62 research outputs found
A simple approach for the fabrication of 3D microelectrodes for impedimetric sensing
In this paper, we present a very simple method to fabricate three-dimensional (3D) microelectrodes integrated with microfluidic devices. We form the electrodes by etching a microwire placed across a microchannel. For precise control of the electrode spacing, we employ a hydrodynamic focusing microfluidic device and control the width of the etching solution stream. The focused widths of the etchant solution and the etching time determine the gap formed between the electrodes. Using the same microfluidic device, we can fabricate integrated 3D electrodes with different electrode gaps. We have demonstrated the functionality of these electrodes using an impedimetric particle counting setup. Using 3D microelectrodes with a diameter of 25 Όm, we have detected 6 Όm-diameter polystyrene beads in a buffer solution as well as erythrocytes in a PBS solution. We study the effect of electrode spacing on the signal-to-noise ratio of the impedance signal and we demonstrate that the smaller the electrode spacing the higher the signal obtained from a single microparticle. The sample stream is introduced to the system using the same hydrodynamic focusing device, which ensures the alignment of the sample in between the electrodes. Utilising a 3D hydrodynamic focusing approach, we force all the particles to go through the sensing region of the electrodes. This fabrication scheme not only provides a very low-cost and easy method for rapid prototyping, but which can also be used for applications requiring 3D electric field focused through a narrow section of the microchannel. © 2015 IOP Publishing Ltd
Rapid fabrication of microfluidic PDMS devices from reusable PDMS molds using laser ablation
The conventional fabrication methods for microfluidic devices require cleanroom processes that are costly and time-consuming. We present a novel, facile, and low-cost method for rapid fabrication of polydimethylsiloxane (PDMS) molds and devices. The method consists of three main fabrication steps: female mold (FM), male mold (MM), and chip fabrication. We use a CO2 laser cutter to pattern a thin, spin-coated PDMS layer for FM fabrication. We then obtain reusable PDMS MM from the FM using PDMS/PDMS casting. Finally, a second casting step is used to replicate PDMS devices from the MM. Demolding of one PDMS layer from another is carried out without any potentially hazardous chemical surface treatment. We have successfully demonstrated that this novel method allows fabrication of microfluidic molds and devices with precise dimensions (thickness, width, length) using a single material, PDMS, which is very common across microfluidic laboratories. The whole process, from idea to device testing, can be completed in 1.5 h in a standard laboratory. © 2016 IOP Publishing Ltd
Mutant induced pluripotent stem cell lines recapitulate aspects of TDP-43 proteinopathies and reveal cell-specific vulnerability
Transactive response DNA-binding (TDP-43) protein is the dominant disease protein in amyotrophic lateral sclerosis (ALS) and a subgroup of frontotemporal lobar degeneration (FTLD-TDP). Identification of mutations in the gene encoding TDP-43 (TARDBP) in familial ALS confirms a mechanistic link between misaccumulation of TDP-43 and neurodegeneration and provides an opportunity to study TDP-43 proteinopathies in human neurons generated from patient fibroblasts by using induced pluripotent stem cells (iPSCs). Here, we report the generation of iPSCs that carry the TDP-43 M337V mutation and their differentiation into neurons and functional motor neurons. Mutant neurons had elevated levels of soluble and detergent-resistant TDP-43 protein, decreased survival in longitudinal studies, and increased vulnerability to antagonism of the PI3K pathway. We conclude that expression of physiological levels of TDP-43 in human neurons is sufficient to reveal a mutation-specific cell-autonomous phenotype and strongly supports this approach for the study of disease mechanisms and for drug screening
Comment on "Drug Screening for ALS Using Patient-Specific Induced Pluripotent Stem Cells"
Egawa et al. recently showed the value of patient-specific induced pluripotent stem cells (iPSCs) for modeling amyotrophic lateral sclerosis in vitro. Their study and our work highlight the need for complementary assays to detect small, but potentially important, phenotypic differences between control iPSC lines and those carrying disease mutations
Induction of Olig2+ Precursors by FGF Involves BMP Signalling Blockade at the Smad Level
During normal development oligodendrocyte precursors (OPCs) are generated in the ventral spinal cord in response to Sonic hedgehog (Shh) signalling. There is also a second, late wave of oligodendrogenesis in the dorsal spinal cord independent of Shh activity. Two signalling pathways, controlled by bone morphogenetic protein and fibroblast growth factor (FGF), are active players in dorsal spinal cord specification. In particular, BMP signalling from the roof plate has a crucial role in setting up dorsal neural identity and its inhibition is sufficient to generate OPCs both in vitro and in vivo. In contrast, FGF signalling can induce OPC production from dorsal spinal cord cultures in vitro. In this study, we examined the cross-talk between mitogen-activated protein kinase (MAPK) and BMP signalling in embryonic dorsal spinal cord cultures at the SMAD1/5/8 (SMAD1) transcription factor level, the main effectors of BMP activity. We have previously shown that FGF2 treatment of neural precursor cells (NPCs) derived from rat E14 dorsal spinal cord is sufficient to generate OPCs in vitro. Utilising the same system, we now show that FGF prevents BMP-induced nuclear localisation of SMAD1-phosphorylated at the C-terminus (C-term-pSMAD1). This nuclear exclusion of C-term-pSMAD1 is dependent on MAPK activity and correlates with OLIG2 upregulation, the obligate transcription factor for oligodendrogenesis. Furthermore, inhibition of the MAPK pathway abolishes OLIG2 expression. We also show that SMAD4, which acts as a common partner for receptor-regulated Smads including SMAD1, associates with a Smad binding site in the Olig2 promoter and dissociates from it upon differentiation. Taken together, these results suggest that FGF can promote OPC generation from embryonic NPCs by counteracting BMP signalling at the Smad1 transcription factor level and that Smad-containing transcriptional complexes may be involved in direct regulation of the Olig2 promoter
Efficient derivation of NPCs, spinal motor neurons and midbrain dopaminergic neurons from hESCs at 3% oxygen
This protocol has been designed to generate neural precursor cells (NPCs) from human embryonic stem cells (hESCs) using a physiological oxygen (O(2)) level of 3% and chemically defined conditions. The first stage involves suspension culture of hESC colonies at 3% O(2), where they acquire a neuroepithelial identity over two weeks. This timescale is comparable to that at 20% O(2), but survival is enhanced. Sequential application of retinoic acid (RA) and purmorphamine (PM), from day 14 to 28, directs differentiation towards spinal motor neurons. Alternatively, addition of FGF-8 and PM generates midbrain dopaminergic neurons. OLIG2 induction in motor neuron precursors is 2-fold greater than at 20% O(2), whereas EN1 is 5-fold enhanced. 3% NPCs can be differentiated into all three neural lineages, and such cultures can be maintained long-term in the absence of neurotrophins. The ability to generate defined cell types at 3% O(2) should represent a significant advance for in vitro disease modelling and potentially cell-based therapies
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2D versus 3D human induced pluripotent stem cell-derived cultures for neurodegenerative disease modelling
Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and amyotrophic lateral sclerosis (ALS), affect millions of people every year and so far, there are no therapeutic cures available. Even though animal and histological models have been of great aid in understanding disease mechanisms and identifying possible therapeutic strategies, in order to find disease-modifying solutions there is still a critical need for systems that can provide more predictive and physiologically relevant results. One possible avenue is the development of patient-derived models, e.g. by reprogramming patient somatic cells into human induced pluripotent stem cells (hiPSCs), which can then be differentiated into any cell type for modelling. These systems contain key genetic information from the donors, and therefore have enormous potential as tools in the investigation of pathological mechanisms underlying disease phenotype, and progression, as well as in drug testing platforms. hiPSCs have been widely cultured in 2D systems, but in order to mimic human brain complexity, 3D models have been proposed as a more advanced alternative. This review will focus on the use of patient-derived hiPSCs to model AD, PD, HD and ALS. In brief, we will cover the available stem cells, types of 2D and 3D culture systems, existing models for neurodegenerative diseases, obstacles to model these diseases in vitro, and current perspectives in the field
Real-time quantitative monitoring of hiPSC-based model of macular degeneration on Electric Cell-substrate Impedance Sensing microelectrodes
AbstractAge-related macular degeneration (AMD) is the leading cause of blindness in the developed world. Humanized disease models are required to develop new therapies for currently incurable forms of AMD.In this work, a tissue-on-a-chip approach was developed through combining human induced pluripotent stem cells, Electric Cellâsubstrate Impedance Sensing (ECIS) and reproducible electrical wounding assays to model and quantitatively study AMD. Retinal Pigment Epithelium (RPE) cells generated from a patient with an inherited macular degeneration and from an unaffected sibling were used to test the model platform on which a reproducible electrical wounding assay was conducted to model RPE damage. First, a robust and reproducible real-time quantitative monitoring over a 25-day period demonstrated the establishment and maturation of RPE layers on the microelectrode arrays. A spatially controlled RPE layer damage that mimicked cell loss in AMD disease was then initiated. Post recovery, significant differences (P<0.01) in migration rates were found between case (8.6±0.46ÎŒm/h) and control cell lines (10.69±0.21ÎŒm/h). Quantitative data analysis suggested this was achieved due to lower cellâsubstrate adhesion in the control cell line. The ECIS cellâsubstrate adhesion parameter (α) was found to be 7.8±0.28Ω1/2cm for the case cell line and 6.5±0.15Ω1/2cm for the control. These findings were confirmed using cell adhesion biochemical assays. The developed disease model-on-a-chip is a powerful platform for translational studies with considerable potential to investigate novel therapies by enabling real-time, quantitative and reproducible patient-specific RPE cell repair studies
Calibration of the CMS hadron calorimeters using proton-proton collision data at âs = 13 TeV
Methods are presented for calibrating the hadron calorimeter system of the CMS detector at the LHC. The hadron calorimeters of the CMS experiment are sampling calorimeters of brass and scintillator, and are in the form of one central detector and two endcaps. These calorimeters cover pseudorapidities |η| < 3 and are positioned inside the solenoidal magnet. An outer calorimeter, outside the magnet coil, covers |η| < 1.26, and a steel and quartz-fiber Cherenkov forward calorimeter extends the coverage to |η| < 5.19. The initial calibration of the calorimeters was based on results from test beams, augmented with the use of radioactive sources and lasers. The calibration was improved substantially using proton-proton collision data collected at âs = 7, 8, and 13 TeV, as well as cosmic ray muon data collected during the periods when the LHC beams were not present. The present calibration is performed using the 13 TeV data collected during 2016 corresponding to an integrated luminosity of 35.9 fbâ»Âč. The intercalibration of channels exploits the approximate uniformity of energy collection over the azimuthal angle. The absolute energy scale of the central and endcap calorimeters is set using isolated charged hadrons. The energy scale for the electromagnetic portion of the forward calorimeters is set using Zâ ee data. The energy scale of the outer calorimeters has been determined with test beam data and is confirmed through data with high transverse momentum jets. In this paper, we present the details of the calibration methods and accuracy
Physiological normoxia and absence of EGF is required for the long-term propagation of anterior neural precursors from human pluripotent cells
Widespread use of human pluripotent stem cells (hPSCs) to study neuronal physiology and function is hindered by the ongoing need for specialist expertise in converting hPSCs to neural precursor cells (NPCs). Here, we describe a new methodology to generate cryo-preservable hPSC-derived NPCs that retain an anterior identity and are propagatable long-term prior to terminal differentiation, thus abrogating regular de novo neuralization. Key to achieving passagable NPCs without loss of identity is the combination of both absence of EGF and propagation in physiological levels (3%) of O2. NPCs generated in this way display a stable long-term anterior forebrain identity and importantly retain developmental competence to patterning signals. Moreover, compared to NPCs maintained at ambient O2 (21%), they exhibit enhanced uniformity and speed of functional maturation, yielding both deep and upper layer cortical excitatory neurons. These neurons display multiple attributes including the capability to form functional synapses and undergo activity-dependent gene regulation. The platform described achieves long-term maintenance of anterior neural precursors that can give rise to forebrain neurones in abundance, enabling standardised functional studies of neural stem cell maintenance, lineage choice and neuronal functional maturation for neurodevelopmental research and disease-modelling
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