Application of Parylene C thin films in cardiac cell culturing

There are two main challenges when producing in vitro cell systems: first, to reconstitute the in situ cellular microenvironment, thus delivering more representative and reliable cell models for drug screening and disease modelling studies. Second, to record and quantify the electrical and chemical gradients across the culture. Ideally, both challenges should be accomplished within a single platform towards a lab-on-chip implementation. This research work investigates the application of Parylene C in cardiac cell scaffolding and its integrability with electrochemical monitoring technologies for measuring extracellular action potentials and pH. The surface properties of Parylene C in terms of water affinity, chemical composition and nanotopography were characterised before and after modifying the material's inherent hydrophobicity through oxygen plasma. A technology was developed to selectively alter the surface hydrophobicity of Parylene C through standard lithography and oxygen plasma, which is characterised by μm-resolution and long-term pattern stability, and can accurately control the extent of induced hydrophilicity, the pattern layout and 3-D geometry. The micro-engineered Parylene C films were employed as scaffolds for cardiac cells with immature physiological properties, such as neonatal rat ventricular myocytes (NRVM). The scaffolds promoted a more in situ cellular structure and organisation, while they improved important calcium (Ca2+) cycling parameters such as fluorescent amplitude, time to peak (Tp), time to 50% (T50) and 90% (T90) decay at 0.5-2 Hz field stimulation. The thickness of the patterned Parylene C films was found to regulate the shape of the cells by controlling their adhesion area on the Parylene substrate through a thickness-dependent hydrophobicity. NRVM on thin (2 μm) membranes tended to bridge across the hydrophobic areas and adopt a spread-out shape (average contact angle at the level of the nucleus was 64.51o). On the other hand, cells on thick (10 μm) films were mostly constrained on the hydrophilic areas and demonstrated a more elongated, cylindrical (in vivo-like) shape (average contact angle was 84.73o). The cylindrical shape and a significantly (p <0.05) denser microtubule structure in cells on thick films possibly suggest a more mature cardiomyocyte. However, there was no significant effect on the Ca2+ physiology between the two groups. The micro-patterning technology was able to deliver free-standing Parylene C thin films (2-10 μm) to study the effect of substrate elasticity and flexibility on the Ca2+ physiology of NRVM. Preliminary results showed that fluorescent amplitude and time to peak were improved in structured NRVM cultures on stand-alone Parylene films compared to rigid Parylene-coated glass surfaces. However, no such trend was present in Ca2+ release parameters (T50, T90). The flexibility of the culture substrate was also manipulated by employing free-standing micro-patterned Parylene C films of distinct thicknesses (2-10 μm), but did not affect the cellular Ca2+ physiology. Further biological validation is needed with a larger sample size to draw a certain conclusion. The cell patterning technology was transferred to commercially available planar Multi-Electrode arrays (MEAs) to demonstrate integrability of this method with existing monitoring tools. The micro-patterned MEAs induced anisotropic cardiomyocyte cultures, as they substantially increased the longitudinal-to-transverse velocity anisotropy ratios (1.09, n=4 to 1.69, n=2), promoting action potential propagation profiles that closer resembled native cardiac tissue. Furthermore, the micro-engineered MEAs were proven to be reusable, yielding a versatile and low-cost approach that is compatible with state-of-art recording equipment and can be employed as a more reliable, off-the-shelf tool for drug screening studies. Selective hydrophilic modification of Parylene C was also employed to activate locally the H+ sensing capacity of such films, implementing extended-gate pH sensors. The ability of Parylene C to act in a dual way - as an encapsulation material and as an active pH sensing membrane - was demonstrated. The material exhibited a distinguishable sensitivity dependent on the oxygen plasma recipe, relatively low drift rates and excellent encapsulation quality. Based on these principles, flexible Parylene-based high-density miniaturised electrode arrays were fabricated, employing Parylene as a flexible structure material and as a H+ sensing membrane for local detection of pH. The presented Parylene-based technology has the potential to deliver integrated lab-on-chip implementations for growing cells in vitro with controlled microtopography while monitoring the extracellular electrical and pH gradients across the culture in a non-invasive way, with application in drug screening and disease modelling.Open Acces

Mask-free laser lithography for rapid and low-cost microfluidic device fabrication

Copyright © 2018 American Chemical Society. 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

Oxygen plasma induced hydrophilicity of parylene-C thin films

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

Parylene C-Based Flexible Electronics for pH Monitoring Applications

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The dual role of Parylene C in chemical sensing: acting as an encapsulant and as a sensing membrane for pH monitoring applications

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. Towards 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

Surface and electrical characterization of Ag/AgCl pseudo-reference electrodes manufactured with commercially available PCB technologies

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Supplementary Information from 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 to 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 <i>within</i> artificial cells, as a route to increase the capabilities of artificial cells. In this feasibility study, we design and embed genetically engineered microbes (<i>Escherichia coli</i>) 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

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 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 &lt; 0.0001) and more organised sarcomere. The Ca2+ 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) Ca2+ release in response to caffeine (p &lt; 0.0001), suggesting improved SR Ca2+ 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