Lars Lauer

PDMS device for patterned application of microfluids to neuronal cells arranged by microcontact printing.

A microfluidic device in polydimethylsiloxane (PDMS) consisting of an eight lines micro-injection array integrated in a base flow channel has been realized. The device is assembled from multiple PDMS parts, which have been moulded using notably micromachined masters in SU-8 photoresist. In contact with a planar substrate, up to eight independent laminar flow lines with cross-sections of 100 x 200 microm(2) can be generated. Dedicated for the application of pharmaceutical compounds to electrogenic cells in vitro, this device was tested with a neuronal cell line, Mz1-cells. These were cultured on lines of laminin deposited onto polystyrene substrates by microcontact printing. We were able to inject into this culture multiple lines of coloured PBS in parallel to the orientation of cellular growth. No mixing between the individual flow lines did occur.

Modulation of the Growth and Guidance of Rat Brain Stem Neurons Using Patterned Extracellular Matrix Proteins

Dissociated neuronal cultures on substrates patterned with extracellular matrix (ECM) proteins have yielded much information regarding the physiological characteristics of neuronal cells behaviour in vitro. However, neuronal patterning using long term embryonic brain slice cultures has not been comprehensively demonstrated to-date. Structuring was performed by micro contact printing of laminin. The slice cultures were evaluated by means of phase contrast microscopy at 3-22 days in culture. We were able to consistently achieve outgrowth of neurons, neurites and filopodia from brain stem slices cultured on ECM proteins structures of grid- and line-shapes. We believe that brain slice cultures on patterned substrates is a favourable approach to study functional synapses in vitro under defined conditions. The use of appropriate structures and the subsequent cell patterning may help to gain further understanding of axonal, dendritic and synaptic signal transductions and processes.

Micropatterned Substrates for the Growth of Functional Neuronal Networks of Defined Geometry

The in vitro assembly of neuronal networks with control over cell position and connectivity is a fascinating approach not only for topics in basic neuroscience research but also in diverse applications such as biosensors and tissue engineering. We grew rat embryonic cortical neurons on patterned substrates created by microcontact printing. Polystyrene was used as a cell repellent background, onto which a grid pattern of physiological proteins was applied. We printed laminin and a mixture of extracellular matrix proteins and additionally both systems mixed with polylysine. Attachment of cells to the pattern with high fidelity as well as the formation of chemical synapses between neighboring cells on the pattern could be observed in all four cases, but cell attachment was strongly increased on samples containing polylysine. Neurons grown on patterned substrates had a membrane capacity smaller than that of neurons on homogeneously coated controls, which we attributed to the geometrical restrictions, but did not differ either in resting membrane potential or in the quality of synapses they formed. We therefore believe that the cells attach and differentiate normally on the pattern and form functional, mature synapses following the predefined geometry.

Spot compliant neuronal networks by structure optimized micro-contact printing.

Neuronal cell growth in vitro can be controlled with micropatterned structures of extracellular matrix proteins such as laminin. This technique is a powerful tool for studying neuronal cell function in order to increase experimental reproducibility and to specifically design innovative experimental setups. In this paper the correlation between the structural dimensions of the ECM pattern and the shape of the resulting cellular network is analyzed. The aim of the present study was to position neuronal cell bodies as precisely as possible and to induce directed cell differentiation. PCC7-MzN cells were cultured on laminin patterns. The line width, node size and gap size in-between cell adhesion sites was varied systematically. Micrographs of the samples were taken and statistically analyzed using Students t-test and linear correlation methods. Precise cell positioning has successfully been performed and evidence for controlled neuronal polarization has been found. With a structure geometry of 4 microm line width, 20 microm node size and 10 microm gap size a nodal compliance of 86% (+/- 10%) has been achieved.

Electrophysiological recordings of patterned rat brain stem slice neurons

Dissociated neuronal cultures on substrates patterned with extracellular matrix (ECM) proteins have yielded much information in the past. However, although the culture of brain slices has many advantages over dissociated neuronal cultures, its feasibility on patterned substrates has not been demonstrated to date. In the present study, neuronal outgrowth from brain stem slices onto homogeneous control substrates, and onto laminin structures of grid- and line-shape was achieved. Cultures were evaluated by means of phase contrast microscopy, antibody staining, and patch-clamp measurements. Only patterns with line sizes of more than 4 microm yielded satisfactory neuronal outgrowth. The size of the nodes in the pattern influenced the nodal compliance of the spreading cells and the amount of unstructured overgrowth. Best grid patterns were 4 microm lines and 10 microm nodes, best line patterns were 4 microm lines and 20 microm nodes. On patterned substrates, average sodium and potassium currents were reduced by approximately 50% compared to controls, whereas area-normalized ion-currents were in the same order of magnitude. This indicates that as a consequence of the pattern-enforced geometrical confinement, neurons tend to have a smaller surface. In addition, neurons on patterned substrates were rapidly covered with glial overgrowth. This was shown by antibody staining.

Controlled outgrowth and synapse formation of rat brain neurons by microcontact printing

Micro contact printing of biomolecules is known as an efficient approach for guiding neuronal cell migration and outgrowth on artificial substrate surfaces. When appropriate surface chemistry and microstructures are chosen, neurons are growing according to the defined geometry of the pattern. In the present study, cortical and hippocampal neurons of rats (E15-E18) were cultured on laminin, laminin/polylysine, and polylysine patterned substrates, such that small neuronal networks with a defined geometry were obtained. The interconnections between neighbouring pairs of neurons within these artificial networks were assessed electrically by double and triple patch-clamp recordings and optically by phase contrast and fluorescence microscopy. Both functional and ohmic synapses were detected. Based on the recorded data and simulations in PSpice, an electrical model for ohmically coupled cells was derived. The functional synapses were evaluated in regard of the average synaptic transmission, the average excitatory post synaptic potential (EPSP), and the average signal transmission delays of synapses. It could be shown that functional synapses on patterned substrates behave very similar to those on unpatterned, homogeneous cultures.