Bruno LePioufle

Techniques for patterning and guidance of primary culture neurons on micro-electrode arrays

Abstract In this work, two different micro-patterning methods for use in the growth guidance of dissociated primary culture neuron cells are compared: open chemically patterned growth substrates and enclosed micro-fluidic channels. Open, chemically patterned growth substrates are prepared by photolithographically patterning perfluoropolymer barrier structures on glass substrates. Neural pathways are created when poly- l -lysine is selectively adsorbed on the glass to form a cytophilic growth matrix. Adsorption of albumin proteins on the perfluoropolymer regions renders the surface cytophobic. In a second method, a three-dimensional micro-fluidic system, fabricated from PDMS is tested as a way to guide neural growth through total confinement and for cell placement. Both of these methods are tested for alignment and compatibility on a commercially available micro-electrode array (MED64). Biological culture and imaging techniques are considered.

Cell Placement and Neural Guidance Using a Three-Dimensional Microfluidic Array

Several fabrication techniques for making three-dimensional arrays of micro-wells for biological cell patterning and single-neuron guidance are presented. Methods for making complex 3d high-aspect-ratio structures in poly-dimethylsiloxane (PDMS) membrane are explored. In this work, three-dimensional micro-molds are made directly on silicon wafers though inductively coupled plasma reactive ion etching (ICP-RIE), and also using SU-8 negative photoresist. Cell placement is achieved through an array of 50-µm square holes in a 150–100 µm thick PDMS membrane, which is placed on a glass substrate. Vertical holes in the membrane are linked by horizontal tunnels on the glass side of the membrane, for use in neural guidance or delivery of drugs or nutrients. The effectiveness of the membrane for cell placement, growth and guidance was tested using fluorescent yeast cells and PC12 neuronal cells.

Arrayed Micro Needles for Mechanical Gene Insertion

A novel approach to realize arrays of out of plane micro needles is given. The exterior shape of the micro needles is realized by dry etching using a RIE. The exterior shape is realized in silicon by dry etching using a SF6 and Argon plasma that achieve the progressive etching of the mask composed by silicon nitride and photoresist. The needles realized present an outer diameter of 900 nm for a height of about 7 microns. The needle is then realized by wet oxidation using a thin film of silicon nitride to perform a LOCOS at the end of the needle. The last step consists in selectively etch the Si3N4 and the silicon inside the needle by xenon difluoride.

Controlling Cell Development by Microfluidic Techniques: A Step Towards Whole-Cell Biosensors with Defined Biological Features

Several microfluidic systems were designed to accommodate cell cultures and control some aspects of cell development. In particular, the development of neuronal networks was guided over microelectrode arrays, and electrical activity of the cells was recorded over more than four weeks. Further work is currently being carried out to control some topological features of P19 cell populations during their differentiation into either neuron-like cells or cardiac myocytes.

Cell cultures over nanoneedle fields

In this paper a new nanostructured support for the culture of cells is presented. The support consists of fields of sharp and high-aspect-ratio nanoneedles. The support is obtained through a specifically developed process that allows controlling the nanoneedles’s densities and height. The nanoneedles are typically 10 µm high with tip diameters under 200 nm. Cell viability on this support was evaluated through long-term cells cultures. The narrow interface between the cells’ membrane and the nanoneedles has been carefully observed to conclude on the perforation of the cells’ membrane thanks to the sharp nanoneedles. Such a nanostructured chip, allowing specific interaction, opens the door to a large number of exciting and valuable applications such as nanoporation for transfection or internal cell potential recording.

Soft Lithographic Techniques for Guidance of Hippocampal Neurons on Micro-Electrode Arrays

In this work, different micropatterning methods were tested for use in patterning of primary culture neural cells. Etching of Teflon covered glass substrates was used to make shallow neural guides defined by hydrophobic/hydrophilic regions. A three-dimensional microfluidic system, fabricated from PDMS was tested as a way to guide neural growth through total confinement. These methods were tested for alignment on a commercially available microelectrode array (MED64). Biological culture and imaging techniques are considered.