3D Compartmentalised Human Pluripotent Stem Cell-derived Neuromuscular Co-cultures.

Human neuromuscular diseases represent a diverse group of disorders with unmet clinical need, ranging from muscular dystrophies, such as Duchenne muscular dystrophy (DMD), to neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS). In many of these conditions, axonal and neuromuscular synapse dysfunction have been implicated as crucial pathological events, highlighting the need for in vitro disease models that accurately recapitulate these aspects of human neuromuscular physiology. The protocol reported here describes the co-culture of neural spheroids composed of human pluripotent stem cell (PSC)-derived motor neurons and astrocytes, and human PSC-derived myofibers in 3D compartmentalised microdevices to generate functional human neuromuscular circuits in vitro. In this microphysiological model, motor axons project from a central nervous system (CNS)-like compartment along microchannels to innervate skeletal myofibers plated in a separate muscle compartment. This mimics the spatial organization of neuromuscular circuits in vivo. Optogenetics, particle image velocimetry (PIV) analysis, and immunocytochemistry are used to control, record, and quantify functional neuromuscular transmission, axonal outgrowth, and neuromuscular synapse number and morphology. This approach has been applied to study disease-specific phenotypes for DMD and ALS by incorporating patient-derived and CRISPR-corrected human PSC-derived motor neurons and skeletal myogenic progenitors into the model, as well as testing candidate drugs for rescuing pathological phenotypes. The main advantages of this approach are: i) its simple design; ii) the in vivo-like anatomical separation between CNS and peripheral muscle; and iii) the amenability of the approach to high power imaging. This opens up the possibility for carrying out live axonal transport and synaptic imaging assays in future studies, in addition to the applications reported in this study. Graphical abstract Graphical abstract abbreviations: Channelrhodopsin-2 (CHR2+), pluripotent stem cell (PSC), motor neurons (MNs), myofibers (MFs), neuromuscular junction (NMJ)

Can hippocampal neurites and growth cones climb over obstacles?

Guidance molecules, such as Sema3A or Netrin-1, can induce growth cone (GC) repulsion or attraction in the presence of a flat surface, but very little is known of the action of guidance molecules in the presence of obstacles. Therefore we combined chemical and mechanical cues by applying a steady Netrin-1 stream to the GCs of dissociated hippocampal neurons plated on polydimethylsiloxane (PDMS) surfaces patterned with lines 2 \ub5m wide, with 4 \ub5m period and with a height varying from 100 to 600 nm. GC turning experiments performed 24 hours after plating showed that filopodia crawl over these lines within minutes. These filopodia do not show staining for the adhesion marker Paxillin. GCs and neurites crawl over lines 100 nm high, but less frequently and on a longer time scale over lines higher than 300 nm; neurites never crawl over lines 600 nm high. When neurons are grown for 3 days over patterned surfaces, also neurites can cross lines 300 nm and 600 nm high, grow parallel to and on top of these lines and express Paxillin. Axons - selectively stained with SMI 312 - do not differ from dendrites in their ability to cross these lines. Our results show that highly motile structures such as filopodia climb over high obstacle in response to chemical cues, but larger neuronal structures are less prompt and require hours or days to climb similar obstacles

Bidimensional focusing of x rays by refraction in an edge

10.1364/OL.39.001250Optics Letters3951250-1253OPLE

Low-power, low-pressure reactive-ion etching process for silicon etching with vertical and smooth walls for mechanobiology application

10.1117/1.JMM.16.3.034501Journal of Micro/ Nanolithography, MEMS, and MOEMS1633450

Characterisation of the magnetic shielding properties of YBaCuO thick films prepared by electrophoretic deposition on silver substrates

peer reviewedThis communication reports experimental results on the superconducting properties of YBaCuO thick films prepared by electrophoretic deposition on silver substrates. The magnetic shielding properties of the coatings were characterised by various methods. First, the electrical resistance and the transport critical current density, Jc, were determined. Our coatings exhibit a superconducting transition at a temperature of 90 K. Next, shielding characterisations were carried out at 77 K for samples having either a slab or a cylindrical geometry. In both cases, the frequency of the applied magnetic field was 103 Hz; the field behind the shielding wall was measured by a pick-up coil connected to a lock-in amplifier. In the case of cylindrical samples and for an applied induction lower than 1 G, the field inside the shielding enclosure is reduced by a factor greater than 106 (i.e. 120 dB) with respect to the applied field.Blindage magnétique par matériaux supraconducteurs à haute température critiqu

Infrared microspectroscopy of live cells in microfluidic devices (MD-IRMS): Toward a powerful label-free cell-based assay

Until nowadays most infrared microspectroscopy (IRMS) experiments on biological specimens (i.e., tissues or cells) have been routinely carried out on fixed or dried samples in order to circumvent water absorption problems. In this paper, we demonstrate the possibility to widen the range of in-vitro IRMS experiments to vibrational analysis of live cellular samples, thanks to the development of novel biocompatible IR-visible transparent microfluidic devices (MD). In order to highlight the biological relevance of IRMS in MD (MD-IRMS), we performed a systematic exploration of the biochemical alterations induced by different fixation protocols, ethanol 70% and formaldehyde solution 4%, as well as air-drying on U937 leukemic monocytes by comparing their IR vibrational features with the live U937 counterpart. Both fixation and air-drying procedures affected lipid composition and order as well as protein structure at a different extent while they both induced structural alterations in nucleic acids. Therefore, only IRMS of live cells can provide reliable information on both DNA and RNA structure and on their cellular dynamic. In summary, we show that MD-IRMS of live cells is feasible, reliable, and biologically relevant to be recognized as a label-free cell-based assay

Synchrotron radiation infrared microspectroscopy of single living cells in microfluidic devices: Advantages, disadvantages and future perspectives

The possibility to fully exploit the diagnostic capabilities of SR-IRMS for studying single living cells under physiological conditions is limited by several constrains. First of all, the technology for manufacturing materials transparent to both IR and visible light is quite immature, limiting the design of fluidic devices to simple demountable liquid cells. In addition, the water spectral features become prominent in the Mid IR, hiding several cellular bands and therefore limiting the diagnostic capabilities of SR-IRMS. The overcoming of the so called "water absorption barrier" requires the improvement of the protocols for the compensation of buffer spectral contributions, a goal that can be achieved also advancing the quality of IR-suitable fluidic devices. In this paper, the technical solutions employed for microfabricating completely sealed IR-visible transparent fluidic devices for living cell analysis will be presented. Several examples of the results obtained in the study of living U937 monocytes subjected to different stimuli will be selected for highlighting both the advantages and the disadvantages offered by our approach for cellular biologyThe possibility to fully exploit the diagnostic capabilities of SR-IRMS for studying single living cells under physiological conditions is limited by several constrains. First of all, the technology for manufacturing materials transparent to both IR and visible light is quite immature, limiting the design of fluidic devices to simple demountable liquid cells. In addition, the water spectral features become prominent in the Mid IR, hiding several cellular bands and therefore limiting the diagnostic capabilities of SR-IRMS. The overcoming of the so called "water absorption barrier" requires the improvement of the protocols for the compensation of buffer spectral contributions, a goal that can be achieved also advancing the quality of IR-suitable fluidic devices. In this paper, the technical solutions employed for microfabricating completely sealed IR-visible transparent fluidic devices for living cell analysis will be presented. Several examples of the results obtained in the study of living U937 monocytes subjected to different stimuli will be selected for highlighting both the advantages and the disadvantages offered by our approach for cellular biolog

Continuous adsorption in highly ordered porous matrices made by nanolithography

10.1038/ncomms3966Nature Communications4