Single Layer Lift-Off of CSAR62 for Dense Nanostructured Patterns.

Lift-off processing is a common method of pattern transfer for different nanofabrication applications. With the emergence of chemically amplified and semi-amplified resist systems, the possibilities for pattern definition via electron beam lithography has been widened. We report a reliable and simple lift-off process for dense nanostructured pattern in CSAR62.

Miniaturized and multiplexed high-content screening of drug and immune sensitivity in a multichambered microwell chip.

Here, we present a methodology based on multiplexed fluorescence screening of two- or three-dimensional cell cultures in a newly designed multichambered microwell chip, allowing direct assessment of drug or immune cell cytotoxic efficacy. We establish a framework for cell culture, formation of tumor spheroids, fluorescence labeling, and imaging of fixed or live cells at various magnifications directly in the chip together with data analysis and interpretation. The methodology is demonstrated by drug cytotoxicity screening using ovarian and non-small cell lung cancer cells and by cellular cytotoxicity screening targeting tumor spheroids of renal carcinoma and ovarian carcinoma with natural killer cells from healthy donors.

Miniaturized Sulfite-Based Gold Bath for Controlled Electroplating of Zone Plate Nanostructures.

X-ray zone plates made from gold are common optical components used in X-ray imaging experiments. These nanostructures are normally fabricated using a combination of electron-beam lithography and gold electroplating with cyanide gold baths. In this study, we present a gold electroplating process in a miniaturized gold-suplphite bath.

NK cells integrate signals over large areas when building immune synapses but require local stimuli for degranulation.

Immune synapses are large-scale, transient molecular assemblies that serve as platforms for antigen presentation to B and T cells and for target recognition by cytotoxic T cells and natural killer (NK) cells. The formation of an immune synapse is a tightly regulated, stepwise process in which the cytoskeleton, cell surface receptors, and intracellular signaling proteins rearrange into supramolecular activation clusters (SMACs). We generated artificial immune synapses (AIS) consisting of synthetic and natural ligands for the NK cell-activating receptors LFA-1 and CD16 by microcontact printing the ligands into circular-shaped SMAC structures.

Metal-Assisted Chemical Etching and Electroless Deposition for Fabrication of Hard X-ray Pd/Si Zone Plates.

Zone plates are diffractive optics commonly used in X-ray microscopes. Here, we present a wet-chemical approach for fabricating high aspect ratio Pd/Si zone plate optics aimed at the hard X-ray regime. A Si zone plate mold is fabricated via metal-assisted chemical etching (MACE) and further metalized with Pd via electroless deposition (ELD).

Admission glucose level was associated with increased short-term mortality and length-of-stay irrespective of diagnosis, treating medical specialty or concomitant laboratory values.

Background: Glucose is a routine emergency sample. General guidelines for inpatient hyperglycemia are scarce, except in myocardial infarction, stroke, and perioperative/ICU. Previous studies found admission glucose associated with increased mortality in specific conditions.

A collagen-based microwell migration assay to study NK-target cell interactions.

Natural killer (NK) cell cytotoxicity in tissue is dependent on the ability of NK cells to migrate through the extracellular matrix (ECM) microenvironment. Traditional imaging studies of NK cell migration and cytotoxicity have utilized 2D surfaces, which do not properly reproduce the structural and mechanical cues that shape the migratory response of NK cells in vivo. Here, we have combined a microwell assay that allows long-term imaging and tracking of small, well-defined populations of NK cells with an interstitial ECM-like matrix.

Microwell-Based Live Cell Imaging of NK Cell Dynamics to Assess Heterogeneity in Motility and Cytotoxic Response.

NK cell heterogeneity has primarily been studied either on the population level, measuring average responses, or on the single cell level by flow cytometry, providing static snapshots. These approaches have certain drawbacks, not enabling dynamic observations of single cells over extended periods of time. One of the primary limitations of single cell imaging has been throughput; it has been challenging to collect data for many cells due to their dynamic nature and migrating out of the field of view.

Microchip Screening Platform for Single Cell Assessment of NK Cell Cytotoxicity.

Here, we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon-glass microchip containing 32,400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis, the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing.

Single-Cell Characterization of in vitro Migration and Interaction Dynamics of T Cells Expanded with IL-2 and IL-7.

T cells are pivotal in the immune defense against cancers and infectious agents. To mount an effector response against cancer cells, T cells need to migrate to the cancer-site, engage in contacts with cancer cells, and perform their effector functions. Adoptive T cell therapy is an effective strategy as treatment of complications such as relapse or opportunistic infections after hematopoietic stem cell transplantations.

Contact poling of Rb:KTiOPO(4) using a micro-structured silicon electrode.

A contact poling technique for domain engineering of ferroelectrics using a micro-structured silicon electrode is demonstrated on Rb:KTiOPO. High quality QPM gratings were reproducibly fabricated. The silicon electrode is reusable and the technique potentially suitable when complex structures with sub-μm features are to be domain engineered, which otherwise is incompatible with conventional photolithography.

Live cell imaging in a micro-array of acoustic traps facilitates quantification of natural killer cell heterogeneity.

Natural killer (NK) cells kill virus-infected or cancer cells through the release of cytotoxic granules into a tight intercellular contact. NK cell populations comprise individual cells with varying sensitivity to distinct input signals, leading to disparate responses. To resolve this NK cell heterogeneity, we have designed a novel assay based on ultrasound-assisted cell-cell aggregation in a multiwell chip allowing high-resolution time-lapse imaging of one hundred NK-target cell interactions in parallel.

Novel Microchip-Based Tools Facilitating Live Cell Imaging and Assessment of Functional Heterogeneity within NK Cell Populations.

Each individual has a heterogeneous pool of NK cells consisting of cells that may be specialized towards specific functional responses such as secretion of cytokines or killing of tumor cells. Many conventional methods are not fit to characterize heterogeneous populations as they measure the average response of all cells. Thus, there is a need for experimental platforms that provide single cell resolution.

Development of a novel microfluidic device for long-term in situ monitoring of live cells in 3-dimensional matrices.

Using the latest innovations in microfabrication technology, 3-dimensional microfluidic cell culture systems have been developed as an attractive alternative to traditional 2-dimensional culturing systems as a model for long-term microscale cell-based research. Most microfluidic systems are based on the embedding of cells in hydrogels. However, physiologically realistic conditions based on hydrogels are difficult to obtain and the systems are often too complicated.

A silicon-glass microwell platform for high-resolution imaging and high-content screening with single cell resolution.

We present a novel microwell array platform suited for various cell-imaging assays where single cell resolution is important. The platform consists of an exchangeable silicon-glass microchip for cell biological applications and a custom made holder that fits in conventional microscopes. The microchips presented here contain arrays of miniature wells, where the well sizes and layout have been designed for different applications, including single cell imaging, studies of cell-cell interactions or ultrasonic manipulation of cells.

Imaging immune surveillance of individual natural killer cells confined in microwell arrays.

New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation.

Ultrasound-controlled cell aggregation in a multi-well chip.

We demonstrate a microplate platform for parallelized manipulation of particles or cells by frequency-modulated ultrasound. The device, consisting of a silicon-glass microchip and a single ultrasonic transducer, enables aggregation, positioning and high-resolution microscopy of cells distributed in an array of 100 microwells centered on the microchip. We characterize the system in terms of temperature control, aggregation and positioning efficiency, and cell viability.

Mechanical properties of primary cilia regulate the response to fluid flow.

The primary cilium is a ubiquitous organelle present on most mammalian cells. Malfunction of the organelle has been associated with various pathological disorders, many of which lead to cystic disorders in liver, pancreas, and kidney. Primary cilia have in kidney epithelial cells been observed to generate intracellular calcium in response to fluid flow, and disruption of proteins involved in this calcium signaling lead to autosomal dominant polycystic kidney disease, implying a direct connection between calcium signaling and cyst formation.

An integrated QCM-based narcotics sensing microsystem.

We present the design, fabrication and successful testing of a 14x14x4 mm3 integrated electronic narcotics sensing system which consists of only four parts. The microsystem absorbs airborne narcotics molecules and performs a liquid assay using an integrated quartz crystal microbalance (QCM). A vertically conductive double-sided adhesive foil (VCAF) was used and studied as a novel material for LOC and MEMS applications and provides easy assembly, electrical contacting and liquid containment.

Microfluidic devices for studies of primary cilium mediated cellular response to dynamic flow conditions.

We present the first microfabricated microfluidic devices designed specifically for studies of primary cilium mediated cellular response to dynamic flow. The primary cilium functions as a mechano-sensor in renal tubular epithelium, sensing the extracellular fluid flow. Malfunction of cilia has been implicated in e.

A microfluidic device for parallel 3-D cell cultures in asymmetric environments.

We demonstrate a concept for how a miniaturized 3-D cell culture in biological extracellular matrix (ECM) or synthetic gels bridges the gap between organ-tissue culture and traditional 2-D cultures. A microfluidic device for 3-D cell culture including microgradient environments has been designed, fabricated, and successfully evaluated. In the presented system stable diffusion gradients can be generated by application of two parallel fluid flows with different composition against opposite sides of a gel plug with embedded cells.

Off-line integration of CE and MALDI-MS using a closed-open-closed microchannel system.

In this work, a new technique for off-line hyphenation between CE and MALDI-MS is presented. Two closed fused-silica capillaries were connected via a silicon chip comprising an open microcanal. The EOF in the system was evaluated using mesityloxide or leucine-enkephalin as a sample and with a running buffer that rendered the analyte neutrally charged.

A micromachined interface for airborne sample-to-liquid transfer and its application in a biosensor system.

A novel micromachined interface for airborne sample-to-liquid adsorption and droplet-to-liquid transfer was designed and fabricated. It enables a robust sheet liquid flow serving as an adsorption site. The interface was characterised for flow and pressure properties and tested successfully for the transfer/adsorption of different samples.

A concept for miniaturized 3-D cell culture using an extracellular matrix gel.

This paper presents a novel method to embed, anchor, and cultivate cells in a controlled 3-D flow-through microenvironment. This is realized using an etched silicon pillar flow chamber filled with extracellular matrix (ECM) gel mixed with cells. At 4 degrees C, while in liquid form, ECM gel is mixed with cells and injected into the chamber.