Cell Membrane Disruption by Vertical Micro-/Nanopillars: Role of Membrane Bending and Traction Forces.

Gaining access to the cell interior is fundamental for many applications, such as electrical recording and drug and biomolecular delivery. A very promising technique consists of culturing cells on micro-/nanopillars. The tight adhesion and high local deformation of cells in contact with nanostructures can promote the permeabilization of lipids at the plasma membrane, providing access to the internal compartment. However, there is still much experimental controversy regarding when and how the intracellular environment is targeted and the role of the geometry and interactions with surfaces. Consequently, we investigated, by coarse-grained molecular dynamics simulations of the cell membrane, the mechanical properties of the lipid bilayer under high strain and bending conditions. We found out that a high curvature of the lipid bilayer dramatically lowers the traction force necessary to achieve membrane rupture. Afterward, we experimentally studied the permeabilization rate of the cell membrane by pillars with comparable aspect ratios but different sharpness values at the edges. The experimental data support the simulation results: even pillars with diameters in the micron range may cause local membrane disruption when their edges are sufficiently sharp. Therefore, the permeabilization likelihood is connected to the local geometric features of the pillars rather than diameter or aspect ratio. The present study can also provide significant contributions to the design of three-dimensional biointerfaces for tissue engineering and cellular growth.

Emergence of bursting activity in connected neuronal sub-populations.

Uniform and modular primary hippocampal cultures from embryonic rats were grown on commercially available micro-electrode arrays to investigate network activity with respect to development and integration of different neuronal populations. Modular networks consisting of two confined active and inter-connected sub-populations of neurons were realized by means of bi-compartmental polydimethylsiloxane structures. Spontaneous activity in both uniform and modular cultures was periodically monitored, from three up to eight weeks after plating. Compared to uniform cultures and despite lower cellular density, modular networks interestingly showed higher firing rates at earlier developmental stages, and network-wide firing and bursting statistics were less variable over time. Although globally less correlated than uniform cultures, modular networks exhibited also higher intra-cluster than inter-cluster correlations, thus demonstrating that segregation and integration of activity coexisted in this simple yet powerful in vitro model. Finally, the peculiar synchronized bursting activity shown by confined modular networks preferentially propagated within one of the two compartments ('dominant'), even in cases of perfect balance of firing rate between the two sub-populations. This dominance was generally maintained during the entire monitored developmental frame, thus suggesting that the implementation of this hierarchy arose from early network development.

Planar patch clamp for neuronal networks--considerations and future perspectives.

The patch-clamp technique is generally accepted as the gold standard for studying ion channel activity allowing investigators to either "clamp" membrane voltage and directly measure transmembrane currents through ion channels, or to passively monitor spontaneously occurring intracellular voltage oscillations. However, this resulting high information content comes at a price. The technique is labor-intensive and requires highly trained personnel and expensive equipment. This seriously limits its application as an interrogation tool for drug development. Patch-clamp chips have been developed in the last decade to overcome the tedious manipulations associated with the use of glass pipettes in conventional patch-clamp experiments. In this chapter, we describe some of the main materials and fabrication protocols that have been developed to date for the production of patch-clamp chips. We also present the concept of a patch-clamp chip array providing high resolution patch-clamp recordings from individual cells at multiple sites in a network of communicating neurons. On this chip, the neurons are aligned with the aperture-probes using chemical patterning. In the discussion we review the potential use of this technology for pharmaceutical assays, neuronal physiology and synaptic plasticity studies.

Deformation and adhesion of elastomer poly(dimethylsiloxane) colloidal AFM probes.

We report on the synthesis and characterization of elastomer colloidal AFM probes. Poly(dimethylsiloxane) microparticles, obtained by water emulsification and cross-linking of viscous prepolymers, are glued to AFM cantilevers and used for contact mechanics investigations on smooth substrates: in detail cyclic loading-unloading experiments are carried on ion-sputtered mica, the deformation rate and dwell time being separately controlled. We analyze load-penetration curves and pull-off forces with models due respectively to Zener; Maugis and Barquins; and Greenwood and Johnson and account for bulk creep, interfacial viscoelasticity, and structural rearrangements at the polymer-substrate interface. A good agreement is found between experiments and theory, with a straightforward estimation of colloidal probes' material parameters. We suggest the use of such probes for novel contact mechanics experiments involving fully reversible deformations at the submicrometer scale.