Philipp Julian Koester

Microneedle arrays for intracellular recording applications

This paper reports on the fabrication and application of microneedles for the electroporation of adherently growing cells and intracellular recording with focus on the influence on external factors on the cell behavior. Patch-on-chip methods such as patch-clamp have been applied mostly to individual cells in suspension. However, in the human body most of cells are adherently growing cells, which motivated the development of a new chip design. The chip contains an array of 64 microneedles occupying a total area of approximately 1 mm2. The microneedles are fabricated using dry etching of silicon, followed by an insulation, metallization and passivation. The passivation layer is opened at the tip of the needles in order to expose the metal for cell positioning via dielectrophoresis, cell electroporation, as well as intracellular recording. Various needles with diameters in the sub-micron range and heights below 10 mum have been fabricated. Heart muscle cells, fibroblasts, and primary neuronal cells of mice were grown on these microneedle arrays. To electrically access the intracellular space, the cells were electroporated with a voltage of plusmn2 V. Preliminary tests show that more than 80% of the cells could successfully be porated.

Recording electric potentials from single adherent cells with 3D microelectrode arrays after local electroporation

This short communication reports on the innovative method of the local micro-invasive needle electroporation (LOMINE) of single adherent cells. The investigation of cellular reactions in living cell cultures represents a fundamental method, e.g. for drug development and environmental monitoring. Existing classical methods for intracellular measurements using, e.g. patch clamp techniques are time-consuming and complex. Present patch-on-chip systems are limited to the investigation of single cells in suspension. Nevertheless, the most part of the cells of the human body is adherently growing. Therefore, we develop a new chip system for the growth of adherent cells with 64 micro-structured needle electrodes as well as 128 dielectrophoretic electrodes, located within a measuring area of 1 mm(2). With this analytical chip, the intracellular investigation of electro-chemical changes and processes in adherently growing cells will become possible in the near future. Here, we present first intracellular measurements with this chip system.

FIB Preparation and SEM Investigations for Three-Dimensional Analysis of Cell Cultures on Microneedle Arrays

We report the investigation of the interfaces between microneedle arrays and cell cultures in patch-on-chip systems by using Focused Ion Beam (FIB) preparation and Scanning Electron Microscopy (SEM). First, FIB preparations of micro chips are made to determine the size and shape of the designed microneedles. In this essay, we investigate the cell-substrate interaction, especially the cell adhesion, and the microneedles potential cell penetration. For this purpose, cross-sectional preparation of these hard/soft hybrid structures is performed by the FIB technology. By applying the FIB technology followed by high-resolution imaging with SEM, new insights into the cell-substrate interface can be received. One can clearly distinguish between cells that are only in contact with microneedles and cells that are penetrated by microneedles. A stack of slice images is collected by the application of the slice-and-view setup during FIB preparation and is used for three-dimensional reconstruction of cells and micro-needles.

Hollow Microneedle Electrode Arrays for Intracellular Recording Applications

This paper reports on the fabrication of hollow microneedle electrodes with fluidic channels arranged in 8×8 arrays. Features of these electrodes include (i) an increased surface area for improved intracellular potential measurements with simultaneous membrane cell poration capabilities, (ii) their potential use in highly parallel patch-clamp applications and (iii) the ability to efficiently inject reagents and extract cytoplasm into and from the cell interior, respectively. Three different fabrication processes to realize hollow microneedle electrode arrays with incorporated microfluidic components, as well as initial experiments with cell cultures, are presented.