A neuron-benign microfluidic gradient generator for studying the response of mammalian neurons towards axon guidance factors.
Investigation of biochemical cues in isolation or in combinations in cell culture systems is crucial for unraveling the mechanisms that govern neural development and repair. The most widely used experimental paradigms that elicit axon guidance in vitro utilize as the source of the gradient a pulsatile pipette, transfected cells, or a loaded gel, producing time-varying gradients of poor reproducibility which are not well suited for studying slow-growing mammalian cells. Although microfluidic device design have allowed for generating stable, complex gradients of diffusible molecules, the flow-induced shear forces in a microchannel has made it impossible to maintain viable mammalian neuronal cultures for sufficiently long times. In this paper, we describe axonal responses of mouse cortical neurons in a "neuron-benign" gradient-generator device based on an open chamber that can establish highly stable gradients of diffusible molecules for at least 6 h with negligible shear stress, and also allows the neurons to thrive for at least 2 weeks. Except for the period when the gradient is on, the cells in the gradient are under the same conditions as the cells on the control surfaces, which ensure a consistent set of micro-environmental variables. The gradient stability and uniformity over the cell culture surface achieved by the device, together with our software platform for acquiring, post-processing and quantitatively analyzing the large number of images allowed us to extract valuable information even from small datasets. We report a directed response of primary mammalian neurons (from E14 embryonic mice cortex) to a diffusible gradient of netrin in vitro. We infer from our studies that a large majority (∼73%) of the neurons that extend axons during the gradient application grow towards the netrin source, and our data analysis also indicates that netrin acts as a growth factor for this same population of neurons.
Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.
Nirveek
N
Bhattacharjee
Nianzhen
N
Li
Thomas M
TM
Keenan
Albert
A
Folch
D000818:Animals; D001369:Axons / Q000187:drug effects / Q000502:physiology; D018929:Cell Culture Techniques; D058948:Cell Tracking; D004867:Equipment Design; D005260:Female; D051379:Mice; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D009414:Nerve Growth Factors / Q000494:pharmacology; D000075388:Netrin-1; D055495:Neurogenesis; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D011247:Pregnancy; D015398:Signal Transduction; D025521:Tumor Suppressor Proteins / Q000494:pharmacology
D009414:Nerve Growth Factors; D025521:Tumor Suppressor Proteins; D000075388:Netrin-1