Growth factor gradients have been implicated in many biological phenomena, including cell migration, neurite outgrowth and guidance in the nervous system. Current in vitro assays to study the effect of growth factors on neurite guidance are limited by their suitability for only substrate-bound gradients and 2D cultures. Here we describe a novel three-channel microfluidic device to study the role of chemogradients on neurite outgrowth and guidance in 3D scaffolds. Neurons packed in the bottom channel onto the collagen gel surface extended their neurites into the gel in three dimensions, and the growing neurites were exposed to a chemogradient orthogonal to the direction of their growth to quantify their response and turning. Experimental and computational studies showed that a linear stable chemogradient could be established in these devices within 30 min, and lasting for up to 48 h. Cell culture studies revealed the dramatic effect of chemoattractive (netrin-1, brain pulp) and chemorepulsive (slit-2) gradients on the migration and neurite guidance of hippocampal and dorsal root ganglion neurons cultured in these devices. The stable chemogradients in these 3-channel devices could not only be used to screen potential drugs suitable for neuron pathway regeneration under disease/ injury conditions, but also to study cancer cell migration and cell-cell interactions.
AbstractStudying neurite guidance by diffusible or substrate bound gradients is challenging with current techniques. In this study, we present the design, fabrication and utility of a microfluidic device to study neurite guidance under chemogradients. Experimental and computational studies demonstrated the establishment of a steep gradient of guidance cue within 30 min and stable for up to 48 h. The gradient was found to be insensitive to external perturbations such as media change and movement of device. The effects of netrin-1 (0.1-10 µg/mL) and brain pulp (0.1 µL/mL) were evaluated for their chemoattractive potential on neurite turning, while slit-2 (62.5 or 250 ng/mL) was studied for its chemorepellant properties. Hippocampal or dorsal root ganglion (DRG) neurons were seeded into a micro-channel and packed onto the surface of a 3D collagen gel. Neurites grew into the matrix in three dimensions, and a gradient of guidance cue was created orthogonal to the direction of neurite growth to impact guidance. The average turning angle of each neurite was measured and averaged across multiple devices cultured under similar conditions to quantify the effect of guidance cue gradient. Significant positive turning towards gradient were measured in the presence of brain pulp and netrin-1 (1 µg/mL), relative to control cultures which received no external guidance cue (p < 0.001). Netrin-1 released from transfected fibroblasts had the most positive turning effect of all the chemoattractive cues tested (p
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