Mechanotransduction and growth factor signalling to engineer cellular microenvironments

Engineering cellular microenvironments involves biochemical factors, the extracellular matrix (ECM) and the interaction with neighbouring cells. This progress report provides a critical overview of key studies that incorporate growth factor (GF) signalling and mechanotransduction into the design of advanced microenvironments. Materials systems have been developed for surface-bound presentation of GFs, either covalently tethered or sequestered through physico-chemical affinity to the matrix, as an alternative to soluble GFs. Furthermore, some materials contain both GF and integrin binding regions and thereby enable synergistic signalling between the two. Mechanotransduction refers to the ability of the cells to sense physical properties of the ECM and to transduce them into biochemical signals. Various aspects of the physics of the ECM, i.e. stiffness, geometry and ligand spacing, as well as time-dependent properties, such as matrix stiffening, degradability, viscoelasticity, surface mobility as well as spatial patterns and gradients of physical cues are discussed. To conclude, various examples illustrate the potential for cooperative signalling of growth factors and the physical properties of the microenvironment for potential applications in regenerative medicine, cancer research and drug testing

Design, manufacture and test for reliable 3D printed electronics packaging

The development of a surrogate modelling approach to aid design of 3D printed electronics packaging structures is presented, alongside a detailed overview of manufacture and reliability of a representative test structure. An overview of the current status in 3D printing in the electronics packaging sector is provided. Subsequently, a surrogate modelling approach for correlating thermomechanical stresses within a package to a number of design parameters is presented. This approach enables the design of a package to be considered in a more insightful manner and can additionally be integrated into condition based monitoring tools capable of enhancing product robustness. An overview of an advanced electronics packaging system capable of 3D printing electronics packages is presented. The system combines inkjet printing and curing of multiple materials, including conductive silver inks, with precision component placement, multi-material dispensing and 3D inspection systems to provide a highly flexible solution for rapid manufacture of electronics packages. Test structures manufactured using the system were subjected to a vigorous set of reliability tests. Details of the test regime and related results are presented. All tests were passed, indicating the robustness of the described manufacturing process. The key originality of the work is that it provides a comprehensive overview of the journey from design assessment an optimisation, through the manufacturing process and on to reliability testing. Areas of novelty in this work are associated with the development of fast, accurate surrogate models able to predict key reliability factors in response to a range of design parameters and insight into the development of a 3D manufacturing system for electronics packaging

Contour models of cellular adhesion

The development of traction-force microscopy, in the past two decades, has created the unprecedented opportunity of performing direct mechanical measurements on living cells as they adhere or crawl on uniform or micro-patterned substrates. Simultaneously, this has created the demand for a theoretical framework able to decipher the experimental observations, shed light on the complex biomechanical processes that govern the interaction between the cell and the extracellular matrix and offer testable predictions. Contour models of cellular adhesion, represent one of the simplest and yet most insightful approach in this problem. Rooted in the paradigm of active matter, these models allow to explicitly determine the shape of the cell edge and calculate the traction forces experienced by the substrate, starting from the internal and peripheral contractile stresses as well as the passive restoring forces and bending moments arising within the actin cortex and the plasma membrane. In this chapter I provide a general overview of contour models of cellular adhesion and review the specific cases of cells equipped with isotropic and anisotropic actin cytoskeleton as well as the role of bending elasticity.Comment: 24 pages, 9 figures. arXiv admin note: text overlap with arXiv:1304.107

Non-photolithographic plastic-mold-based fabrication of cylindrical and multi-tiered poly(dimethylsiloxane) microchannels for biomimetic lab-on-a-chip applications

To overcome the limitations of conventional lithography for generating cylindrical and multi-tiered microchannels, we demonstrate a facile and alternative route for non-photolithographic fabrication of plastic molds via micro-milling combined with hot embossing. First, semi-cylindrical negative channels were engraved on poly(methylmethacrylate) (PMMA) using a ball mill, and the obtained semi-cylindrical negative channel structure was transferred onto poly(ethyleneterephthalate) (PET) via hot embossing performed at a temperature intermediate between the glass transition temperature (Tg) values of the two thermoplastics. In this way, a positive semi-cylindrical channel structure was formed on the PET without distorting the original patterns on the PMMA. The PET mold with positive structures was then replicated onto poly(dimethylsiloxane) (PDMS) to produce negative semi-cylindrical channels, and by aligning two identical PDMS replicas, a cylindrical microchannel with a completely circular cross section was formed. Second, multi-tiered channel structures were readily obtained by controlling the depths of the microchannels in the micro-milling process. The effectiveness of the fabricated cylindrical and multi-tiered microchannels was evaluated by constructing a microvascular network and human liver sinusoid structure as proof-of-concept experiments. The simple fabrication and high precision in the resulting structures will pave the way for the construction of disposable biomimetic Lab-on-a-Chip (LOC) platforms with low manufacturing cost in a simple and facile manner feasible for mass production

Micropost Arrays to Advance Cell Handling

Mechanical engineering methods and microfabrication techniques offer powerful means for meeting biological challenges. In particular, microfabrication processes enable researchers to develop technologies at scales that are biologically relevant and advantageous. In this work, microfabricated posts were employed to advance cell handling capabilities in both static and dynamic (i.e., microfluidic) systems. Static, substrate-based biophysical properties influence diverse cellular processes. Methods for engineering micropost arrays enable microscale control over the biophysical characteristics of discrete topographic features. Here, unidirectional micropost array gradients of variable micropost stiffness and variable interpost spacing were constructed to regulate cell motility using two distinct biophysical cues: (i) gradients in substrate rigidity (i.e., via durotaxis - a subset of mechanotaxis), and (ii) variable spacing of substrate binding sites - via a phenomenon herein referred to as spatiotaxis. Micropost array stiffness gradients were designed with post-to-post differences in stiffness of 0.5 nN/µm, 2 nN/µm, 3 nN/µm, and 7.5 nN/µm. Bovine aortic endothelial cells (BAECs) seeded on micropost array gradients with variable micropost stiffness exhibited preferential cell migration in the direction of increasing micropost stiffness. Gradients of elliptical microposts further enhanced unidirectional guidance by limiting cellular movement perpendicular to the direction of increasing micropost stiffness. Micropost array spacing gradients were designed with average post-to-post differences in spacing of 10 nm, 20 nm, and 40 nm. Micropost array gradients with variable interpost spacing were found to promote BAEC migration in the direction of decreasing interpost spacing, which represents the first demonstration of unidirectional spatiotaxis. Higher gradient strengths were observed to enhance the aforementioned migratory behaviors for both biophysical cues. For substrates with simultaneous, anti-parallel stiffness and spatial stimuli, the spatial cues were found to dominate the migratory response. The micropost array gradient methodology offers a powerful technique for investigating the biophysical cellular response, while also providing the basis for new classes of passive substrates capable of directing cell motility in biological fields, such as biomaterials, tissue engineering, and regenerative medicine.In order to create high-speed lab-on-a-chip devices for quantitative cell biology, drug discovery, and molecular diagnostics, precision hydrodynamic controls of microparticles (e.g., cells and microbeads) are in critical demand. The ability to achieve multi-stage fluidic reaction processes for microparticles is integral to diverse chemical and biological applications; however, microfluidic particulate-based systems remain limited due to particle handling issues. In contrast to suspended cells, which are experimentally complex, microbeads offer a simplified example for initial demonstrations of microfluidic particulate handling. Thus, the ability to manipulate microbeads in microfluidic systems represents a fundamental first step toward advancing microfluidic cell handling. In this dissertation, microposts (15×15 µm2) were arrayed within microfluidic architectures (18 µm in height) to enhance microparticle handling and enable multi-stage fluidic reactions and analyses for suspended particles. The presented microfluidic systems were first characterized using suspended microbeads (15 µm in diameter); thereafter, the potential of employing the platforms for cell handling applications was also investigated using suspended BAECs. A resettable, hydrodynamic microparticle trapping system - termed micropost array trapping (µPAT) - was designed and demonstrated to accomplish controlled particulate arraying and microarray resettability by trapping-and-releasing both microbeads and cells. The µPAT technique was integrated into: (i) a dynamic microarray to detect multiple bio-molecules in parallel via molecular beacon probes conjugated to microbead substrates, and (ii) a "Microfluidic Ping Pong" (MPP) system to achieve multi-stage fluidic reactions under discontinuous flow conditions. As a demonstrative example, the MPP technique was employed to detect an inflammatory cytokine at 100 pM concentrations via an 11-stage aptamer beacon-based sandwich assay performed using microbeads. Additionally, a microfluidic micropost array railing (µPAR) system was developed to rapidly transport both microbeads and cells into adjacent flow streams under continuous flow conditions. To demonstrate the µPAR technique, a multiplexed layer-by-layer (LbL) molecular synthesis process (i.e., consisting of up to 18 fluidic stages) was accomplished on microbead substrates. This work represents the first demonstration of a microfluidic platform capable of railing either microbeads or cells into adjacent flow streams. Through improved microparticle handling in microfluidic systems, the presented methodologies could further extend the efficacy of dynamic cell-based and bead-based microarrays for applications in diverse chemical and biological fields

Micropost Arrays to Advance Cell Handling

Mechanical engineering methods and microfabrication techniques offer powerful means for meeting biological challenges. In particular, microfabrication processes enable researchers to develop technologies at scales that are biologically relevant and advantageous. In this work, microfabricated posts were employed to advance cell handling capabilities in both static and dynamic (i.e., microfluidic) systems. Static, substrate-based biophysical properties influence diverse cellular processes. Methods for engineering micropost arrays enable microscale control over the biophysical characteristics of discrete topographic features. Here, unidirectional micropost array gradients of variable micropost stiffness and variable interpost spacing were constructed to regulate cell motility using two distinct biophysical cues: (i) gradients in substrate rigidity (i.e., via durotaxis - a subset of mechanotaxis), and (ii) variable spacing of substrate binding sites - via a phenomenon herein referred to as spatiotaxis. Micropost array stiffness gradients were designed with post-to-post differences in stiffness of 0.5 nN/µm, 2 nN/µm, 3 nN/µm, and 7.5 nN/µm. Bovine aortic endothelial cells (BAECs) seeded on micropost array gradients with variable micropost stiffness exhibited preferential cell migration in the direction of increasing micropost stiffness. Gradients of elliptical microposts further enhanced unidirectional guidance by limiting cellular movement perpendicular to the direction of increasing micropost stiffness. Micropost array spacing gradients were designed with average post-to-post differences in spacing of 10 nm, 20 nm, and 40 nm. Micropost array gradients with variable interpost spacing were found to promote BAEC migration in the direction of decreasing interpost spacing, which represents the first demonstration of unidirectional spatiotaxis. Higher gradient strengths were observed to enhance the aforementioned migratory behaviors for both biophysical cues. For substrates with simultaneous, anti-parallel stiffness and spatial stimuli, the spatial cues were found to dominate the migratory response. The micropost array gradient methodology offers a powerful technique for investigating the biophysical cellular response, while also providing the basis for new classes of passive substrates capable of directing cell motility in biological fields, such as biomaterials, tissue engineering, and regenerative medicine.In order to create high-speed lab-on-a-chip devices for quantitative cell biology, drug discovery, and molecular diagnostics, precision hydrodynamic controls of microparticles (e.g., cells and microbeads) are in critical demand. The ability to achieve multi-stage fluidic reaction processes for microparticles is integral to diverse chemical and biological applications; however, microfluidic particulate-based systems remain limited due to particle handling issues. In contrast to suspended cells, which are experimentally complex, microbeads offer a simplified example for initial demonstrations of microfluidic particulate handling. Thus, the ability to manipulate microbeads in microfluidic systems represents a fundamental first step toward advancing microfluidic cell handling. In this dissertation, microposts (15×15 µm2) were arrayed within microfluidic architectures (18 µm in height) to enhance microparticle handling and enable multi-stage fluidic reactions and analyses for suspended particles. The presented microfluidic systems were first characterized using suspended microbeads (15 µm in diameter); thereafter, the potential of employing the platforms for cell handling applications was also investigated using suspended BAECs. A resettable, hydrodynamic microparticle trapping system - termed micropost array trapping (µPAT) - was designed and demonstrated to accomplish controlled particulate arraying and microarray resettability by trapping-and-releasing both microbeads and cells. The µPAT technique was integrated into: (i) a dynamic microarray to detect multiple bio-molecules in parallel via molecular beacon probes conjugated to microbead substrates, and (ii) a "Microfluidic Ping Pong" (MPP) system to achieve multi-stage fluidic reactions under discontinuous flow conditions. As a demonstrative example, the MPP technique was employed to detect an inflammatory cytokine at 100 pM concentrations via an 11-stage aptamer beacon-based sandwich assay performed using microbeads. Additionally, a microfluidic micropost array railing (µPAR) system was developed to rapidly transport both microbeads and cells into adjacent flow streams under continuous flow conditions. To demonstrate the µPAR technique, a multiplexed layer-by-layer (LbL) molecular synthesis process (i.e., consisting of up to 18 fluidic stages) was accomplished on microbead substrates. This work represents the first demonstration of a microfluidic platform capable of railing either microbeads or cells into adjacent flow streams. Through improved microparticle handling in microfluidic systems, the presented methodologies could further extend the efficacy of dynamic cell-based and bead-based microarrays for applications in diverse chemical and biological fields