Search

Hendrik Reynders,1 Indra Van Zundert,1 Rui Silva,1– 3 Bram Carlier,3 Olivier Deschaume,4 Carmen Bartic,4 Susana Rocha,1 Sergey Basov,5 Margriet J Van Bael,5 Uwe Himmelreich,3 Thierry Verbiest,1 Ana Zamora1,3 1Molecular Imaging and Photonics, KU Leuven, Leuven, Belgium; 2Engineering Department, Oporto University, Porto, Portugal; 3Biomedical MRI, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium; 4Laboratory for Soft Matter and Biophysics, KU Leuven, Leuven, Belgium; 5Quantum Solid State Physics, KU Leuven, Leuven, BelgiumCorrespondence: Ana Zamora Email anamaria.zamoramartinez@kuleuven.beIntroduction: The inherent fluorescence properties of iron oxide nanoparticles (IONPs) were characterized, and their applicability for multiphoton imaging in cells was tested in combination with their magnetic resonance imaging (MRI) capabilities.Methods: Superparamagnetic iron oxide nanoparticles were synthesized and subsequently coated with polyethylene glycol to make them water-dispersible. Further characterization of the particles was performed using Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), dynamic light scattering (DLS), superconducting quantum interference device (SQUID) and magnetic resonance relaxivity measurements. MRI and fluorescence properties of bare IONPs were first studied in solution and subsequently in A549-labeled cells.Results: The particles, with a core size of 11.3 ± 4.5 nm, showed a good negative MRI contrast in tissue-mimicking phantoms. In vitro studies in mammalian A549 cells demonstrate that these IONPs are biocompatible and can also produce significant T2/T2* contrast enhancement in IONPs-labeled cells. Furthermore, excitation-wavelength dependent photoluminescence was observed under one- and two-photon excitation.Discussion: The obtained results indicated that IONPs could be used for fluorescence label-free bioimaging at multiple wavelengths, which was proven by multiphoton imaging of IONPs internalization in A549 cancer cells.Keywords: iron oxide nanoparticles, photoluminescence, biomedical imaging, MRI, cancer cells, multimodal imaging

Different microscopy and scattering methods used in the literature to determine the dimensions of cellulose nanocrystals derived from cotton and bacterial cellulose were compared to investigate potential bias and discrepancies. Atomic force microscopy (AFM), small-angle X-ray scattering (SAXS), depolarized dynamic light scattering (DDLS), and static light scattering (SLS) were compared. The lengths, widths, and heights of the particles and their respective distributions were determined by AFM. In agreement with previous work, the CNCs were found to have a ribbon-like shape, regardless of the source of cellulose or the surface functional groups. Tip broadening and agglomeration of the particles during deposition cause AFM-derived lateral dimensions to be systematically larger those obtained from SAXS measurements. The radius of gyration determined by SLS showed a good correlation with the dimensions obtained by AFM. The hydrodynamic lateral dimensions determined by DDLS were found to have the same magnitude as either the width or height obtained from the other techniques; however, the precision of DDLS was limited due to the mismatch between the cylindrical model and the actual shape of the CNCs, and to constraints in the fitting procedure. Therefore, the combination of AFM and SAXS, or microscopy and small-angle scattering, is recommended for the most accurate determination of CNC dimensions.

Foreign body response (FBR), inflammation, and fibrotic encapsulation of neural implants remain major problems affecting the impedance of the electrode-tissue interface and altering the device performance. Adhesion of proteins and cells (e.g., pro-inflammatory macrophages, and fibroblasts) triggers the FBR cascade and can be diminished by applying antifouling coatings onto the implanted devices. In this paper, we report the deposition and characterization of a thin (±6 nm) sulfobetaine-based coating onto microfabricated platinum electrodes and cochlear implant (CI) electrode arrays. We found that this coating has stable cell and protein-repellent properties, for at least 31 days in vitro, not affected by electrical stimulation protocols. Additionally, its effect on the electrochemical properties relevant to stimulation (i.e., impedance, charge injection capacity) was negligible. When applied to clinical CI electrode arrays, the film was successful at inhibiting fibroblast adhesion on both the silicone packaging and the platinum/iridium electrodes. In vitro, in fibroblast cultures, coated CI electrode arrays maintained impedance values up to five times lower compared to non-coated devices. Our studies demonstrate that such thin sulfobetaine containing layers are stable and prevent protein and cell adhesion in vitro and are compatible for use on CI electrode arrays. Future in vivo studies should be conducted to investigate its ability to mitigate biofouling, fibrosis, and the resulting impedance changes upon long-term implantation in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Over recent years, nano-engineered materials have become an important component of artificial extracellular matrices. On one hand, these materials enable static enhancement of the bulk properties of cell scaffolds, for instance, they can alter mechanical properties or electrical conductivity, in order to better mimic the in vivo cell environment. Yet, many nanomaterials also exhibit dynamic, remotely tunable optical, electrical, magnetic, or acoustic properties, and therefore, can be used to non-invasively deliver localized, dynamic stimuli to cells cultured in artificial ECMs in three dimensions. Vice versa, the same, functional nanomaterials, can also report changing environmental conditions-whether or not, as a result of a dynamically applied stimulus-and as such provide means for wireless, long-term monitoring of the cell status inside the culture. In this review article, we present an overview of the technological advances regarding the incorporation of functional nanomaterials in artificial extracellular matrices, highlighting both passive and dynamically tunable nano-engineered components.

Disease research and drug screening platforms require in vitro model systems with cellular cues resembling those of natural tissues. Fibrillar alignment, occurring naturally in extracellular matrices, is one of the crucial attributes in tissue development. Obtaining fiber alignment in 3D, in vitro remains an important challenge due to non-linear material characteristics. Here, we report a cell-compatible, shear stress-based method allowing to obtain 3D homogeneously aligned fibrillar collagen hydrogels. Controlling the shear-stress during gelation results in low strain rates, with negligible effects on the viability of embedded SH-SY5Y cells. Our approach offers reproducibility and tunability through a paradigm shift: The shear-stress initiation moment, being the critical optimization parameter in the process, is related to the modulus of the developing gel, whereas state of the art methods often rely on a predefined time to initiate the alignment procedure. After curing, the induced 3D alignment is maintained after the release of stress, with a linear relation between the total acquired strain and the fiber alignment. This method is generally applicable to 3D fibrillar materials and stress/pressure-controlled setups, making it a valuable addition to the fast-growing field of tissue engineering. STATEMENT OF SIGNIFICANCE: Controlling fiber alignment in vitro 3D hydrogels is crucial for developing physiologically relevant model systems. However, it remains challenging due to the non-linear material characteristics of fibrillar hydrogels, limiting the scalability and repeatability. Our approach tackles these challenges by utilizing a stress-controlled rheometer allowing us to monitor structural changes in situ and determine the optimal moment for applying a shear-stress inducing alignment. By careful parameter control, we infer the relationship between time, induced strain, alignment and biocompatibility. This tunable and reproducible method is both scalable and generally applicable to any fibrillar hydrogel, therefore, we believe it is useful for research investigating the link between matrix anisotropy and cell behavior in 3D systems, organ-on-chip technologies and drug research., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Despite the importance of cell characterization and identification for diagnostic and therapeutic applications, developing fast and label-free methods without (bio)-chemical markers or surface-engineered receptors remains challenging. Here, we exploit the natural cellular response to mild thermal stimuli and propose a label- and receptor-free method for fast and facile cell characterization. Cell suspensions in a dedicated sensor are exposed to a temperature gradient, which stimulates synchronized and spontaneous cell-detachment with sharply defined time-patterns, a phenomenon unknown from literature. These patterns depend on metabolic activity (controlled through temperature, nutrients, and drugs) and provide a library of cell-type-specific indicators, allowing to distinguish several yeast strains as well as cancer cells. Under specific conditions, synchronized glycolytic-type oscillations are observed during detachment of mammalian and yeast-cell ensembles, providing additional cell-specific signatures. These findings suggest potential applications for cell viability analysis and for assessing the collective response of cancer cells to drugs., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

To overcome the problems of refractive index matching and increased disorder when working with traditional heterostructure colloidal photonic crystals (CPCs) with dual or multiple photonic bandgaps (PBGs) for fluorescence enhancement in water, we propose the use of a chemical heterostructure in hollow sphere CPCs (HSCPCs). A partial chemical modification of the HSCPC creates a large contrast in wettability to induce the heterostructure, while the hollow spheres increase the refractive index difference when used in aqueous environment. With the platform, fluorescence enhancement reaches around 160 times in solution, and 72 times (signal-to-background ratio ~7 times) in cells during proof-of-concept live cardiomyocyte contractility experiments. Such photonic platform can be further exploited for chemical sensing, bioassays, and environmental monitoring. Moreover, the introduction of chemical heterostructures provides new design principles for functionalized photonic devices., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The application of antibodies in nanomedicine is now standard practice in research since it represents an innovative approach to deliver chemotherapy agents selectively to tumors. The variety of targets or markers that are overexpressed in different types of cancers results in a high demand for antibody conjugated-nanoparticles, which are versatile and easily customizable. Considering up-scaling, the synthesis of antibody-conjugated nanoparticles should be simple and highly reproducible. Here, we developed a facile coating strategy to produce antibody-conjugated nanoparticles using 'click chemistry' and further evaluated their selectivity towards cancer cells expressing different markers. Our approach was consistently repeated for the conjugation of antibodies against CD44 and EGFR, which are prominent cancer cell markers. The functionalized particles presented excellent cell specificity towards CD44 and EGFR overexpressing cells, respectively. Our results indicated that the developed coating method is reproducible, versatile, and non-toxic, and can be used for particle functionalization with different antibodies. This grafting strategy can be applied to a wide range of nanoparticles and will contribute to the development of future targeted drug delivery systems.

Flocculation is a widely used technology in industry including for wastewater treatment and microalgae harvesting. To increase the sustainability of wastewater treatment, and to avoid contamination of the harvested microalgal biomass, there is a need for bio-based flocculants to replace synthetic polymer flocculants or metal salt coagulants. We developed the first cellulose nanocrystalline flocculant with a grafted cationic point charge, i.e. glycine betaine ( i.e. N , N , N -trimethylglycine) grafted cellulose nanocrystals (CNCs) effective for the flocculation of kaolin (a model system for wastewater treatment), the freshwater microalgae Chlorella vulgaris , and the marine microalgae Nannochloropsis oculata . We successfully grafted glycine betaine onto CNCs using a one-pot reaction using a tosyl chloride activated esterification reaction with a degree of substitution ranging from 0.078 ± 0.003 to 0.152 ± 0.002. The degree of substitution is controlled by the reaction conditions. Flocculation of kaolin (0.5 g L -1 ) required a dose of 2 mg L -1 , a comparable dose to commercial polyacrylamide-based flocculants. Flocculation was also successful for freshwater as well as marine microalgae (biomass concentration about 300 mg L -1 dry matter), although the flocculation efficiency of the latter remained below 80%. The dose to induce flocculation (DS = 0.152 ± 0.002) was 20 mg L -1 for the freshwater Chlorella vulgaris and 46 mg L -1 for the marine Nannochloropsis oculata , comparable to other bio-based flocculants such as chitosan or TanFloc., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

Tailored hydrogels mimicking the native extracellular environment could help overcome the high variability in outcomes within regenerative endodontics. This study aimed to evaluate the effect of the chemokine-binding and antimicrobial polymer, chlorite-oxidized oxyamylose (COAM), on the microstructural properties of fibrin and self-assembling peptide (SAP) hydrogels. A further goal was to assess the influence of the microstructural differences between the hydrogels on the in vitro behavior of human dental pulp stem cells (hDPSCs). Structural and mechanical characterization of the hydrogels with and without COAM was performed by atomic force microscopy and scanning electron microscopy to characterize their microstructure (roughness and fiber length, diameter, straightness, and alignment) and by nanoindentation to measure their stiffness (elastic modulus). Then, hDPSCs were encapsulated in hydrogels with and without COAM. Cell viability and circularity were determined using confocal microscopy, and proliferation was determined using DNA quantification. Inclusion of COAM did not alter the microstructure of the fibrin hydrogels at the fiber level while affecting the SAP hydrogel microstructure (homogeneity), leading to fiber aggregation. The stiffness of the SAP hydrogels was sevenfold higher than the fibrin hydrogels. The viability and attachment of hDPSCs were significantly higher in fibrin hydrogels than in SAP hydrogels. The DNA content was significantly affected by the hydrogel type and the presence of COAM. The microstructural stability after COAM inclusion and the favorable hDPSCs' response observed in fibrin hydrogels suggest this system as a promising carrier for COAM and application in endodontic regeneration.

Understanding microbial adhesion and retention is crucial for controlling many processes, including biofilm formation, antimicrobial therapy as well as cell sorting and cell detection platforms. Cell detachment is inextricably linked to cell adhesion and retention and plays an important part in the mechanisms involved in these processes. Physico-chemical and biological forces play a crucial role in microbial adhesion interactions and altering the medium ionic strength offers a potential means for modulating these interactions. Real-time studies on the effect of ionic strength on microbial adhesion are often limited to short-term bacterial adhesion. Therefore, there is a need, not only for long-term bacterial adhesion studies, but also for similar studies focusing on eukaryotic microbes, such as yeast. Hereby, we monitored, in real-time, S. cerevisiae adhesion on gold and silica as examples of surfaces with different surface charge properties to disclose long-term adhesion, retention and detachment as a function of ionic strength using quartz crystal microbalance with dissipation monitoring. Our results show that short- and long-term cell adhesion levels in terms of mass-loading increase with increasing ionic strength, while cells dispersed in a medium of higher ionic strength experience longer retention and detachment times. The positive correlation between the cell zeta potential and ionic strength suggests that zeta potential plays a role on cell retention and detachment. These trends are similar for measurements on silica and gold, with shorter retention and detachment times for silica due to strong short-range repulsions originating from a high electron-donicity. Furthermore, the results are comparable with measurements in standard yeast culture medium, implying that the overall effect of ionic strength applies for cells in nutrient-rich and nutrient-deficient media., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A simple and cost-effective method based on multi-photon microscopy is presented for the preliminary screening of the general morphology, size range and heterogeneity of Ir(iii) nano-aggregate formulations., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

Poly(thiophene)s have an inherently large third-order nonlinear optical (TONO) response, but applications are not straightforward due to unoptimized materials. Therefore, several structure-property relationships (molar mass, branching, regioregularity) are investigated to unravel which structural modifications give the highest TONO response. Poly(3-hexylthiophene) with different molar masses, poly[3-(2-ethylhexyl)thiophene] with different molar masses, and random copolymers with different degrees of regioregularity are synthesized and measured by UV-vis spectroscopy and the third harmonic scattering technique. Every structural modification that leads to an increase in π-π interactions in poly(thiophene)s leads to an increase in the TONO response of the material. Therefore, a material with a high molar mass, an unbranched alkyl side chain, and a high regioregularity degree is preferably tested as a promising TONO material.

Hematopoietic stem/progenitor cells (HSPCs) are responsible for the generation of blood cells throughout life. It is believed that, in addition to soluble cytokines and niche cells, biophysical cues like elasticity and oxygen tension are responsible for the orchestration of stem cell fate. Although several studies have examined the effects of bone marrow (BM) niche elasticity on HSPC behavior, no study has yet investigated the effects of the elasticity of other niche sites like the fetal liver (FL), where HSPCs expand more extensively. In this study, we evaluated the effect of matrix stiffness values similar to those of the FL on BM-derived HSPC expansion. We first characterized the elastic modulus of murine FL tissue at embryonic day E14.5. Fibrin hydrogels with similar stiffness values as the FL (soft hydrogels) were compared with stiffer fibrin hydrogels (hard hydrogels) and with suspension culture. We evaluated the expansion of total nucleated cells (TNCs), Lin - /cKit + cells, HSPCs (Lin - /Sca + /cKit + (LSK) cells), and hematopoietic stem cells (HSCs: LSK- Signaling Lymphocyte Activated Molecule (LSK-SLAM) cells) when cultured in 5% O 2 (hypoxia) or in normoxia. After 10 days, there was a significant expansion of TNCs and LSK cells in all culture conditions at both levels of oxygen tension. LSK cells expanded more in suspension culture than in both fibrin hydrogels, whereas TNCs expanded more in suspension culture and in soft hydrogels than in hard hydrogels, particularly in normoxia. The number of LSK-SLAM cells was maintained in suspension culture and in the soft hydrogels but not in the hard hydrogels. Our results indicate that both suspension culture and fibrin hydrogels allow for the expansion of HSPCs and more differentiated progeny whereas stiff environments may compromise LSK-SLAM cell expansion. This suggests that further research using softer hydrogels with stiffness values closer to the FL niche is warranted.

Structured illumination microscopy (SIM) is a widely used super resolution imaging technique that can down-modulate a sample's high-frequency information into objective recordable frequencies to enhance the resolution below the diffraction limit. However, classical SIM image reconstruction methods often generate poor results under low illumination conditions, which are required for reducing photobleaching and phototoxicity in cell imaging experiments. Although denoising methods or auxiliary items improved SIM image reconstruction in low signal level situations, they still suffer from decreased reconstruction quality and significant background artifacts, inevitably limiting their practical applications. In order to improve the reconstruction quality, second-order optimized regularized SIM (sorSIM) is designed specifically for image reconstruction in low signal level situations. In sorSIM, a second-order regularization term is introduced to suppress noise effect, and the penalty factor in this term is selected to optimize the resolution enhancement and noise resistance. Compared to classical SIM image reconstruction algorithms as well as to those previously used in low illumination cases, the proposed sorSIM provides images with enhanced resolution and fewer background artifacts. Therefore, sorSIM can be a potential tool for high-quality and rapid super resolution imaging, especially for low signal images.

We report on a novel biomimetic sensor that allows sensitive and specific detection of Escherichia coli (E. coli) bacteria in a broad concentration range from 10 2 up to 10 6  CFU/mL in both buffer fluids and relevant food samples (i.e. apple juice). The receptors are surface-imprinted polyurethane layers deposited on stainless-steel chips. Regarding the transducer principle, the sensor measures the increase in thermal resistance between the chip and the liquid due to the presence of bacteria captured on the receptor surface. The low noise level that enables the low detection limit originates from a planar meander element that serves as both a heater and a temperature sensor. Furthermore, the experiments show that the presence of bacteria in a liquid enhances the thermal conductivity of the liquid itself. Reference tests with a set of other representative species of Enterobacteriaceae, closely related to E. coli, indicate a very low cross-sensitivity with a sensor response at or below the noise level., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Building further upon the high spatial resolution offered by ultrasonic imaging and the high optical contrast yielded by laser excitation of photoacoustic imaging, and exploiting the temperature dependence of photoacoustic signal amplitudes, this paper addresses the question whether the rich information given by multispectral optoacoustic tomography (MSOT) allows to obtain 3D temperature images. Numerical simulations and experimental results are reported on agarose phantoms containing gold nanoparticles and the effects of shadowing, reconstruction flaws, etc. on the accuracy are determined.

Recent developments in nonlinear optical light scattering techniques have opened a window into morphological and structural characteristics for a variety of supramolecular systems. However, for the study of dynamic processes, the current way of measuring is often too slow. Here we present an alternative measurement scheme suitable for following dynamic processes. Fast acquisition times are achieved through Fourier imaging, allowing simultaneous detection at multiple scattering angles for different polarization combinations. This allows us to follow the crystal growth of the metal organic framework ZIF-8 in solution. The angle dependence of the signal provides insight into the growth mechanism by probing the evolution of size, shape and concentration, while polarization analysis yields structural information in terms of point group symmetry. Our findings highlight the potential of dynamic angle-resolved harmonic light scattering to probe crystal growth processes, assembly-disassembly of biological systems, adsorption, transport through membranes and myriad other applications.

Small soluble species of amyloid-beta (Aβ) formed during early peptide aggregation stages are responsible for several neurotoxic mechanisms relevant to the pathology of Alzheimer's disease (AD), although their interaction with the neuronal membrane is not completely understood. This study quantifies the changes in the neuronal membrane elasticity induced by treatment with the two most common Aβ isoforms found in AD brains: Aβ40 and Aβ42. Using quantitative atomic force microscopy (AFM), we measured for the first time the static elastic modulus of living primary hippocampal neurons treated with pre-aggregated Aβ40 and Aβ42 soluble species. Our AFM results demonstrate changes in the elasticity of young, mature and aged neurons treated for a short time with the two Aβ species pre-aggregated for 2 hours. Neurons aging under stress conditions, showing aging hallmarks, are the most susceptible to amyloid binding and show the largest decrease in membrane stiffness upon Aβ treatment. Membrane stiffness defines the way in which cells respond to mechanical forces in their environment and has been shown to be important for processes such as gene expression, ion-channel gating and neurotransmitter vesicle transport. Thus, one can expect that changes in neuronal membrane elasticity might directly induce functional changes related to neurodegeneration.

Several questions about the role of the oxygen sensor prolyl-hydroxylase 2 (PHD2) in cancer have not been addressed. First, the role of PHD2 in metastasis has not been studied in a spontaneous tumor model. Here, we show that global PHD2 haplodeficiency reduced metastasis without affecting tumor growth. Second, it is unknown whether PHD2 regulates cancer by affecting cancer-associated fibroblasts (CAFs). We show that PHD2 haplodeficiency reduced metastasis via two mechanisms: (1) by decreasing CAF activation, matrix production, and contraction by CAFs, an effect that surprisingly relied on PHD2 deletion in cancer cells, but not in CAFs; and (2) by improving tumor vessel normalization. Third, the effect of concomitant PHD2 inhibition in malignant and stromal cells (mimicking PHD2 inhibitor treatment) is unknown. We show that global PHD2 haplodeficiency, induced not only before but also after tumor onset, impaired metastasis. These findings warrant investigation of PHD2's therapeutic potential., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Missense mutations in alanine 673 of the amyloid precursor protein (APP), which corresponds to the second alanine of the amyloid β (Aβ) sequence, have dramatic impact on the risk for Alzheimer disease; A2V is causative, and A2T is protective. Assuming a crucial role of amyloid-Aβ in neurodegeneration, we hypothesized that both A2V and A2T mutations cause distinct changes in Aβ properties that may at least partially explain these completely different phenotypes. Using human APP-overexpressing primary neurons, we observed significantly decreased Aβ production in the A2T mutant along with an enhanced Aβ generation in the A2V mutant confirming earlier data from non-neuronal cell lines. More importantly, thioflavin T fluorescence assays revealed that the mutations, while having little effect on Aβ42 peptide aggregation, dramatically change the properties of the Aβ40 pool with A2V accelerating and A2T delaying aggregation of the Aβ peptides. In line with the kinetic data, Aβ A2T demonstrated an increase in the solubility at equilibrium, an effect that was also observed in all mixtures of the A2T mutant with the wild type Aβ40. We propose that in addition to the reduced β-secretase cleavage of APP, the impaired propensity to aggregate may be part of the protective effect conferred by A2T substitution. The interpretation of the protective effect of this mutation is thus much more complicated than proposed previously., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The establishment of neuronal connectivity depends on the correct initial polarization of the young neurons. In vivo, developing neurons sense a multitude of inputs and a great number of molecules are described that affect their outgrowth. In vitro, many studies have shown the possibility to influence neuronal morphology and growth by biophysical, i.e. topographic, signaling. In this work we have taken this approach one step further and investigated the impact of substrate topography in the very early differentiation stages of developing neurons, i.e. when the cell is still at the round stage and when the first neurite is forming. For this purpose we fabricated micron sized pillar structures with highly reproducible feature sizes, and analyzed neurons on the interface of flat and topographic surfaces. We found that topographic signaling was able to attract the polarization markers of mouse embryonic neurons -N-cadherin, Golgi-centrosome complex and the first bud were oriented towards topographic stimuli. Consecutively, the axon was also preferentially extending along the pillars. These events seemed to occur regardless of pillar dimensions in the range we examined. However, we found differences in neurite length that depended on pillar dimensions. This study is one of the first to describe in detail the very early response of hippocampal neurons to topographic stimuli.

Embryonic cells are very robust in surviving dissection and culturing protocols and easily adapt to their in vitro environment. Despite these advantages, research in the olfactory field on cultured embryonic olfactory neurons is sparse. In this study, two primary rat olfactory explant cultures of different embryonic d (E17 and E20) were established, comprising epithelium and bulb. The functionality of these neurons was tested by measuring intracellular calcium responses to cAMP-inducing agents forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) with fluorescence microscopy. For E17, the responsive cell fraction increased over time, from an initial 3% at the 1 d in vitro (DIV) to a maximum of 19% at 11 DIV. The response of E20 neurons fluctuated over time around a more or less stable 13%. A logistic regression analysis indicated a significant difference between both embryonic d in the response to FSK + IBMX. In addition, of these functional neurons, 23.3% of E17 and 54.3% of E20 cultures were responsive to the odorant isoamyl acetate.

The investigation of single-neuron parameters is of great interest because many aspects in the behavior and communication of neuronal networks still remain unidentified. However, the present available techniques for single-cell measurements are slow and do not allow for a high-throughput approach. We present here a CMOS compatible microelectrode array with 84 electrodes (with diameters ranging from 1.2 to 4.2 μm) that are smaller than the size of cell, thereby supporting single-cell addressability. We show controllable electroporation of a single cell by an underlying electrode while monitoring changes in the intracellular membrane potential. Further, by applying a localized electrical field between two electrodes close to a neuron while recording changes in the intracellular calcium concentration, we demonstrate activation of a single cell (∼270%, DF/F(0)), followed by a network response of the neighboring cells. The technology can be easily scaled up to larger electrode arrays (theoretically up to 137,000 electrodes/mm(2)) with active CMOS electronics integration able to perform high-throughput measurements on single cells., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The amyloid peptides Aβ(40) and Aβ(42) of Alzheimer's disease are thought to contribute differentially to the disease process. Although Aβ(42) seems more pathogenic than Aβ(40), the reason for this is not well understood. We show here that small alterations in the Aβ(42):Aβ(40) ratio dramatically affect the biophysical and biological properties of the Aβ mixtures reflected in their aggregation kinetics, the morphology of the resulting amyloid fibrils and synaptic function tested in vitro and in vivo. A minor increase in the Aβ(42):Aβ(40) ratio stabilizes toxic oligomeric species with intermediate conformations. The initial toxic impact of these Aβ species is synaptic in nature, but this can spread into the cells leading to neuronal cell death. The fact that the relative ratio of Aβ peptides is more crucial than the absolute amounts of peptides for the induction of neurotoxic conformations has important implications for anti-amyloid therapy. Our work also suggests the dynamic nature of the equilibrium between toxic and non-toxic intermediates.

Interfacing neurons with micro- and nano-electronic devices has been a subject of intense study over the last decade. One of the major problems in assembling efficient neuro-electronic hybrid systems is the weak electrical coupling between the components. This is mainly attributed to the fundamental property of living cells to form and maintain an extracellular cleft between the plasma membrane and any substrate to which they adhere. This cleft shunts the current generated by propagating action potentials and thus reduces the signal-to-noise ratio. Reducing the cleft thickness, and thereby increasing the seal resistance formed between the neurons and the sensing surface, is thus a challenge and could improve the electrical coupling coefficient. Using electron microscopic analysis and field potential recordings, we examined here the use of gold micro-structures that mimic dendritic spines in their shape and dimensions to improve the adhesion and electrical coupling between neurons and micro-electronic devices. We found that neurons cultured on a gold-spine matrix, functionalized by a cysteine-terminated peptide with a number of RGD repeats, readily engulf the spines, forming tight apposition. The recorded field potentials of cultured Aplysia neurons are significantly larger using gold-spine electrodes in comparison with flat electrodes.

The use of organic thin-film transistors (OTFTs) in sensorics is relatively new. Although electronic noses, electronic textiles and disposable biochemical sensors appear to be viable applications for this type of devices, the benefits of the technology still have to be proven. This paper aims to provide a review of the recent advances in the area of chemically sensitive field-effect devices based on organic thin-film transistors (OTFTs), with emphasis on bioanalytical applications. Detection principle, device configuration, materials and fabrication processes as well as sensor performances will be discussed, with emphasis on the potential for implementation in real applications and the important challenges ahead.