Tatiana Trantidou

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 < 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 < 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.

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.

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.

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.

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.

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.

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.

A lab-on-chip approach for monitoring the electrochemical activity of biorealistic cell cultures

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.

Free-standing parylene C thin films as flexible pH sensing membranes

Parylene C has been extensively used as a biocompatible encapsulation material of implantable microdevices. Towards a new understanding of the materials 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 Parylenes 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.

Sensing H+ with conventional neural probes

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.