits both behaviors. Overexpression of the formin Bni1, a component of the polarisome, makes both bending-growth projections and second projections at low and high α-factor concentrations, suggesting a role for Bni1 downstream of the heterotrimeric G-protein and Cdc42 during gradient sensing and response. Thus we demonstrate that G-proteins modulate in a ligand-dependent manner two fundamental cell-polarity behaviors in response to gradient directional change.
PMCID: PMC3571874  PMID: 23242998
7.  Axonal mRNA in uninjured and regenerating cortical mammalian axons 
Using a novel microfluidic chamber that allows the isolation of axons without contamination by non-axonal material, we have for the first time purified mRNA from naïve, matured CNS axons, and identified the presence of >300 mRNA transcripts. We demonstrate that the transcripts are axonal in nature, and that many of the transcripts present in uninjured CNS axons overlap with those previously identified in PNS injury-conditioned DRG axons. The axonal transcripts detected in matured cortical axons are enriched for protein translational machinery, transport, cytoskeletal components, and mitochondrial maintenance. We next investigated how the axonal mRNA pool changes after axotomy, revealing that numerous gene transcripts related to intracellular transport, mitochondria and the cytoskeleton show decreased localization two days after injury. In contrast, gene transcripts related to axonal targeting and synaptic function show increased localization in regenerating cortical axons, suggesting that there is an increased capacity for axonal outgrowth and targeting, and increased support for synapse formation and presynaptic function in regenerating CNS axons after injury. Our data demonstrate that CNS axons contain many mRNA species of diverse functions, and suggest that, like invertebrate and PNS axons, CNS axons synthesize proteins locally, maintaining a degree of autonomy from the cell body.
PMCID: PMC3632375  PMID: 19369540
Axon; Axon Terminal; Axoplasm; Axotomy; Presynaptic; Presynaptic mechanisms
8.  Reprogramming axonal behavior by axon-specific viral transduction 
Gene therapy  2012;19(9):947-955.
The treatment of axonal disorders, such as diseases associated with axonal injury and degeneration, is limited by the inability to directly target therapeutic protein expression to injured axons. Current gene therapy approaches rely on infection and transcription of viral genes in the cell body. Here we describe an approach to target gene expression selectively to axons. Using a genetically engineered mouse containing epitope-labeled ribosomes, we find that neurons in adult animals contain ribosomes in distal axons. To use axonal ribosomes to alter local protein expression, we utilized a Sindbis virus containing an RNA genome that has been modified so that it can be directly used as a template for translation. Selective application of this virus to axons leads to local translation of heterologous proteins. Furthermore, we demonstrate that selective axonal protein expression can be used to modify axonal signaling in cultured neurons, enabling axons to grow over inhibitory substrates typically encountered following axonal injury. We also show that this viral approach also can be used to achieve heterologous expression in axons of living animals, indicating that this approach can be used to alter the axonal proteome in vivo. Together, these data identify a novel strategy to manipulate protein expression in axons, and provides a novel approach for using gene therapies for disorders of axonal function.
PMCID: PMC3426632  PMID: 22278412
viral vector gene transfer; Sindbis; axon regeneration
9.  Chemical genetic-mediated spatial regulation of protein expression in neurons reveals an axonal function for WldS 
Chemistry & biology  2012;19(2):179-187.
The degeneration of axons is the underlying pathological process of several neurological disorders. The Wallerian degeneration (WldS) slow protein, which is primarily nuclear, markedly inhibits axonal degeneration. Contradictory models have been proposed to explain its mechanism, including a role in the nucleus where it affects gene transcription, and roles outside the nucleus where it regulates unknown effectors. To determine which pool of WldS accounts for its axon protective effects, we developed a strategy to control the spatial expression of proteins within neurons. This strategy couples a chemical genetic method to control protein stability with microfluidic culturing. Using neurons that are selectively deficient in WldS in axons, we show that the axonal pool of WldS is necessary for protection from axon degeneration. These results implicate an axonal pathway regulated by WldS that controls axon degeneration.
PMCID: PMC3292772  PMID: 22365601
10.  Dielectrophoresis switching with vertical sidewall electrodes for microfluidic flow cytometry 
Lab on a chip  2007;7(9):1114-1120.
A novel dielectrophoresis switching with vertical electrodes in the sidewall of microchannels for multiplexed switching of objects has been designed, fabricated and tested. With appropriate electrode design, lateral DEP force can be generated so that one can dynamically position particulates along the width of the channel. A set of interdigitated electrodes in the sidewall of the microchannels is used for the generation of non-uniform electrical fields to generate negative DEP forces that repel beads/cells from the sidewalls. A countering DEP force is generated from another set of electrodes patterned on the opposing sidewall. These lateral negative DEP forces can be adjusted by the voltage and frequency applied. By manipulating the coupled DEP forces, the particles flowing through the microchannel can be positioned at different equilibrium points along the width direction and continue to flow into different outlet channels. Experimental results for switching biological cells and polystyrene microbeads to multiple outlets (up to 5) have been achieved. This novel particle switching technique can be integrated with other particle detection components to enable microfluidic flow cytometry systems.
PMCID: PMC3437924  PMID: 17713608
11.  HDAC6 Inhibitor Blocks Amyloid Beta-Induced Impairment of Mitochondrial Transport in Hippocampal Neurons 
PLoS ONE  2012;7(8):e42983.
Even though the disruption of axonal transport is an important pathophysiological factor in neurodegenerative diseases including Alzheimer's disease (AD), the relationship between disruption of axonal transport and pathogenesis of AD is poorly understood. Considering that α-tubulin acetylation is an important factor in axonal transport and that Aβ impairs mitochondrial axonal transport, we manipulated the level of α-tubulin acetylation in hippocampal neurons with Aβ cultured in a microfluidic system and examined its effect on mitochondrial axonal transport. We found that inhibiting histone deacetylase 6 (HDAC6), which deacetylates α-tubulin, significantly restored the velocity and motility of the mitochondria in both anterograde and retrograde axonal transports, which would be otherwise compromised by Aβ. The inhibition of HDAC6 also recovered the length of the mitochondria that had been shortened by Aβ to a normal level. These results suggest that the inhibition of HDAC6 significantly rescues hippocampal neurons from Aβ-induced impairment of mitochondrial axonal transport as well as mitochondrial length. The results presented in this paper identify HDAC6 as an important regulator of mitochondrial transport as well as elongation and, thus, a potential target whose pharmacological inhibition contributes to improving mitochondrial dynamics in Aβ treated neurons.
PMCID: PMC3425572  PMID: 22937007
12.  Examination of Axonal Injury and Regeneration in Microfluidic Neuronal Culture Using Pulsed Laser Microbeam Dissection 
Lab on a Chip  2010;10(16):2083-2092.
We describe the integrated use of pulsed laser microbeams and microfluidic cell culture to examine the dynamics of axonal injury and regeneration in vitro. Microfabrication methods are used to place high purity dissociated central nervous system neurons in specific regions that allow the axons to interact with permissive and inhibitory substrates. Acute injury to neuron bundles is produced via the delivery of single 180 ps duration, λ=532 nm laser pulses. Laser pulse energies of 400 nJ and 800 nJ produce partial and complete transection of the axons, respectively, resulting in elliptical lesions 25 μm and 50 μm in size. The dynamics of the resulting degeneration and regrowth of proximal and distal axonal segments are examined for up to 8 h using time-lapse microscopy. We find the proximal and distal dieback distances from the site of laser microbeam irradiation to be roughly equal for both partial and complete transection of the axons. In addition, distinct growth cones emerge from the proximal neurite segments within 1–2 h post-injury, followed by a uniform front of regenerating axons that originate from the proximal segment and traverse the injury site within 8 h. We also examine the use of EGTA to chelate the extracellular calcium and potentially reduce the severity of the axonal degeneration following injury. While we find the addition of EGTA to reduce the severity of the initial dieback, it also hampers neurite repair and interfere with the formation of neuronal growth cones to traverse the injury site. This integrated use of laser microbeam dissection within a microfluidic cell culture system to produce precise zones of neuronal injury shows potential for high-throughput screening of agents to promote neuronal regeneration.
PMCID: PMC3380453  PMID: 20532390
13.  Recreating the Perivascular Niche Ex Vivo Using a Microfluidic Approach 
Biotechnology and Bioengineering  2010;107(6):1020-1028.
Stem cell niches are composed of numerous microenvironmental features, including soluble and insoluble factors, cues from other cells, and the extracellular matrix (ECM), which collectively serve to maintain stem cell quiescence and promote their ability to support tissue homeostasis. A hallmark of many adult stem cell niches is their proximity to the vasculature in vivo, a feature common to neural stem cells, mesenchymal stem cells (MSCs) from bone marrow and adipose tissue, hematopoietic stem cells, and many tumor stem cells. In this study, we describe a novel 3D microfluidic device (MFD) as a model system in which to study the molecular regulation of perivascular stem cell niches. Endothelial cells (ECs) suspended within 3D fibrin gels patterned in the device adjacent to stromal cells (either fibroblasts or bone marrow-derived mesenchymal stem cells) executed a morphogenetic process akin to vasculogenesis, forming a primitive vascular plexus and maturing into a robust capillary network with hollow well-defined lumens. Both MSCs and fibroblasts formed pericytic associations with the ECs, but promoted capillary morphogenesis with distinct kinetics. Biochemical assays within the niche revealed that the perivascular association of MSCs required interaction between their α6β1 integrin receptor and EC-deposited laminin. These studies demonstrate the potential of this physiologically relevant ex vivo model system to study how proximity to blood vessels may influence stem cell multipotency.
PMCID: PMC3367510  PMID: 20672286
Stem cell niche; capillary; mesenchymal stem cell; pericyte; integrin; 3D culture
14.  Two Distinct Filopodia Populations at the Growth Cone Allow to Sense Nanotopographical Extracellular Matrix Cues to Guide Neurite Outgrowth 
PLoS ONE  2010;5(12):e15966.
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.
Methodology/Principal Findings
We show that, when the ECM protein laminin is presented on a line pattern with nanometric size features, it leads to orientation of neurite outgrowth along the line pattern. This is also coupled with a robust increase in neurite length. The sensing mechanism that allows neurite orientation occurs through a highly stereotypical growth cone behavior involving two filopodia populations. Non-aligned filopodia on the distal part of the growth cone scan the pattern in a lateral back and forth motion and are highly unstable. Filopodia at the growth cone tip align with the line substrate, are stabilized by an F-actin rich cytoskeleton and enable steady neurite extension. This stabilization event most likely occurs by integration of signals emanating from non-aligned and aligned filopodia which sense different extent of adhesion surface on the line pattern. In contrast, on the 2D substrate only unstable filopodia are observed at the growth cone, leading to frequent neurite collapse events and less efficient outgrowth.
We propose that a constant crosstalk between both filopodia populations allows stochastic sensing of nanotopographical ECM cues, leading to oriented and steady neurite outgrowth. Our work provides insight in how neuronal growth cones can sense geometric ECM cues. This has not been accessible previously using routine 2D culture systems.
PMCID: PMC3012734  PMID: 21209862
15.  Microfluidic Multicompartment Device for Neuroscience Research† 
This paper describes and characterizes a novel microfabricated neuronal culture device. This device combines microfabrication, microfluidic, and surface micropatterning techniques to create a multicompartment neuronal culturing device that can be used in a number of neuroscience research applications. The device is fabricated in poly(dimethylsiloxane), PDMS, using soft lithography techniques. The PDMS device is placed on a tissue culture dish (polystyrene) or glass substrate, forming two compartments with volumes of less than 2 μL each. These two compartments are separated by a physical barrier in which a number of micron-size grooves are embedded to allow growth of neurites across the compartments while maintaining fluidic isolation. Cells are plated into the somal (cell body) compartment, and after 3–4 days, neurites extend into the neuritic compartment via the grooves. Viability of the neurons in the devices is between 50 and 70% after 7 days in culture; this is slightly lower than but comparable to values for a control grown on tissue culture dishes. Healthy neuron morphology is evident in both the devices and controls. We demonstrate the ability to use hydrostatic pressure to isolate insults to one compartment and, thus, expose localized areas of neurons to insults applied in soluble form. Due to the high resistance of the microgrooves for fluid transport, insults are contained in the neuritic compartment without appreciable leakage into the somal compartment for over 15 h. Finally, we demonstrate the use of polylysine patterning in combination with the microfabricated device to facilitate identification and visualization of neurons. The ability to direct sites of neuronal attachment and orientation of neurite outgrowth by micropatterning techniques, combined with fluidically isolated compartments within the culture area, offers significant advantages over standard open culture methods and other conventional methods for manipulating distinct neuronal microenvironments.
PMCID: PMC2923462  PMID: 20725530
16.  Pre-synaptic regulation of astroglial excitatory neurotransmitter transporter GLT1 
Neuron  2009;61(6):880-894.
The neuron-astrocyte synaptic complex is a fundamental operational unit of the nervous system. Astroglia play a central role in the regulation of synaptic glutamate, via neurotransmitter transport by GLT1/EAAT2. The astroglial mechanisms underlying this essential neuron-glial communication are not known. Here we show that presynaptic terminals are sufficient and necessary for GLT1/EAAT2 transcriptional activation and have identified the molecular pathway that regulates astroglial responses to presynaptic input. Presynaptic terminals regulate astroglial GLT1/EAAT2 via kappa B-motif binding phosphoprotein (KBBP), the mouse homologue of human heterogeneous nuclear ribonucleoprotein K (hnRNP K), which binds to an essential element of GLT1/EAAT2 promoter. This neuron-stimulated factor is required for GLT1/EATT2 transcriptional activation and is responsible for astroglial alterations in neural injury. Denervation of neuron-astrocyte signaling in vivo, by acute corticospinal tract transection, ricin-induced motor neuron death, or chronic neurodegeneration in amyotrophic lateral sclerosis (ALS) all result in reduced astroglial KBBP expression and transcriptional dysfunction of astroglial transporter expression. Our studies indicate that presynaptic elements dynamically coordinate normal astroglial function and also provide a fundamental signaling mechanism by which altered neuronal function and injury leads to dysregulated astroglia in CNS disease.
PMCID: PMC2743171  PMID: 19323997
18.  Robust Spatial Sensing of Mating Pheromone Gradients by Yeast Cells 
PLoS ONE  2008;3(12):e3865.
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.
PMCID: PMC2586657  PMID: 19052645
19.  A Microfluidic Chamber for Analysis of Neuron-to-Cell Spread and Axonal Transport of an Alpha-Herpesvirus 
PLoS ONE  2008;3(6):e2382.
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.
PMCID: PMC2426917  PMID: 18560518
20.  A microfluidic culture platform for CNS axonal injury, regeneration and transport 
Nature methods  2005;2(8):599-605.
Investigation of axonal biology in the central nervous system (CNS) is hindered by a lack of an appropriate in vitro method to probe axons independently from cell bodies. Here we describe a microfluidic culture platform that polarizes the growth of CNS axons into a fluidically isolated environment without the use of targeting neurotrophins. In addition to its compatibility with live cell imaging, the platform can be used to (i) isolate CNS axons without somata or dendrites, facilitating biochemical analyses of pure axonal fractions and (ii) localize physical and chemical treatments to axons or somata. We report the first evidence that presynaptic (Syp) but not postsynaptic (Camk2a) mRNA is localized to developing rat cortical and hippocampal axons. The platform also serves as a straightforward, reproducible method to model CNS axonal injury and regeneration. The results presented here demonstrate several experimental paradigms using the microfluidic platform, which can greatly facilitate future studies in axonal biology.
PMCID: PMC1558906  PMID: 16094385
21.  A hybrid microfluidic-vacuum device for direct interfacing with conventional cell culture methods 
BMC Biotechnology  2007;7:60.
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.
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.
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.
PMCID: PMC2071914  PMID: 17883868
22.  Reconstituting ring-rafts in bud-mimicking topography of model membranes 
Nature Communications  2014;5:4507.
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.
Budding processes on cell membranes involve distortions of surface geometry, driven by the sorting of membrane components. Here, the authors model a budding process and observe spontaneous reordering of materials in the areas of high curvature, especially around bud-necks, producing ring-raft type structures.
PMCID: PMC4124864  PMID: 25058275

Results 1-22 (22)