Wrinkled Thermo-Electric Meander-Shaped Element on a Thin Freestanding PDMS Membrane.

Natural wrinkling of metal films on silicone substrates can appear by means of the metal sputtering process and can be described by the continuous elastic theory and non-linear wrinkling model. Here, we report the fabrication technology and behavior of thin freestanding Polydimethylsiloxane (PDMS) membranes equipped with thermo-electric meander-shaped elements. The Cr/Au wires were obtained on the silicone substrate by magnetron sputtering. We observe wrinkle formation and suppose furrows appear once PDMS returns to its initial state after the thermo-mechanical expansion during sputtering. Although the substrate thickness is usually a negligible parameter in the theory of wrinkle formation, we found that the self-assembled wrinkling architecture of the PDMS/Cr/Au varies due to the membrane thickness of 20 µm and 40 µm PDMS. We also demonstrate that the wrinkling of the meander wire affects its length, and it causes a 2.7 times higher resistance compared to a calculated value. Therefore, we investigate the influence of the PDMS mixing ratio on the thermo-electric meander-shaped elements. For the stiffer PDMS with a mixing ratio of 10:4, the resistance due to wrinkle amplitude alterations is 25% higher compared to the PDMS of ratio 10:1. Additionally, we observe and describe a thermo-mechanically induced motion behavior of the meander wires on completely freestanding PDMS membrane under applied current. These results can improve the understanding of wrinkle formation, which influences thermo-electric characteristics and may promote the integration of this technology in applications.

Two-Dimensional Phononic Crystal Based Sensor for Characterization of Mixtures and Heterogeneous Liquids.

We show new approaches to developing acoustic liquid sensors based on phononic crystals. The proposed phononic crystal integrates fluidic elements. A solid block with periodic cylindrical holes contains a defect-a liquid-filled cylindrical cavity. We pay attention to acoustic excitation and the readout of the axisymmetric cylindrical resonator eigenmode of the liquid-filled defect in the middle of the phononic crystal structure. This mode solves the challenge of mechanical energy losses due to liquid viscosity. We also analyze the coupling effects between oscillations of liquid and solid systems and consider coupling issues between piezoelectric transducers and the liquid-filled cavity resonator. The numerical simulation of the propagation of acoustic waves through the phononic crystal sensor was carried out in COMSOL Multiphysics Software. The phononic crystal was made of stainless steel with mechanically drilled holes and was fabricated for experimental verification. We show that a tuning of the solid-liquid vibrational modes coupling is the key to an enhanced level of sensitivity to liquid properties. Besides (homogeneous) water-propanol mixtures, experimental studies were carried out on (disperse) water-fuel emulsions.

On-a-Chip-Based Sensitive Detection of Drug-Induced Apoptosis in Polarized Gastric Epithelial Cells.

Microfluidic devices for culturing cells have been successfully utilized for biomedical applications, including drug screening. Several cell lines could be cultivated in microengineered environments with promising results, but gastric cell lines have not yet been widely used or studied. Therefore, this study focuses on establishing a polarized gastric epithelial monolayer on-a-chip and describes a general-purpose methodology applicable for bonding any porous material to PDMS through an adhesive sublayer. The fully transparent microfluidic chip consists of two microfluidic channels separated by a collagen-coated porous membrane and lined by human polarized gastric epithelial (NCI-N87) cells. We present considerations on how to ensure continuous and stable flow through the channels. The continuous flow rate was achieved using a pressure-driven pump. Media flow at a constant rate (0.5 µL/min) rapidly led the gastric epithelial cells to develop into a polarized monolayer. The barrier integrity was assessed by the FITC-dextran test. The generation of a monolayer was faster than in the static Boyden chamber. Moreover, fluorescence microscopy was used to monitor the apoptotic cell death of gastric epithelial monolayers on-a-chip in response to camptothecin, a therapeutic gastric cancer drug.

From 3D Back to 2D Monolayer Stomach Organoids-on-a-Chip.

2D monolayer gastric organoids (2DMGOs)-on-a-chip have consistent structures and can live for more than a year in culture. This state-of-the-art cell physiological system in a microfluidic device provides a way to investigate biomedically relevant, stimuli-dependent cellular responses in a variety of differentiated 2DMGOs.

Femtosecond Laser-Based Integration of Nano-Membranes into Organ-on-a-Chip Systems.

Organ-on-a-chip devices are gaining popularity in medical research due to the possibility of performing extremely complex living-body-resembling research in vitro. For this reason, there is a substantial drive in developing technologies capable of producing such structures in a simple and, at the same time, flexible manner. One of the primary challenges in producing organ-on-chip devices from a manufacturing standpoint is the prevalence of layer-by-layer bonding techniques, which result in limitations relating to the applicable materials and geometries and limited repeatability. In this work, we present an improved approach, using three dimensional (3D) laser lithography for the direct integration of a functional part-the membrane-into a closed-channel system. We show that it allows the freely choice of the geometry of the membrane and its integration into a complete organ-on-a-chip system. Considerations relating to sample preparation, the writing process, and the final preparation for operation are given. Overall, we consider that the broader application of 3D laser lithography in organ-on-a-chip fabrication is the next logical step in this field's evolution.

Narrow Band Solid-Liquid Composite Arrangements: Alternative Solutions for Phononic Crystal-Based Liquid Sensors.

Periodic elastic composite structures attract great attention. They offer the ability to design artificial properties to advance the control over the propagation of elastic/acoustic waves. In previous work, we drew attention to composite periodic structures comprising liquids. It was shown that the transmission spectrum of the structure, specifically a well-isolated peak, follows the material properties of liquid constituent in a distinct manner. This idea was realized in several liquid sensor concepts that launched the field of phononic crystal liquid sensors. In this work we introduce a novel concept-narrow band solid-liquid composite arrangements. We demonstrate two different concepts to design narrow band structures, and show the results of theoretical studies and results of experimental investigations that confirm the theoretical predictions. This work extends prior studies in the field of phononic crystal liquid sensors with novel concepts and results that have a high potential in a field of volumetric liquid properties evaluation.

Neisseria meningitidis elicits a pro-inflammatory response involving IκBζ in a human blood-cerebrospinal fluid barrier model.

BACKGROUND: The human-specific, Gram-negative bacterium Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis worldwide. The blood-cerebrospinal fluid barrier (BCSFB), which is constituted by the epithelial cells of the choroid plexus (CP), has been suggested as one of the potential entry sites of Nm into the CSF and can contribute to the inflammatory response during infectious diseases of the brain. Toll-like receptors (TLRs) are involved in mediating signal transduction caused by the pathogens. METHODS: Using a recently established in vitro model of the human BCSFB based on human malignant CP papilloma (HIBCPP) cells we investigated the cellular response of HIBCPP cells challenged with the meningitis-causing Nm strain, MC58, employing transcriptome and RT-PCR analysis, cytokine bead array, and enzyme-linked immunosorbent assay (ELISA). In comparison, we analyzed the answer to the closely related unencapsulated carrier isolate Nm α14. The presence of TLRs in HIBCPP and their role during signal transduction caused by Nm was studied by RT-PCR and the use of specific agonists and mutant bacteria. RESULTS: We observed a stronger transcriptional response after infection with strain MC58, in particular with its capsule-deficient mutant MC58siaD-, which correlated with bacterial invasion levels. Expression evaluation and Gene Set Enrichment Analysis pointed to a NFκB-mediated pro-inflammatory immune response involving up-regulation of the transcription factor IκBζ. Infected cells secreted significant levels of pro-inflammatory chemokines and cytokines, including, among others, IL8, CXCL1-3, and the IκBζ target gene product IL6. The expression profile of pattern recognition receptors in HIBCPP cells and the response to specific agonists indicates that TLR2/TLR6, rather than TLR4 or TLR2/TLR1, is involved in the cellular reaction following Nm infection. CONCLUSIONS: Our data show that Nm can initiate a pro-inflammatory response in human CP epithelial cells probably involving TLR2/TLR6 signaling and the transcriptional regulator IκBζ.

Transmigration of polymorphnuclear neutrophils and monocytes through the human blood-cerebrospinal fluid barrier after bacterial infection in vitro.

BACKGROUND: Bacterial invasion through the blood-cerebrospinal fluid barrier (BCSFB) during bacterial meningitis causes secretion of proinflammatory cytokines/chemokines followed by the recruitment of leukocytes into the CNS. In this study, we analyzed the cellular and molecular mechanisms of polymorphonuclear neutrophil (PMN) and monocyte transepithelial transmigration (TM) across the BCSFB after bacterial infection. METHODS: Using an inverted transwell filter system of human choroid plexus papilloma cells (HIBCPP), we studied leukocyte TM rates, the migration route by immunofluorescence, transmission electron microscopy and focused ion beam/scanning electron microscopy, the secretion of cytokines/chemokines by cytokine bead array and posttranslational modification of the signal regulatory protein (SIRP) α via western blot. RESULTS: PMNs showed a significantly increased TM across HIBCPP after infection with wild-type Neisseria meningitidis (MC58). In contrast, a significantly decreased monocyte transmigration rate after bacterial infection of HIBCPP could be observed. Interestingly, in co-culture experiments with PMNs and monocytes, TM of monocytes was significantly enhanced. Analysis of paracellular permeability and transepithelial electrical resistance confirmed an intact barrier function during leukocyte TM. With the help of the different imaging techniques we could provide evidence for para- as well as for transcellular migrating leukocytes. Further analysis of secreted cytokines/chemokines showed a distinct pattern after stimulation and transmigration of PMNs and monocytes. Moreover, the transmembrane glycoprotein SIRPα was deglycosylated in monocytes, but not in PMNs, after bacterial infection. CONCLUSIONS: Our findings demonstrate that PMNs and monoctyes differentially migrate in a human BCSFB model after bacterial infection. Cytokines and chemokines as well as transmembrane proteins such as SIRPα may be involved in this process.

Culture models to study leukocyte trafficking across the choroid plexus.

BACKGROUND: A critical point during the course of central nervous system infection is the influx of leukocytes from the blood into the brain across the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB). However, experimental in vitro models to investigate leukocyte transmigration across cultured choroid plexus epithelial cells have been lacking so far. METHODS: We have developed a porcine and human "inverted" culture insert system that enables leukocyte transmigration specifically from the physiologically relevant basolateral side. The models use primary porcine choroid plexus epithelial cells (PCPEC) and human choroid plexus papilloma cells (HIBCPP). As a prerequisite for a functional barrier, we optimized culture conditions in which cells are maintained in serum-containing medium until high barrier function is reached. Leukocyte transmigration through the plexus epithelial cells is analysed by three-dimensional Apotome®-imaging and electron microscopy, and the route of transmigration through the plexus epithelial cells, i.e. transcellular as well as paracellular, can be determined. DISCUSSION: As a functionally relevant porcine and human BCSFB model, PCPEC and HIBCPP respectively, offer a wide range of options for analysis of disease-related mechanisms at the choroid plexus epithelium, especially involving human pathogens. Moreover, our in vitro models facilitate the investigation of leukocyte entry into the CNS via the blood-CSF barrier.

Chemotaxis of T-cells after infection of human choroid plexus papilloma cells with Echovirus 30 in an in vitro model of the blood-cerebrospinal fluid barrier.

Enterovirus is the most common pathogen causing viral meningitis especially in children. Besides the blood-brain barrier (BBB) the choroid plexus, which forms the blood-cerebrospinal-fluid (CSF) barrier (BCSFB), was shown to be involved in the pathogenesis of enteroviral meningitis. In a human in vitro model of the BCSFB consisting of human choroid plexus papilloma cells (HIBCPP), the permissiveness of plexus epithelial cells for Echovirus 30 (EV30) was analyzed by immunoblotting and quantitative real-time PCR (Q-PCR). HIBCPP could be directly infected by EV30 from the apical as well as from the physiological relevant basolateral side. During an infection period of 5h no alterations of barrier function and cell viability could be observed. Analysis of the cytokine/chemokine-profile following enteroviral infection with a cytometric bead array (CBA) and Q-PCR revealed an enhanced secretion of PanGRO (CXCL1, CXCL2 and CXCL3), IL8 and CCL5. Q-PCR showed a significant upregulation of CXCL1, CXCL2 and CXCL3 in a time dependant manner. However, there was only a minor effect of HIBCPP-infection with EV30 on transepithelial T lymphocyte migration with or without the chemoattractant CXCL12. Moreover, CXCL3 did not significantly enhance T cell migrations. Therefore additional factors must be involved for the in vivo reported enhanced T cell migration into the CNS in the context of enteroviral meningitis. As HIBCPP are permissive for infection with EV30, they constitute a valuable human in vitro model to study viral infection at the BCSFB.

Polar invasion and translocation of Neisseria meningitidis and Streptococcus suis in a novel human model of the blood-cerebrospinal fluid barrier.

Acute bacterial meningitis is a life-threatening disease in humans. Discussed as entry sites for pathogens into the brain are the blood-brain and the blood-cerebrospinal fluid barrier (BCSFB). Although human brain microvascular endothelial cells (HBMEC) constitute a well established human in vitro model for the blood-brain barrier, until now no reliable human system presenting the BCSFB has been developed. Here, we describe for the first time a functional human BCSFB model based on human choroid plexus papilloma cells (HIBCPP), which display typical hallmarks of a BCSFB as the expression of junctional proteins and formation of tight junctions, a high electrical resistance and minimal levels of macromolecular flux when grown on transwell filters. Importantly, when challenged with the zoonotic pathogen Streptococcus suis or the human pathogenic bacterium Neisseria meningitidis the HIBCPP show polar bacterial invasion only from the physiologically relevant basolateral side. Meningococcal invasion is attenuated by the presence of a capsule and translocated N. meningitidis form microcolonies on the apical side of HIBCPP opposite of sites of entry. As a functionally relevant human model of the BCSFB the HIBCPP offer a wide range of options for analysis of disease-related mechanisms at the choroid plexus epithelium, especially involving human pathogens.