(a) Elastomeric membrane concept. The microwells confine the neurons while the short microtunnels allow only the neurites to pass through. (b) Directed microstructure design. (c) Snag microstructure design. (d) Hook microstructure design.
Fluorescence microscope images of neurons 72 h after deposition into snag microstructures: Red indicates MAP2 abundant in dendrites, green indicates neurofilaments abundant in axons, and yellow areas indicate an overlap of the two stains. White arrows point to neuron cell bodies and striped arrows point to axons. (a) A circuit connecting two neurons. (b) An incomplete circuit where an obscured neuron (left) extended its axon down the intended microtunnel direction toward a clearly identifiable neuron (right) extending an axon back in the unintended direction toward the first neuron.
Classifications of neurite growth. Phase contrast micrographs of neurons 24 h after deposition into snag microstructures. White arrows point to neuron cell bodies and striped arrows point to axons. (a) A typical neuron with a neurite (here split in two directions) that did not extend far enough down either microtunnel to be ascribed to either direction (neither). (b) A typical neuron with multiple neurites that extended significantly into both microtunnels (both). (c) A typical neuron extending a single neurite into the intended microtunnel/direction . (d) A typical neuron extending a neurite into the unintended microtunnel/direction (−).
Time-lapse phase contrast micrographs of cells patterned into a hook microstructure. White arrows point to neuron cell bodies, striped arrows point to axons, and black arrows point to astrocytes. (a) Neuron 23 h after deposition with laser cell patterning system. Two neurites are visible. (b) Neuron and astrocyte 1 h after astrocyte deposition with laser patterning system (24 h after neuron deposition). Neurites are missing or obscured by the astrocyte. (c) Neuron and astrocyte 24 h after astrocyte deposition (48 h after neuron deposition). The astrocyte has changed morphology and a single neurite is visible. (d) Neuron and astrocyte 72 h after astrocyte deposition (96 h after neuron deposition). The astrocyte has proliferated and migrated down the microtunnel and into the narrow perpendicular tunnel. The neuron has extended a neurite down the channel but it is blocked by the astrocyte barrier and directed along the side of the astrocyte.
Neurite growth behavior. The four classifications (as depicted in Fig. 3) are neither direction (neither), both directions (both), the unintended direction (−), and the intended direction . The table shows the results for only the 87 neurons that exhibited neurite outgrowth after 24 h.
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