Design for a lithographically patterned bioartificial endocrine pancreas.

Cell encapsulation provides a means to transplant therapeutic cells for a variety of diseases including diabetes. However, due to the large numbers of cells, approximately on the order of a billion, that need to be transplanted for human diabetes therapy, adequate mass transport of nutrients such as oxygen presents a major challenge. Proof-of-concept for the design of a bioartificial endocrine pancreas (BAEP) that is optimized to minimize hypoxia in a scalable and precise architecture is demonstrated using a combination of simulations and experiments. The BAEP is composed of an array of porous, lithographically patterned polyhedral capsules arrayed on a rolled-up alginate sheet. All the important structural variables such as the capsule dimensions, pore characteristics, and spacing can be precisely engineered and tuned. Further, all cells are encapsulated within a single device with a volume not much greater than the total volume of the encapsulated cells, and no cell within the device is located more than 200 µm from the surrounding medium that facilitates efficient mass transport with the surroundings. Compared with gel-based encapsulation methods, our approach offers unprecedented precision and tunability of structural parameters as well as the volume of the encapsulated cells and consequently the amount of secreted insulin. Our work highlights the utility of lithography and self-assembly in the fabrication of micro- and nanostructured three-dimensional structures that simulate the function of natural endocrine organs.

Chemistry with spatial control using particles and streams().

Spatial control of chemical reactions, with micro- and nanometer scale resolution, has important consequences for one pot synthesis, engineering complex reactions, developmental biology, cellular biochemistry and emergent behavior. We review synthetic methods to engineer this spatial control using chemical diffusion from spherical particles, shells and polyhedra. We discuss systems that enable both isotropic and anisotropic chemical release from isolated and arrayed particles to create inhomogeneous and spatially patterned chemical fields. In addition to such finite chemical sources, we also discuss spatial control enabled with laminar flow in 2D and 3D microfluidic networks. Throughout the paper, we highlight applications of spatially controlled chemistry in chemical kinetics, reaction-diffusion systems, chemotaxis and morphogenesis.

Voltage-gated ion transport through semiconducting conical nanopores formed by metal nanoparticle-assisted plasma etching.

Nanopores with conical geometries have been found to rectify ionic current in electrolytes. While nanopores in semiconducting membranes are known to modulate ionic transport through gated modification of pore surface charge, the fabrication of conical nanopores in silicon (Si) has proven challenging. Here, we report the discovery that gold (Au) nanoparticle (NP)-assisted plasma etching results in the formation of conical etch profiles in Si. These conical profiles result due to enhanced Si etch rates in the vicinity of the Au NPs. We show that this process provides a convenient and versatile means to fabricate conical nanopores in Si membranes and crystals with variable pore-diameters and cone-angles. We investigated ionic transport through these pores and observed that rectification ratios could be enhanced by a factor of over 100 by voltage gating alone, and that these pores could function as ionic switches with high on-off ratios of approximately 260. Further, we demonstrate voltage gated control over protein transport, which is of importance in lab-on-a-chip devices and biomolecular separations.

Self-folding immunoprotective cell encapsulation devices.

Cell encapsulation therapy (CET) provides an attractive means to transplant cells without the need for immunosuppression. The cells are immunoisolated by surrounding them with a synthetic, semipermeable nanoporous membrane that allows selective permeation of nutrients and therapeutics while isolating the cells from hostile immune components. This communication describes the fabrication and in vitro characterization of lithographically structured and self-folded containers for immunoprotective cell encapsulation. Lithographic patterning ensured identical shapes, sizes, tunable porosity, and precise volumetric control, whereas self-folding enabled transformation of two-dimensional porous membranes into cubes, ensuring that pores were present in all three dimensions for adequate diffusion of O(2) and other nutrients to encapsulated cells. We fabricated containers with varying pore sizes and observed that pores sizes of approximately 78 nm were sufficient to significantly inhibit diffusion of IgG (the smallest antibody) and permit adequate diffusion of insulin, highlighting the possibility to utilize these containers to develop a lithographically structured bioartificial pancreas. FROM THE CLINICAL EDITOR: In this paper, a novel immunoisolation technique is presented to enable cell transplant survival by surrounding them with a synthetic, semipermeable nanoporous membrane that allows selective permeation of nutrients and therapeutics while isolating the cells from hostile immune components. This method may pave the way to effective pancreatic islet cell transplantation.

Three-dimensional microwell arrays for cell culture.

We propose the concept of three-dimensional (3D) microwell arrays for cell culture applications and highlight the importance of oxygen diffusion through pores in all three dimensions to enhance cell viability.

Responses of Escherichia coli bacteria to two opposing chemoattractant gradients depend on the chemoreceptor ratio.

Escherichia coli chemotaxis has long served as a simple model of environmental signal processing, and bacterial responses to single chemical gradients are relatively well understood. Less is known about the chemotactic behavior of E. coli in multiple chemical gradients. In their native environment, cells are often exposed to multiple chemical stimuli. Using a recently developed microfluidic chemotaxis device, we exposed E. coli cells to two opposing but equally potent gradients of major attractants, methyl-aspartate and serine. The responses of E. coli cells demonstrated that chemotactic decisions depended on the ratio of the respective receptor number of Tar/Tsr. In addition, the ratio of Tar to Tsr was found to vary with cells' growth conditions, whereby it depended on the culture density but not on the growth duration. These results provide biological insights into the decision-making processes of chemotactic bacteria that are subjected to multiple chemical stimuli and demonstrate the importance of the cellular microenvironment in determining phenotypic behavior.

An agarose-based microfluidic platform with a gradient buffer for 3D chemotaxis studies.

The current state-of-art in 3D microfluidic chemotaxis device (microFCD) is limited by the inherent coupling of the fluid flow and chemical concentration gradients. Here, we present an agarose-based 3D microFCD that decouples these two important parameters, in that the flow control channels are separated from the cell compartment by an agarose gel wall. This decoupling is enabled by the transport property of the agarose gel, which-in contrast to the conventional microfabrication material such as polydimethylsiloxane (PDMS)-provides an adequate physical barrier for convective fluid flow while at the same time readily allowing protein diffusion. We demonstrate that in this device, a gradient can be pre-established in an agarose layer above the cell compartment (a gradient buffer) before adding the 3D cell-containing matrix, and the dextran (10 kDa) concentration gradients can be re-established within 10 min across the cell-containing matrix and remain stable indefinitely. We successfully quantified the chemotactic response of murine dendritic cells to a gradient of CCL19, an 8.8 kDa lymphoid chemokine, within a type I collagen matrix. This model system is easy to set up, highly reproducible, and will benefit research on 3D chemoinvasion studies, for example with cancer cells or immune cells. Because of its gradient buffering capacity, it is particularly suitable for studying rapidly migrating cells like mature dendritic cells and neutrophils.

Contact line pinning by microfabricated patterns: effects of microscale topography.

We study how the microscale topography of a solid surface affects the apparent advancing and receding angles at the contact line of a liquid drop pinned to this surface. Photolithographic methods are used to produce continuous circular polymer rings of varying cross-sectional size and shape on hydrophilic silicon wafer surfaces. Drops of water and glycerol are dispensed into the areas bounded by these rings, and critical apparent advancing and receding angles are measured and correlated with the parameters that characterize the ring cross section. For much of the examined parameter space, the apparent critical angles are independent of ring height and width and are determined primarily by the slope of the ring's sidewalls, consistent with a model by Gibbs. For ring heights below a few micrometers, the critical angles decrease below the values predicted by the sidewall slopes alone. These results provide data for calculation of hysteresis on naturally rough surfaces and demonstrate a simple method for controlling and enhancing contact line pinning on solid surfaces.

Logarithmic sensing in Escherichia coli bacterial chemotaxis.

We studied the response of swimming Escherichia coli (E. coli) bacteria in a comprehensive set of well-controlled chemical concentration gradients using a newly developed microfluidic device and cell tracking imaging technique. In parallel, we carried out a multi-scale theoretical modeling of bacterial chemotaxis taking into account the relevant internal signaling pathway dynamics, and predicted bacterial chemotactic responses at the cellular level. By measuring the E. coli cell density profiles across the microfluidic channel at various spatial gradients of ligand concentration grad[L] and the average ligand concentration [L] near the peak chemotactic response region, we demonstrated unambiguously in both experiments and model simulation that the mean chemotactic drift velocity of E. coli cells increased monotonically with grad [L]/[L] or approximately grad(log[L])--that is E. coli cells sense the spatial gradient of the logarithmic ligand concentration. The exact range of the log-sensing regime was determined. The agreements between the experiments and the multi-scale model simulation verify the validity of the theoretical model, and revealed that the key microscopic mechanism for logarithmic sensing in bacterial chemotaxis is the adaptation kinetics, in contrast to explanations based directly on ligand occupancy.

Controlling microdrop shape and position for biotechnology using micropatterned rings.

Photolithographic micropatterning is used to achieve topographic rather than chemical control of the static shape and position of microdrops on solid substrates in a gaseous ambient. Micrometer cross-section, millimeter-diameter circular rings with steep sidewalls strongly and robustly pin contact lines of nanoliter to 100 microliter liquid drops, increasing the maximum stable drop volume and eliminating contact line motion due to transient accelerations. Physical and chemical processes involving two-phase transport within these drops are more reproducible, and automated image analysis of the evolving drop contents is greatly simplified. This technique has particular promise for high-throughput protein solution screening in structural genomics and drug discovery.

Quantifying X-ray radiation damage in protein crystals at cryogenic temperatures.

The dependence of radiation damage to protein crystals at cryogenic temperatures upon the X-ray absorption cross-section of the crystal has been examined. Lysozyme crystals containing varying heavy-atom concentrations were irradiated and diffraction patterns were recorded as a function of the total number of incident photons. An experimental protocol and a coefficient of sensitivity to absorbed dose, proportional to the change in relative isotropic B factor, are defined that together yield a sensitive and robust measure of damage. Radiation damage per incident photon increases linearly with the absorption coefficient of the crystal, but damage per absorbed photon is the same for all heavy-atom concentrations. Similar damage per absorbed photon is observed for crystals of three proteins with different molecular sizes and solvent contents.

Crystal growth in X-ray-transparent plastic tubing: an alternative for high-throughput applications.

A modified capillary-growth method is described that has substantial advantages for standard and high-throughput protein crystal growth. Protein-containing drops are injected into vapor-permeable flexible X-ray-transparent polyester tubing. The protein concentration in the drop increases over time by water transport through the tubing wall at a rate controlled by the wall thickness and ambient relative humidity. Unlike in conventional vapor-diffusion growth, the evaporation rate from the drop is constant over a longer time period, providing more suitable conditions for nucleation, and can be controlled by varying the tubing thickness and surrounding humidity. In situ X-ray diffraction can be performed at room temperature or, by flash-cooling, at low temperatures. Compared with glass capillaries or thick-wall plastic tubing, sealing and handling the tubing and extracting crystals are much easier.