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Microfluidics has become recognized as a powerful platform technology associated with a constantly increasing array of applications across the life sciences. This surge of interest over recent years has led to an increased demand for microfluidic chips, resulting in more time being spent in the cleanroom fabricating devices using soft lithography—a slow and expensive process that requires extensive materials, training and significant engineering resources. This bottleneck limits platform complexity as a byproduct of lengthy delays between device iterations and affects the time spent developing the final application. To address this problem, we report a new, rapid, and economical approach to microfluidic device fabrication using dry resist films to laminate laser cut sheets of acrylic. We term our method laser lithography and show that our technique can be used to engineer 200 μm width channels for assembling droplet generators capable of generating monodisperse water droplets in oil and micromixers designed to sustain chemical reactions. Our devices offer high transparency, negligible device to device variation, and low X-ray background scattering, demonstrating their suitability for real-time X-ray-based characterization applications. Our approach also requires minimal materials and apparatus, is cleanroom free, and at a cost of around $1.00 per chip could significantly democratize device fabrication, thereby increasing the interdisciplinary accessibility of microfluidics.

Microfluidics has has enabled the generation of a range of single compartment and multicompartment vesicles and bilayer-delineated droplets that can be assembled in 2D and 3D. These model systems are becoming increasingly used as artificial cell chassis and as biomimetic constructs for assembling tissue models, engineering therapeutic delivery systems, and screening drugs. One bottleneck in developing this technology is the time, expertise, and equipment required for device fabrication. This has led to interest across the microfluidics community in using rapid prototyping to engineer microfluidic devices from computer-aided-design (CAD) drawings. We highlight how this rapid-prototyping revolution is transforming the fabrication of microfluidic devices for artificial cell construction in bottom-up synthetic biology. We provide an outline of the current landscape and present how advances in the field may give rise to the next generation of multifunctional biodevices, particularly with Industry 4.0 on the horizon. Successfully developing this technology and making it open-source could pave the way for a new generation of citizen-led science, fueling the possibility that the next multibillion-dollar start-up could emerge from an attic or a basement.

The design of membrane-based constructs with multiple compartments is of increasing importance given their potential applications as microreactors, as artificial cells in synthetic-biology, as simplified cell models, and as drug delivery vehicles. The emergence of droplet microfluidics as a tool for their construction has allowed rapid scale-up in generation throughput, scale-down of size, and control over gross membrane architecture. This is true on several levels: size, level of compartmentalisation and connectivity of compartments can all be programmed to various degrees. This tutorial review explains and explores the reasons behind this. We discuss microfluidic strategies for the generation of a family of compartmentalised systems that have lipid membranes as the basic structural motifs, where droplets are either the fundamental building blocks, or are precursors to the membrane-bound compartments. We examine the key properties associated with these systems (including stability, yield, encapsulation efficiency), discuss relevant device fabrication technologies, and outline the technical challenges. In doing so, we critically review the state-of-play in this rapidly advancing field.

There is increasing interest in constructing artificial cells by functionalising lipid vesicles with biological and synthetic machinery. Due to their reduced complexity and lack of evolved biochemical pathways, the capabilities of artificial cells are limited in comparison to their biological counterparts. We show that encapsulating living cells in vesicles provides a means for artificial cells to leverage cellular biochemistry, with the encapsulated cells serving organelle-like functions as living modules inside a larger synthetic cell assembly. Using microfluidic technologies to construct such hybrid cellular bionic systems, we demonstrate that the vesicle host and the encapsulated cell operate in concert. The external architecture of the vesicle shields the cell from toxic surroundings, while the cell acts as a bioreactor module that processes encapsulated feedstock which is further processed by a synthetic enzymatic metabolism co-encapsulated in the vesicle.

The effect of O2 and Ar plasma etching on poly(chloro‐p‐xylylene) (Parylene C) is thoroughly studied by atomic force microscopy, X‐ray photoelectron spectroscopy, and static contact angle measurements. Results indicate that O2 plasma changes the topography more drastically than Ar plasma. Furthermore, despite the fact that Ar plasma is expected to be chemically inert, both plasmas introduce O2 to the surface of the Parylene C films, while Ar plasma additionally reduces the amount of Cl present in the polymer. The effect on the viability of cultured cardiomyocytes is also examined, indicating that cells attach and survive both on Ar and O2 treated films in contrast to untreated Parylene. These observations can provide useful insight into the field of material science and tissue engineering.

Compartmentalization of biological content and function is a key architectural feature in biology, where membrane bound micro- and nanocompartments are used for performing a host of highly specialized and tightly regulated biological functions. The benefit of compartmentalization as a design principle is behind its ubiquity in cells and has led to it being a central engineering theme in construction of artificial cell-like systems. In this review, we discuss the attractions of designing compartmentalized membrane-bound constructs and review a range of biomimetic membrane architectures that span length scales, focusing on lipid-based structures but also addressing polymer-based and hybrid approaches. These include nested vesicles, multicompartment vesicles, large-scale vesicle networks, as well as droplet interface bilayers, and double-emulsion multiphase systems (multisomes). We outline key examples of how such structures have been functionalized with biological and synthetic machinery, for example, to manufacture and deliver drugs and metabolic compounds, to replicate intracellular signaling cascades, and to demonstrate collective behaviors as minimal tissue constructs. Particular emphasis is placed on the applications of these architectures and the state-of-the-art microfluidic engineering required to fabricate, functionalize, and precisely assemble them. Finally, we outline the future directions of these technologies and highlight how they could be applied to engineer the next generation of cell models, therapeutic agents, and microreactors, together with the diverse applications in the emerging field of bottom-up synthetic biology.

Polydimethylsiloxane (PDMS) is a dominant material in the fabrication of microfluidic devices to generate water-in-oil droplets, particularly lipid-stabilized droplets, because of its highly hydrophobic nature. However, its key property of hydrophobicity has hindered its use in the microfluidic generation of oil-in-water droplets, which requires channels to have hydrophilic surface properties. In this article, we developed, optimized, and characterized a method to produce PDMS with a hydrophilic surface via the deposition of polyvinyl alcohol following plasma treatment and demonstrated its suitability for droplet generation. The proposed method is simple, quick, effective, and low cost and is versatile with respect to surfactants, with droplets being successfully generated using both anionic surfactants and more biologically relevant phospholipids. This method also allows the device to be selectively patterned with both hydrophilic and hydrophobic regions, leading to the generation of double emulsions and inverted double emulsions.

Cell micropatterning has certainly proved to improve the morphological and physiological properties of cardiomyocytes in vitro; however, there is little knowledge on the single cell-scaffold interactions that influence the cells' development and differentiation in culture. In this study, we employ hydrophobic/hydrophilic micropatterned Parylene C thin films (2-10 μm) as cell microscaffolds that can control the morphology and microtubule density of neonatal rat ventricular myocytes (NRVM) by regulating their adhesion area on Parylene through a thickness-dependent hydrophobicity. Structured NRVM on thin films tend to bridge across the hydrophobic areas, demonstrating a more spread-out shape and sparser microtubule organization, while cells on thicker films adopt a cylindrical (in vivo-like) shape (contact angles at the level of the nucleus are 64.51° and 84.73°, respectively) and a significantly (p

This paper investigates the surface modification of Parylene-C thin films under various oxygen plasma treatment conditions, such as power intensity (50:400 W) and exposure time (1:20 min). The extent of hydrophilicity was investigated through contact angle measurements, and correlations between treatment parameters, film thickness, restoration of hydrophobicity and etching rates were experimentally established. We also demonstrate the selective modification of Parylene-C films, facilitating distinct hydrophilic and hydrophobic areas with μm-resolution that can be exploited in self-alignment applications.

The design of vesicle microsystems as artificial cells (bottom-up synthetic biology) has traditionally relied on the incorporation of molecular components to impart functionality. These cell mimics have reduced capabilities compared with their engineered biological counterparts (top-down synthetic biology), as they lack the powerful metabolic and regulatory pathways associated with living systems. There is increasing scope for using whole intact cellular components as functional modules within artificial cells, as a route to increase the capabilities of artificial cells. In this feasibility study, we design and embed genetically engineered microbes ( Escherichia coli ) in a vesicle-based cell mimic and use them as biosensing modules for real-time monitoring of lactate in the external environment. Using this conceptual framework, the functionality of other microbial devices can be conferred into vesicle microsystems in the future, bridging the gap between bottom-up and top-down synthetic biology.

Compartmentalised structures based on lipid-stabilised double emulsions (multisomes) have recently attracted great interest in translational healthcare as potential micro-bioreactors for the synthesis of high-end materials, in situ drug synthesis and delivery, encapsulation of small molecules and cells, and as building blocks for cell-like structures in bottom-up synthetic biology. In contrast to surfactant-stabilised systems, lipid-stabilised systems are particularly more challenging to fabricate, mainly because many materials and surface modification techniques are incompatible with lipids. This paper demonstrates a robust and versatile microfluidic technology for the automated generation of miniaturised multisomes of pL volume in high-throughput using biologically relevant phospholipids.

Lab-on-Chip is a technology that coul d potentially revolutionize medical Point-of-Care diagnostics. Cons iderable research effort is focused towards innovating production technologies that will make commer cial upscaling financially viable. Printed circuit board manufacturing techni ques offer several prospects in this field. Here, we present a novel approach to manufacturing Printed Ci rcuit Board (PCB)-based Ag/AgCl reference electrodes, an essential com ponent of biosensors. Our prot otypes were characterized both structurally and electri cally. Scanning Electron Mi croscopy (SEM) and X-Ray Photoelectron Spectroscopy (XPS) were empl oyed to evaluate the electrode surface characteristics. Electrical characterization was performed to determine stability and pH dependency. Finally, we demonstrate utilizat ion along with PCB pH sensors, as a step towards a fully integrated PCB platform, comp aring performance with discrete commercial reference electrodes.

Background: Scaffolds are a key component of tissue-engineered heart valves (TEHVs). Several approaches had been adopted in the design of scaffolds using both natural and synthetic resources. We have investigated the suitability of parylene C (PC), a vapor deposited polymeric material, for the use as a scaffold in TEHV. Aims: To evaluate the adsorption of extracellular matrix components onto plasma-activated PC and study the biocompatibility of PC by measuring cellular adhesion, viability, apoptosis, and phenotypic expression of valve endothelial and interstitial cells. Finally, the mechanical properties of PC were compared with those of native aortic valve cusp tissue. Methods: PC slides were plasma activated and then coated with gelatin, type I collagen, or fibronectin. Porcine pulmonary valve endothelial and interstitial cells were then grown on plasma oxidized PC with different types of coatings and their adhesion was observed after 20 h of incubation. Cell viability was tested using the MTS assay, and apoptosis was estimated using TUNEL staining. The mechanical properties of PC and valve tissue were measured using a Bose Mechanical Tester. Finally, cell-seeded PC films were exposed to pulsatile pressure and aortic shear stress, respectively, to test their durability in a dynamic environment. Results: Our findings show that collagen and fibronectin could bind to plasma oxidized PC. Both valve endothelial and interstitial cells adhered to protein-coated ECM. PC had a profile of mechanical stiffness and ultimate tensile strength that were comparable with or in excess of those seen in porcine aortic valve cusps. Cells were still attached to PC films after 3 days of exposure to up to 50 mmHg pulsatile pressure or aortic levels of shear stress. Conclusion: PC is a promising candidate for use as a scaffold in tissue engineering heart valves. Additional studies are required to determine both the durability and long-term performance of cell-seeded PC when in a similar hemodynamic environment to that of the aortic valve.

Current platforms for in vitro drug development utilize confluent, unorganized monolayers of heart cells to study the effect on action potential propagation. However, standard cell cultures are of limited use in cardiac research, as they do not preserve important structural and functional properties of the myocardium. Here we present a method to integrate a scaffolding technology with multi-electrode arrays and deliver a compact, off-the-shelf monitoring platform for growing biomimetic cardiac tissue. Our approach produces anisotropic cultures with conduction velocity (CV) profiles that closer resemble native heart tissue; the fastest impulse propagation is along the long axis of the aligned cardiomyocytes (CVL) and the slowest propagation is perpendicular (CVT), in contrast to standard cultures where action potential propagates isotropically (CVL ≈ CVT). The corresponding anisotropy velocity ratios (CVL/CVT = 1.38 – 2.22) are comparable with values for healthy adult rat ventricles (1.98 – 3.63). The main advantages of this approach are that (i) it provides ultimate pattern control, (ii) it is compatible with automated manufacturing steps and (iii) it is utilized through standard cell culturing protocols. Our platform is compatible with existing read-out equipment and comprises a prompt method for more reliable CV studies.

The objective of any cell culturing platform is to decipher the in vivo functionality of native tissue in order to deliver reliable cell models for disease and pharmacological studies and eventually in patient-specific tissue engineering. We present a new perspective in lab-on-chip implementations for cell culturing, emphasizing on a versatile technology for cell micropatterning that can integrate electrical and pH monitoring modalities to record extracellular activity. We employ Parylene C, a highly biocompatible material, as a flexible culture substrate that controls the cellular microtopography and promotes a more in vivo-like morphology of neonatal rat ventricular myocytes. Moreover, we transfer the patterning technology on commercially available Multi-Electrode arrays to highlight the potential of integration with products customly used for extracellular electrical recordings. Finally, we implement flexible Parylene sensors for spatiotemporal pH monitoring, using the material both as a support medium and as a sensing membrane. Integration of these three attributes may deliver a compact solution with high scientific and commercial impact.

We demonstrate a simple, accurate and versatile method to manipulate Parylene C, a material widely known for its high biocompatibility, and transform it to a substrate that can effectively control the cellular microenvironment and consequently affect the morphology and function of the cells in vitro. The Parylene C scaffolds are fabricated by selectively increasing the material's surface water affinity through lithography and oxygen plasma treatment, providing free bonds for attachment of hydrophilic biomolecules. The micro-engineered constructs were tested as culture scaffolds for rat ventricular fibroblasts and neonatal myocytes (NRVM), toward modeling the unique anisotropic architecture of native cardiac tissue. The scaffolds induced the patterning of extracellular matrix compounds and therefore of the cells, which demonstrated substantial alignment compared to typical unstructured cultures. Ca(2+) cycling properties of the NRVM measured at rates of stimulation 0.5-2 Hz were significantly modified with a shorter time to peak and time to 90% decay, and a larger fluorescence amplitude (p < 0.001). The proposed technique is compatible with standard cell culturing protocols and exhibits long-term pattern durability. Moreover, it allows the integration of monitoring modalities into the micro-engineered substrates for a comprehensive interrogation of physiological parameters.

Cardiac fibroblasts influence cardiomyocyte structure and function through direct physical interaction and/or by the secretion of soluble factors. A role for cardiac fibroblasts in cardiomyopathies has been proposed but clear mechanisms are still lacking. This in vitro study set out to characterise the influence of cardiac fibroblasts from patients with dilated cardiomyopathy on cardiomyocyte Ca2+ cycling, a fundamental mechanism of cardiac function universally altered in cardiac disease. Human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) were cultured with human DCM ventricular fibroblasts at a ratio of 2:1 (120,000 fibroblasts to 60,000 iPS-CMs) for 24 hours in three groups: iPS-CMs with fibroblast conditioned medium, co-cultured in transwells to allow bi-directional paracrine communication but prevent physical contact, and iPS-CMs in direct contact with fibroblasts. iPS-CMs alone were used as a baseline. iPS-CMs were field-stimulated at 1Hz and calcium transients were recorded optically. TGF-β, a cytokine previously shown to be important in fibroblast-myocyte interaction, was measured in culture supernate using an ELISA assay. Versus iPS-CMs alone, Ca2+ transient amplitude was reduced in the conditioned medium group but increased in co-culture (p

Parylene C has been extensively used as a biocompatible encapsulation material of implantable microdevices. Towards a new understanding of the material's potential, we demonstrate a versatile method that enables the deployment of the material both as an encapsulant and as a H002B; sensing membrane in a single flexible platform using discrete MOSFETs to evaluate its chemical sensing performance. A 40-electrode array was implemented through standard microfabrication techniques on a free-standing 5 μm Parylene film. A thin film (1 μm) of Parylene was finally deposited on top of the array to passivate the electrode tracks. O2 plasma treatment was employed to selectively functionalize Parylene's H002B; sensing capacity. Measured results indicate a chemical sensitivity of 22.8 mV/pH, while the device exhibits relatively low leakage currents (1.2-13.9 nA) and chemical drifts (10-32 mV/h) over a wide pH range (4-10), rendering Parylene a promising material in the field of flexible bio-sensors.

In this work, we demonstrate a new property of Parylene C emphasizing on its application in pH sensing technologies. For many decades the material has been extensively used as a biocompatible inert encapsulant of implantable micro-devices. Toward a new understanding of the material's potential, we explore the transformation of Parylene C from a passive encapsulation membrane into an active H + sensing membrane using discrete MOSFETs to evaluate its chemical sensing performance. We employ oxygen plasma treatment to functionalize Parylene's H + sensing capacity and enhance the chemical sensitivity, drift rates, and reliability of the sensing devices. Moreover, we demonstrate a versatile technique that enables the deployment of the material both as an encapsulant and as a sensing membrane in a single platform, in order to benefit from distinguishable and consistent sensitivities, and low leakage currents during pH measurements. Our investigation reveals that the selective modification of Parylene's surface chemistry yields reliable pH sensing devices, ensuring the best combination of sensitivity (16.3 mV/pH) and leakage currents (6–10 nA) over a reasonably wide pH range (4–10), while drift rates remain in low levels (2.5–20 mV/h). We believe that this study opens up new application horizons for Parylene, which is a new promising material in the emerging field of flexible electronics able to deliver low film thicknesses and high biocompatibility, while facilitating the application of mechanical stimulus.

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) have been widely proposed as in vitro models of myocardial physiology and disease. A significant obstacle, however, is their immature phenotype. We hypothesised that Ca2+ cycling of iPSC-CM is influenced by culture conditions and can be manipulated to obtain a more mature cellular behaviour. To test this hypothesis we seeded iPSC-CM onto fibronectin coated microgrooved polydimethylsiloxane (PDMS) scaffolds fabricated using photolithography, or onto unstructured PDMS membrane. After two weeks in culture, the structure and function of iPSC-CM were studied. PDMS microgrooved culture substrates brought about cellular alignment (p

Neonatal and stem cell-derived cardiomyocytes differ from adult heart cells in their structure and function, limiting their use in pharmacological studies. Establishment of anisotropic cultures can improve the functional properties of cardiomyocytes; however, the mechanism that links functional improvement with ultrastructural reorganisation is unclear. Microtubules act as a link between mechanical stimuli and functional response in cardiac cells but whether they are affected during cellular alignment in culture is unknown. In this study neonatal rat ventricular myocytes were cultured on micropatterned Parylene C constructs, with alternating hydrophilic-hydrophobic parallel lines, to induce cellular alignment. The cells formed an anisotropic monolayer, those on the 10μm thick constructs were more constrained within the lines and cylindrical in shape compared with the cells on 2μm thick constructs (p

In this paper, we demonstrate a technique for transforming commercially available neural probes used for electrical recordings, into chemical sensing devices for detection of ionic concentrations in electrolytes, with particular emphasis to pH. This transformation requires a single post-processing step to incorporate a thin indium tin oxide membrane for sensing H+. Measured results indicate a chemical sensitivity of 28 mV/pH, and relatively low leakage currents (2–10 nA) and drifts (1–10 mV/h). The proposed sensing device demonstrates the possibility of a low-cost implementation that can be reusable and thus versatile, with potential applications in real-time extracellular but mainly intracellular chemical monitoring.