New Directions for Artificial Cells Using Prototyped Biosystems.

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.

Mask-Free Laser Lithography for Rapid and Low-Cost Microfluidic Device Fabrication.

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.

Functionalizing cell-mimetic giant vesicles with encapsulated bacterial biosensors.

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.

Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules.

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.

Engineering Compartmentalized Biomimetic Micro- and Nanocontainers.

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.

Hydrophilic surface modification of PDMS for droplet microfluidics using a simple, quick, and robust method via PVA deposition.

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.

Surface Chemistry and Microtopography of Parylene C Films Control the Morphology and Microtubule Density of Cardiac Myocytes.

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 < 0.05) denser microtubule structure. Ion scanning microscopy on NRVM revealed that cells on thicker membranes were significantly (p < 0.05) smaller in volume, but more elongated. The cylindrical shape and a significantly denser microtubule structure indicate the ability to influence cardiomyocyte phenotype using patterning and manipulation of hydrophilicity. These combined bioengineering strategies are promising tools in the generation of more representative cardiomyocytes in culture.

Surface and Electrical Characterization of Ag/AgCl Pseudo-Reference Electrodes Manufactured with Commercially Available PCB Technologies.

Lab-on-Chip is a technology that could potentially revolutionize medical Point-of-Care diagnostics. Considerable research effort is focused towards innovating production technologies that will make commercial upscaling financially viable. Printed circuit board manufacturing techniques offer several prospects in this field. Here, we present a novel approach to manufacturing Printed Circuit Board (PCB)-based Ag/AgCl reference electrodes, an essential component of biosensors. Our prototypes were characterized both structurally and electrically. Scanning Electron Microscopy (SEM) and X-Ray Photoelectron Spectroscopy (XPS) were employed to evaluate the electrode surface characteristics. Electrical characterization was performed to determine stability and pH dependency. Finally, we demonstrate utilization along with PCB pH sensors, as a step towards a fully integrated PCB platform, comparing performance with discrete commercial reference electrodes.

Assessment of Parylene C Thin Films for Heart Valve Tissue Engineering.

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.

Biorealistic cardiac cell culture platforms with integrated monitoring of extracellular action potentials.

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.

Parylene C-based flexible electronics for pH monitoring applications.

Emerging materials in the field of implantable sensors should meet the needs for biocompatibility; transparency; flexibility and integrability. In this work; we present an integrated approach for implementing flexible bio-sensors based on thin Parylene C films that serve both as flexible support substrates and as active H(+) sensing membranes within the same platform. Using standard micro-fabrication techniques; a miniaturized 40-electrode array was implemented on a 5 µm-thick Parylene C film. A thin capping film (1 µm) of Parylene on top of the array was plasma oxidized and served as the pH sensing membrane. The sensor was evaluated with the use of extended gate discrete MOSFETs to separate the chemistry from the electronics and prolong the lifetime of the sensor. The chemical sensing array spatially maps the local pH levels; providing a reliable and rapid-response (<5 s) system with a sensitivity of 23 mV/pH. Moreover; it preserves excellent encapsulation integrity and low chemical drifts (0.26-0.38 mV/min). The proposed approach is able to deliver hybrid flexible sensing platforms that will facilitate concurrent electrical and chemical recordings; with application in real-time physiological recordings of organs and tissues.

The effect of microgrooved culture substrates on calcium cycling of cardiac myocytes derived from human induced pluripotent stem cells.

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 Ca(2+) 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 < 0.0001) and more organised sarcomere. The Ca(2+) cycling properties of iPSC-CM cultured on these substrates were significantly altered with a shorter time to peak amplitude (p = 0.0002 at 1 Hz), and more organised sarcoplasmic reticulum (SR) Ca(2+) release in response to caffeine (p < 0.0001), suggesting improved SR Ca(2+) cycling. These changes were not associated with modifications in gene expression. Whilst structured tissue culture may make iPSC-CM more representative of adult myocardium, further construct development and characterisation is required to optimise iPSC-CM as a model of adult myocardium.