Nanoscale membrane actuator for in vitro mechano-stimuli responsive studies of neuronal cell networks on chip

In order to investigate the hypothesis that dynamic nanoscale stimuli can influence the functional response of the brain, in this paper we describe the development of a membrane actuator chip based on polydimethylsiloxane (PDMS) soft lithography. The chip exerts a local nanoscale mechanical load on an in vitro neuronal cell network by microfluidic pneumatic deformation of the membrane. The deformation provides a topographical change in the substrate as an input stimulus for the study of response functions of a neuronal cell network in vitro. Calcium ions (Ca²⁺) imaging within a neuronal cell network grown from dissociated cortical cells of the rat's brain used as a brain model indicates that a neural networks response can be provoked by means of our new method. This actuator chip provides a relatively mild and localised mechanical stimulus by means of a 2% elongation of the membrane's width during the application of a pressure pulse underneath the membrane using a microfluidic channel design. We found an average 50% increase of the intracellular Ca²⁺ flux activity for 2D neuronal cell networks among 4 independent samples cultured on flat membranes. Additionally, we have proven the applicability of the actuator chip for networks on nanogrooved membranes by the observation of Ca²⁺ traces and we also observed the Ca²⁺ waves response upon stimulation in a three dimensional (3D) in vivo-like neuronal cell network using Matrigel on flat membranes. Hence, the chip potentially provides a novel technology platform for the in vitro modelling of brain tissues with topographically and 3D hydrogel-defined network architectures.