Application of Parylene C thin films in cardiac cell culturing

Abstract

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.