Non-Invasive Analyses of Electrophysiological Properties of Primary Cell Culture

Abstract

The model of efficacy and toxicity testing has been largely based on a model of high quantities of low quality data gathered in vitro. Recent advances in nanotechnology offer an alternative strategy. Soft lithography has made microscale tissue engineering possible. It is now feasible to create the cellular microenvironments of healthy and diseased tissues and engineer biological systems with high fidelity. Further, development of multielectrode array (MEA) platforms by precise location of metal electrodes on glass substrates has proven to be a robust innovative tool to use with cells that generate electrical activity. In this study, we first fabricated polydimethylsiloxane (PDMS) stamps for microcontact (µC) printing extra cellular protein along microscale features through soft lithography techniques. Engineering anisotropic cardiac monolayers on glass substrate without an underlying of PDMS coating was achieved to mimic the aligned architecture of myocardium without the hydrophobic polymer. Direct microcontact printing on glass is an important development for engineering anisotropic cellular layers on top of MEAs. Additionally, we investigated physical and protein cues from extracellular matrix to engineer anisotropic cardiac tissues as highly aligned monolayers on top of MEA. Non-invasive measurements of beating rate and conduction velocity were collected over different days of culture to determine the ideal substrate. While anisotropic cardiac tissues started to delaminate in early time point on µC fibronectin and gelatin substrates, the anisotropic tissues stayed intact for a long-culture time on micromolded (µM) gelatin hydrogels and provided synchronized beating and steady conduction velocity that was close to the physiological values. Ultimately, 3D micromolded gelatin hydrogel that recapitulated myocardial stiffness improved the synchronicity and conduction velocity of neonatal rat ventricular myocytes (NRVM) without any stimulation. In a second study, we validated the usage of MEA to measure the electrical activity induced by glucose response in dissociated human islets. First, we solved the obstacle of adhering 3D islets on planar electrodes MEA by successfully dissociating the 3D islets into dissociated singular cells and small cell clusters, which helped the cells to adhere in a single layer for a long culture time. The planar cell contact on the electrodes allowed us to start testing islet functionality from the first day by recording the electrical activity with low glucose and then high glucose incubation. MEA recordings captured higher electrical activities of islet cells under high glucose than low glucose conditions. While traditional functional assay showed an insulin response to glucose challenge at only early time points, spiking activity from MEA recordings corresponding to high and low glucose was consistently recorded up to a week in culture. Overall, we report important developments in primary cardiac and islet cell culture techniques that further enable MEA as a non-invasive real time platform for functional evaluation of electrophysiology.</p