Micro-fabricated Structures Research Articles

Biomimicking the tumor microenvironment for biomechanical study of glioblastoma cells analyzed by single cell traction force microscopy

The structures of the tissue microenvironment influence cell function and behavior, both in physiological and pathological conditions. Cells experience and integrate a multitude of mechanical and physical cues from this 3D tissue microenvironment to adapt to organismal development. In turn, they respond by exerting forces, regulating their shape, internal cytoskeletal tension, and elasticmodulus. Disruption of the cellular forces, as well as variations in subcellular mechanical properties, can lead to altered pathophysiological conditions and the onset of diseases i.e., cancer. Diseases like cancer are characterized by dramatic changes in cell and tissue mechanics, and dysregulation of forcesat the cell and tissue level can activate mechanosensing to compromise tissue integrity and function and promote disease progression. Cells can move by exerting traction forces to their environment. These can be assessed by using microfabricated substrates and improved computational approaches, enabling the characterization of the biomechanical forces generated by single cells cultured in defined microenvironments. This provides valuable insights into the malignancy of cancer cells. However, the conventional microfabricated structures i.e., 2D substrates are far from matching the complexities present in vivo. Hence, there is a need for biomimicking the natural surfaces for cellular applications. We here show the traction forces induced by single glioblastoma cells in 3D tumor microenvironment-inspired scaffolds i.e., collagen in comparison with 2D collagen coating. The dimensionality of cell culture influences cell motility and cellular interaction with the surrounding cells and ECM. Cells grown on 2D scaffolds, adapt to the artificial environment and may no longer display characteristics of the original tumor. An attempt to develop 3D tumor microenvironment-inspired scaffolds and their functionality in unravelling the role of microenvironment on tumor cell behaviors are also examined. Characterizing the cues involved in glioblastoma cell migration could enable the scientific and medical community to develop better strategies to understand and treat brain cancer.

Compensating porosity gradient to produce flat, micromachined porous silicon structures

Abstract Obtaining flat micromachined porous silicon structures is extremely challenging due to the myriad of factors affecting film stress in such structures. In this work, the relationships between the current control during anodization, average porosity, and residual stress within porous silicon thin films and micro-fabricated structures were investigated. Using a combination of electron microscopy, surface profilometry and reflectance spectroscopy, the optimum conditions to produce near zero residual stress in thin films and micro-fabricated structures were determined. The residual stress was adjusted by a continuous variation of the anodization current in order to achieve flat structures. The flattest released porous silicon microbeams were 2.3 μm thick with a peak to valley height variation of only 72 nm over a length of 150 μm. These were achieved by using an initial current density of 20 mA/cm2 that was reduced down to 8 mA/cm2 during anodization. By using this method of reducing the current during anodization, the inherent high porosity at the porous-silicon/silicon interface was reduced, which also enabled a 36% increase in the film thickness before film delamination compared with using a constant current. These results provide a pathway to fabricate thick, optically flat micromachined multi-layer filters using a single base material (silicon) for the structural and released layers.

A Platform for Mechano(-Electrical) Characterization of Free-Standing Micron-Sized Structures and Interconnects.

A device for studying the mechanical and electrical behavior of free-standing micro-fabricated metal structures, subjected to a very large deformation, is presented in this paper. The free-standing structures are intended to serve as interconnects in high-density, highly stretchable electronic circuits. For an easy, damage-free handling and mounting of these free-standing structures, the device is designed to be fabricated as a single chip/unit that is separated into two independently movable parts after it is fixed in the tensile test stage. Furthermore, the fabrication method allows for test structures of different geometries to be easily fabricated on the same substrate. The utility of the device has been demonstrated by stretching the free-standing interconnect structures in excess of 1000% while simultaneously measuring their electrical resistance. Important design considerations and encountered processing challenges and their solutions are discussed in this paper.

Simple agarose micro-confinement array and machine-learning-based classification for analyzing the patterned differentiation of mesenchymal stem cells

The geometrical confinement of small cell colonies gives differential cues to cells sitting at the periphery versus the core. To utilize this effect, for example to create spatially graded differentiation patterns of human mesenchymal stem cells (hMSCs) in vitro or to investigate underpinning mechanisms, the confinement needs to be robust for extended time periods. To create highly repeatable micro-fabricated structures for cellular patterning and high-throughput data mining, we employed here a simple casting method to fabricate more than 800 adhesive patches confined by agarose micro-walls. In addition, a machine learning based image processing software was developed (open code) to detect the differentiation patterns of the population of hMSCs automatically. Utilizing the agarose walls, the circular patterns of hMSCs were successfully maintained throughout 15 days of cell culture. After staining lipid droplets and alkaline phosphatase as the markers of adipogenic and osteogenic differentiation, respectively, the mega-pixels of RGB color images of hMSCs were processed by the software on a laptop PC within several minutes. The image analysis successfully showed that hMSCs sitting on the more central versus peripheral sections of the adhesive circles showed adipogenic versus osteogenic differentiation as reported previously, indicating the compatibility of patterned agarose walls to conventional microcontact printing. In addition, we found a considerable fraction of undifferentiated cells which are preferentially located at the peripheral part of the adhesive circles, even in differentiation-inducing culture media. In this study, we thus successfully demonstrated a simple framework for analyzing the patterned differentiation of hMSCs in confined microenvironments, which has a range of applications in biology, including stem cell biology.

Hydrodynamic Trapping of Swimming Bacteria by Convex Walls.

Swimming bacteria display a remarkable tendency to move along flat surfaces for prolonged times. This behavior may have a biological importance but can also be exploited by using microfabricated structures to manipulate bacteria. The main physical mechanism behind the surface entrapment of swimming bacteria is, however, still an open question. By studying the swimming motion of Escherichia coli cells near microfabricated pillars of variable size, we show that cell entrapment is also present for convex walls of sufficiently low curvature. Entrapment is, however, markedly reduced below a characteristic radius. Using a simple hydrodynamic model, we predict that trapped cells swim at a finite angle with the wall and a precise relation exists between the swimming angle at a flat wall and the critical radius of curvature for entrapment. Both predictions are quantitatively verified by experimental data. Our results demonstrate that the main mechanism for wall entrapment is hydrodynamic in nature and show the possibility of inhibiting cell adhesion, and thus biofilm formation, using convex features of appropriate curvature.

Finite Element Modelling To Evaluate the Cross-plane Thermal conductivity and Seebeck Coefficient of Ge/SiGe Heterostructure

Finite element modelling (FEM) is used to evaluate a new experimental procedure for extracting the thermal conductivity and Seebeck coefficients from micro-fabricated structures with integrated heaters, thermometers and Ohmic contacts. This experimental technique was designed to simultaneously produce and measure the differential temperature and voltage output for the determination of both thermoelectric properties of Ge/SiGeheterostructures.This study demonstrates the reliability of the FEM modelling by comparing the simulation results with experiment. It demonstrates how FEM can be implemented to complement the experiments: it can be used to observe the heat distribution in the heterostructure. Therefore all the mechanisms of parasitic heat loss in the devices can be evaluated in order to improve the accuracy of the extraction of thermal coefficients and especially the thermal conductivity. This will allow for further analysis to be taken to optimize the ZT and power factor for the Ge/SiGeheterostructures.The micro-fabricated structures consisted of an etched mesa of the Ge/SiGeheterostructure with Ohmic contacts, for measuring the voltages, at the top and bottom. On the top of the mesa, a 30nm thick Si3N4 insulator was applied followed by a Ti/Pt thermometer, another 70nm of Si3N4 and then a NiCr heater. A second thermometer was micro-fabricated at the bottom of the mesa to allow the ▵T across the material to be measured as a function of the heater power [1]. The results obtained from the FEM studies closely matches the experimental results in [1].

Shaping living tissues using microfabricated structures

In this paper, we present a technique for shaping the morphology of living Multi Cellular Tumour Spheroids (MCTS) by using micro-engineered structures. To that aim we created biocompatible, high aspect ratio, polydimethylsiloxane (PDMS) microstructures, that are compatible with the size of MCTS. We show that these microstructures can conform MCTS into pre-determined shapes by confining them, under cell culture conditions. This moulding of a living growing tissue by PDMS microstructures needs optimization of surface conditions, in order to favour the wetting of the mould by the cells, while preventing their attachment to the confining structures. An adequate treatment of PDMS with non-adhesive coating turned out to be mandatory for combining a successful shaping and a soft unmoulding of the engineered tissue. After unmoulding, the tissues are still viable and can be further cultured. Our work demonstrates that very sharp corners can be moulded on originally spherical aggregates of cells and that freestanding cell architectures of arbitrary shapes can be generated. This bio-moulding process can therefore be used for the investigation of the rheological properties of tissues as well as morphogenesis mechanisms by measuring the post-moulding evolution of the engineered shape.

Theme Issue in memory of Tom Duke

This Theme Issue is dedicated to the memory of Tom Duke, who passed away in 2012 at the age of 48. Tom made lasting contributions to a broad range of topics and was highly regarded for his very elegant approaches that unravel key physical principles underlying the function of biological systems.

2D12 微細加工ハイドロゲル構造を用いた血管組織モデルの作製(OS9-1:バイオMEMS(1))

2D12 微細加工ハイドロゲル構造を用いた血管組織モデルの作製(OS9-1:バイオMEMS(1))

Combined microfabrication and electrospinning to produce 3-D architectures for corneal repair

Corneal stem cell niches are located within the limbus of the eye and are believed to play an important role in corneal regeneration. These niches are often lost in corneal disease or trauma. Our work explores the design of artificial limbal stem cell niches by the fabrication of biodegradable electrospun rings containing bespoke microfeatures. In creating artificial niches, we seek to provide a physically protective environment for limbal cells to act as a cell reservoir for tissue regeneration purposes. This study describes the first step in this challenge to produce structures which structurally approximate to the limbal niches. This was achieved using a combination of electrospinning and microfabrication. Initial microfabricated structures were developed using microstereolithography via a layer-by-layer photocuring approach based on the patterning of photocurable polymers, in this case polyethylene glycol diacrylate. This was then used as a template on which to electrospin a biodegradable membrane of poly(lactic-co-glycolic acid) 50:50, which incorporates the features of the underlying microfabricated structures. The study describes preliminary evaluation of these constructs using rabbit limbal epithelial and stromal cells.

Controlled Magnetic Propulsion of Floating Polymeric Two-Dimensional Nano-Objects

Soft-bodied magnetically actuated microrobots could be developed by including controlled dispersions of magnetic nanoparticles into polymeric micro-fabricated structures. The characterization and actuation of magnetically active soft-bodied microrobots by tailored magnetic fields is, thus, a key issue for the design, full control and further development of these mobile micro-systems. In this work, the authors demonstrate the predictable and controllable transportation of polymeric flexible nanofilms embedding super-paramagnetic nanoparticles, by developing and quantitatively validating a model of the magnetic force acting on such structures, thus paving the way towards the wireless magnetic actuation of soft-bodied microrobots. The magnetic forces generated in our experimental conditions range from about 10 –9 to 10–6 N, with typical velocities for the nanofilms ranging between about 0.1 and 2.3 mm/s. For the entire range, a good agreement between theoretical model predictions and measured data is obtained (average normalized error δ =3. 65%). The proposed approach for microrobotic development targets challenging environments, where keywords are liquid or wet micro-structured environments, like in biomedical applications.

A microprobe technique for simultaneously measuring thermal conductivity and Seebeck coefficient of thin films

We demonstrate a microprobe technique that can simultaneously measure thermal conductivity κ and Seebeck coefficient α of thin films. In this technique, an alternative current joule-heated V-shaped microwire that serves as heater, thermometer and voltage electrode, locally heats the thin film when contacted with the surface. The κ is extracted from the thermal resistance of the microprobe and α from the Seebeck voltage measured between the probe and unheated regions of the film by modeling heat transfer in the probe, sample and their contact area, and by calibrations with standard reference samples. Application of the technique on sulfur-doped porous Bi2Te3 and Bi2Se3 films reveals α=−105.4 and 1.96 μV/K, respectively, which are within 2% of the values obtained by independent measurements carried out using microfabricated test structures. The respective κ values are 0.36 and 0.52 W/mK, which are significantly lower than the bulk values due to film porosity, and are consistent with effective media theory. The dominance of air conduction at the probe-sample contact area determines the microscale spatial resolution of the technique and allows probing samples with rough surfaces.

Theoretical and experimental investigation of spatial temperature gradient effects on cells using a microfabricated microheater platform

While the cellular interaction with the chemical microenvironment has been studied in detail in the past, the effect of thermal signals and gradients on cells is not well understood. While almost all biological macrosystems exhibit temperature sensitivity, this has not been studied much at the cellular level. The capability of precise control of temperature and heat flux at the scale of a few microns using microfabricated structures makes microelectromechanical systems (MEMS) ideal for investigating these effects. This work describes a MEMS-based microheater platform capable of subjecting surface-adhered cells to microscale temperature gradients. This microdevice comprises a free-standing thin film based microheater platform. The design and microfabrication of this microdevice are described. A thermal conduction model is developed in order to thermally characterize the microheater platform. Protocols for culturing cells on the microheater platform are developed, making it possible to adhere cells on the microheater surface and subject them to a desired spatial temperature gradient. Experiments demonstrating the spatial control of cell viability using the microheater device are described. This is followed by experiments that explore the possibility of cellular growth in response to thermal gradients, similar to chemotaxis. Thermotactic effect is well known at the organismal level. However, cells used in this work did not exhibit any thermotactic effect. A theoretical model for predicting cell response to a spatial temperature gradient in its microenvironment is proposed. The model is based on the effect of the temperature gradient on the chemical sensory process of the cell. The model predicts that in addition to the thermal gradient, the thermotactic effect also depends on a number of other parameters that have not been well characterized in the past. The microheater platform described in this work offers a fundamental new experimental tool with which to explore the interaction of cellular systems with their thermal microenvironment. The theoretical model of thermotaxis developed here furthers the understanding of the function of a cell as a thermal and chemical sensor.

Practical, Microfabrication-Free Device for Single-Cell Isolation

Microfabricated devices have great potential in cell-level studies, but are not easily accessible for the broad biology community. This paper introduces the Microscale Oil-Covered Cell Array (MOCCA) as a low-cost device for high throughput single-cell analysis that can be easily produced by researchers without microengineering knowledge. Instead of using microfabricated structures to capture cells, MOCCA isolates cells in discrete aqueous droplets that are separated by oil on patterned hydrophilic areas across a relatively more hydrophobic substrate. The number of randomly seeded Escherichia coli bacteria in each discrete droplet approaches single-cell levels. The cell distribution on MOCCA is well-fit with Poisson distribution. In this pioneer study, we created an array of 900-picoliter droplets. The total time needed to seed cells in ∼3000 droplets was less than 10 minutes. Compared to traditional microfabrication techniques, MOCCA dramatically lowers the cost of microscale cell arrays, yet enhances the fabrication and operational efficiency for single-cell analysis.

Current Challenges in Cartilage Tissue Engineering: A Review of Current Cellular-Based Therapies

Cellular-based therapies for cartilage tissue engineering have evolved quite significantly in the past decades. The realization that such an endeavor requires the acquisition of an adequate stem or progenitor cell population, techniques to effectively maintain or induce the desired phenotype and efficient culturing and implantation conditions have led researchers to develop a wide variety of protocols to approach the issue. Originally, cells taken from donor grafts of healthy cartilage were used as the cell source for tissue engineering constructs. Since then, however, researchers have moved on to cells from the adipose tissue, embryonic tissues, bone marrow and other sources. Similarly, as the potential for different cell sources to generate functional mesenchymal lineage tissues are discovered, several differentiation and phenotypic maintainance stimuli have been explored to optimize the resulting grafts. Physical stimuli in the form of mechanical compression as well as chemical stimuli in the form of growth factor cocktails have all proven effective in the induction of cells into the chondrogenic lineage to varying degrees. Finally, substrate expansion of these cells in materials ranging from naturally occuring ploymers such as fibrin, to synthetic and micro-fabricated structures is an active field of research. This review will discuss the most recent methods being utilized for the initiation of chondrogenesis from various stem cell sources as well as some methods being explored in our laboratory.

An experimental investigation into micro-fabricated solid oxide fuel cells with ultra-thin La 0.6Sr 0.4Co 0.8Fe 0.2O 3 cathodes and yttria-doped zirconia electrolyte films

Thin-film solid oxide fuel cells (SOFCs) were fabricated with both Pt and mixed conducting oxide cathodes using sputtering, lithography, and etching. Each device consists of a 75–150 nm thick yttria-stabilized zirconia (YSZ) electrolyte, a 40–80 nm porous Pt anode, and a cathode of either 15–150 nm dense La 0.6Sr 0.4Co 0.8Fe 0.2O 3− δ (LSCF) or 130 nm porous Pt. Maximum powers produced by the cells are found to increase with temperature with activation energies of 0.94–1.09 eV. At 500 °C, power densities of 90 and 60 mW cm −2 are observed with Pt and LSCF cathodes, respectively, although in some conditions LSCF outperforms Pt. Several device types were fabricated to systematically investigate electrical properties of components of these fuel cells. Micro-fabricated YSZ structures contacted on opposite edges by Pt electrodes were used to study temperature-dependent in-plane conductivity of YSZ as a function of lateral size and top and bottom interfaces. Si/Si 3N 4/Pt and Si/Si 3N 4/Au capacitor structures are fabricated and found to explain certain features observed in impedance spectra of in-plane and fuel cell devices containing silicon nitride layers. The results are of relevance to micro-scale energy conversion devices for portable applications.

Effect of shape and coating of a subretinal prosthesis on its integration with the retina

Retinal stimulation with high spatial resolution requires close proximity of electrodes to target cells. This study examines the effects of material coatings and 3-dimensional geometries of subretinal prostheses on their integration with the retina. A trans-scleral implantation technique was developed to place microfabricated structures in the subretinal space of RCS rats. The effect of three coatings (silicon oxide, iridium oxide and parylene) and three geometries (flat, pillars and chambers) on the retinal integration was compared using passive implants. Retinal morphology was evaluated histologically 6 weeks after implantation. For 3-dimensional implants the retinal cell phenotype was also evaluated using Computational Molecular Phenotyping. Flat implants coated with parylene and iridium oxide were generally well tolerated in the subretinal space, inducing only a mild gliotic response. However, silicon-oxide coatings induced the formation of a significant fibrotic seal around the implants. Glial proliferation was observed at the base of the pillar electrode arrays and inside the chambers. The non-traumatic penetration of pillar tips into the retina provided uniform and stable proximity to the inner nuclear layer. Retinal cells migrated into chambers with apertures larger than 10 μm. Both pillars and chambers achieved better proximity to the inner retinal cells than flat implants. However, isolation of retinal cells inside the chamber arrays is likely to affect their long-term viability. Pillars demonstrated minimal alteration of the inner retinal architecture, and thus appear to be the most promising approach for maintaining close proximity between the retinal prosthetic electrodes and target neurons.

Microtechnologies for membrane protein studies.

Despite the rapid and enormous progress in biotechnologies, the biochemical analysis of membrane proteins is still a difficult task. The presence of the large hydrophobic region buried in the lipid bilayer membrane (transmembrane domain) makes it difficult to analyze membrane proteins in standard assays developed for water-soluble proteins. To handle membrane proteins, the lipid bilayer membrane may be used as a platform to sustain their functionalities. Relatively slow progress in developing micro total analysis systems (μTAS) for membrane protein analysis directly reflects the difficulty of handling lipid membranes, which is a common problem in bulk measurement technologies. Nonetheless, researchers are continuing to develop efficient and sensitive analytical microsystems for the study of membrane proteins. Here, we review the latest developments, which enable detection of events caused by membrane proteins, such as ion channel current, membrane transport, and receptor/ligand interaction, by utilizing microfabricated structures. High-throughput and highly sensitive detection systems for membrane proteins are now becoming a realistic goal. Although most of these systems are still in the early stages of development, we believe this field will become one of the most important applications of μTAS for pharmaceutical and clinical screenings as well as for basic biochemical research.

Preparation of micro-fabricated biodegradable polymeric structures using NCDS

PLGA scaffolds were prepared using a nano-composite deposition system (NCDS). 5-Fluorouracil (5-FU) was used as a model drug. Hydroxyapatite (HA) was included in the scaffolds to improve the mechanical properties of the scaffolds and modulate the release of 5-FU from the scaffolds. 5-FU and HA were dispersed well in the prepared scaffolds when evaluated with SEM, FT-IR, XRPD and DSC. The release of 5-FU from the prepared scaffolds consisting of different compositions was determined using 40 mL PBS as the medium. The release profiles of 5-FU from PLGA scaffolds followed the typical triphasic release pattern. The addition of HA to the compositions increased the release rate of 5-FU from the scaffolds and improved the mechanical properties of the scaffolds, while it retarded the degradation of PLGA. Therefore, NCDS could be a good system to prepare polymeric implants of various shapes with different drug release patterns.

Elastic–plastic modeling of heat-treated bimorph micro-cantilevers

This paper models the residual stress distributions within micro-fabricated bimorph cantilevers of varying thickness. A contact model is introduced to calculate the influence of contact on the residual stress following a heat treatment process. An analytical modeling approach is adopted to characterize bimorph cantilevers composed of thin Au films deposited on thick poly-silicon or silicon-dioxide beams. A thermal elastic–plastic finite element model (FEM) is utilized to calculate the residual stress distribution across the cantilever cross-section and to determine the beam tip deflection following heat treatment. The influences of the beam material and thickness on the residual stress distribution and tip deflections are thoroughly investigated. The numerical results indicate that a larger beam thickness leads to a greater residual stress difference at the interface between the beam and the film. The residual stress established in the poly-silicon cantilever is greater than that induced in the silicon-dioxide cantilever. The results confirm the ability of the developed thermal elastic–plastic finite element contact model to predict the residual stress distributions within micro-fabricated cantilever structures with high accuracy. As such, the proposed model makes a valuable contribution to the development of micro-cantilevers for sensor and actuator applications.