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This paper describes and characterizes a novel microfabricated neuronal culture device. This device combines microfabrication, microfluidic, and surface micropatterning techniques to create a multicompartment neuronal culturing device that can be used in a number of neuroscience research applications. The device is fabricated in poly(dimethylsiloxane), PDMS, using soft lithography techniques. The PDMS device is placed on a tissue culture dish (polystyrene) or glass substrate, forming two compartments with volumes of less than 2 μL each. These two compartments are separated by a physical barrier in which a number of micron-size grooves are embedded to allow growth of neurites across the compartments while maintaining fluidic isolation. Cells are plated into the somal (cell body) compartment, and after 3−4 days, neurites extend into the neuritic compartment via the grooves. Viability of the neurons in the devices is between 50 and 70% after 7 days in culture; this is slightly lower than but comparable to values for a control grown on tissue culture dishes. Healthy neuron morphology is evident in both the devices and controls. We demonstrate the ability to use hydrostatic pressure to isolate insults to one compartment and, thus, expose localized areas of neurons to insults applied in soluble form. Due to the high resistance of the microgrooves for fluid transport, insults are contained in the neuritic compartment without appreciable leakage into the somal compartment for over 15 h. Finally, we demonstrate the use of polylysine patterning in combination with the microfabricated device to facilitate identification and visualization of neurons. The ability to direct sites of neuronal attachment and orientation of neurite outgrowth by micropatterning techniques, combined with fluidically isolated compartments within the culture area, offers significant advantages over standard open culture methods and other conventional methods for manipulating distinct neuronal microenvironments.

The amyloidogenic peptide beta-amyloid has previously been shown to bind to neurons in the form of fibrillar clusters on the cell surface, which induces neurodegeneration and activates a program of cell death characteristic of apoptosis. To further investigate the mechanism of Abeta neurotoxicity, we synthesized the all-D- and all-L-stereoisomers of the neurotoxic truncated form of Abeta (Abeta25-35) and the full-length peptide (Abeta1-42) and compared their physical and biological properties. We report that the purified peptides exhibit nearly identical structural and assembly characteristics as assessed by high performance liquid chromatography, electron microscopy, circular dichroism, and sedimentation analysis. In addition, both enantiomers induce similar levels of toxicity in cultured hippocampal neurons. These data suggest that the neurotoxic actions of Abeta result not from stereoisomer-specific ligand-receptor interactions but rather from Abeta cellular interactions in which fibril features of the amyloidogenic peptide are a critical feature. The promiscuous nature of these beta-sheet-containing fibrils suggests that the accumulation of amyloidogenic peptides in vivo as extracellular deposits represents a site of bioactive peptides with the ability to provide inappropriate signals to cells leading to cellular degeneration and disease.