Adhesion Stabilized en Masse Intracellular Electrical Recordings from Multicellular Assemblies

Abstract

Coordinated collective electrochemical signals in multicellular assemblies, such as ion fluxes, membrane potentials, electrical gradients, and steady electric fields, play an important role in cell and tissue spatial organization during many physiological processes like wound healing, inflammatory responses, and hormone release. This mass of electric actions cumulates in an en masse activity within cell collectives which cannot be deduced from considerations at the individual cell level. However, continuously sampling en masse collective electrochemical actions of the global electrochemical activity of large-scale electrically coupled cellular assemblies with intracellular resolution over long time periods has been impeded by a lack of appropriate recording techniques. Here we present a bioelectrical interface consisting of low impedance vertical gold nanoelectrode interfaces able to penetrate the cellular membrane in the course of cellular adhesion, thereby allowing en masse recordings of intracellular electrochemical potentials that transverse electrically coupled NRK fibroblast, C2C12 myotube assemblies, and SH-SY5Y neuronal networks of more than 200,000 cells. We found that the intracellular electrical access of the nanoelectrodes correlates with substrate adhesion dynamics and that penetration, stabilization, and sealing of the electrode–cell interface involves recruitment of surrounding focal adhesion complexes and the anchoring of actin bundles, which form a caulking at the electrode base. Intracellular recordings were stable for several days, and monitoring of both basal activity as well as pharmacologically altered electric signals with high signal-to-noise ratios and excellent electrode coupling was performed.

High‐Aspect‐Ratio Nanostructured Surfaces as Biological Metamaterials

“…Porous membranes can be used to define an initial template for nanostructures, a technique sometimes referred to as template synthesis. These membranes can be stochastically patterned, e.g., using track‐etched membranes or can originate from self‐organized processes such as the anodization of aluminum oxide …”

“…Tailoring the surface chemistry to promote cell adhesion to nanostructured surfaces consists of either creating a favorable electrostatic interaction between the cell and surface, or by replicating naturally occurring binding sites. Electrostatically charged synthetic polymers such as polylysines and polyornithines have been used to promote the adhesion of cells to diamond nanoneedles, quartz nanopillars, alumina nanostraws, gold nanoelectrodes, silicon nanowires, and polymer nanopillars (Figure E) . Staufer et al claimed that the cell–substrate adhesion created by coating their gold nanoelectrodes with poly‐ l ‐lysine was strong enough to cause widespread spontaneous membrane penetration .…”

“…Electrostatically charged synthetic polymers such as polylysines and polyornithines have been used to promote the adhesion of cells to diamond nanoneedles, quartz nanopillars, alumina nanostraws, gold nanoelectrodes, silicon nanowires, and polymer nanopillars (Figure E) . Staufer et al claimed that the cell–substrate adhesion created by coating their gold nanoelectrodes with poly‐ l ‐lysine was strong enough to cause widespread spontaneous membrane penetration . Amin et al used poly‐ dl ‐ornithine (a racemic mixture of both d and l forms of polyornithine) in combination with a selectively patterned nanopillar array to achieve controlled guidance of 90% of primary hippocampal neurons ( Figure ) …”

“…Motivations include studying the fundamental cell electrophysiology of cardiac, neural, and skeletal‐myotube cells, as well as developing platforms for high‐throughput drug screening . The spatially resolved, parallel, intimate interface of high‐aspect‐ratio nanostructures with cells and tissue is well suited for sensing complex neural network behaviors . These benefits have been recognized commercially, with a number of micro‐ and nanoneedle‐coated microelectrode arrays already on the market…”

“…There are a number of different nanoelectrode fabrication approaches that are also worth noting, including carbon‐based nanofibers and carbon‐nanotube‐coated micropillars, for the stimulation and sensing of hippocampal cells and slices. Electrodeposited gold nanoelectrodes have similarly been used for sensing and stimulating fibroblast, myotube, and neuronal assemblies . Gonzales et al recently demonstrated an interesting alternative to vertically aligned nanoelectrodes, instead fabricating horizontally orientated, high‐aspect‐ratio (25:1), suspended electrodes (named nano‐SPEARs), which they used to measure the electrophysiology of roundworms and other animals .…”