Large-scale microfluidic gradient arrays reveal axon guidance behaviors in hippocampal neurons

Nirveek Bhattacharjee,Albert Folch

doi:10.1038/micronano.2017.3

Nirveek Bhattacharjee, Albert Folch

Open Access

https://doi.org/10.1038/micronano.2017.3

Copy DOI

Abstract

High-throughput quantitative approaches to study axon growth behaviors have remained a challenge. We have developed a 1024-chamber microfluidic gradient generator array that enables large-scale investigations of axon guidance and growth dynamics from individual primary mammalian neurons, which are exposed to gradients of diffusible molecules. Our microfluidic method (a) generates statistically rich data sets, (b) produces a stable, reproducible gradient with negligible shear stresses on the culture surface, (c) is amenable to the long-term culture of primary neurons without any unconventional protocol, and (d) eliminates the confounding influence of cell-secreted factors. Using this platform, we demonstrate that hippocampal axon guidance in response to a netrin-1 gradient is concentration-dependent—attractive at higher concentrations and repulsive at lower concentrations. We also show that the turning of the growth cone depends on the angle of incidence of the gradient. Our study highlights the potential of microfluidic devices in producing large amounts of data from morphogen and chemokine gradients that play essential roles not only in axonal navigation but also in stem cell differentiation, cell migration, and immune response.

Highlights

The challenges associated with investigating the spatiotemporally complex processes of axon guidance in vivo have long motivated the development of in vitro experimental paradigms, with both explant cultures[1] and dissociated primary neurons[2]
Large-scale 1024 microjet array: Design and principle of operation Based on a previous design[31], we developed a large-scale array of 1024 gradient generators that can be loaded with cells from the top using a pipette
While the analysis in the previous section indicated that a linear gradient of netrin-1 did not appear to direct the extension of the hippocampal axons in a particular direction (Supplementary Figure S5), we investigated whether the absolute concentration observed by the growth cone could have played a role in determining the response to the netrin-1 gradient

Summary

Introduction

The challenges associated with investigating the spatiotemporally complex processes of axon guidance in vivo have long motivated the development of in vitro experimental paradigms, with both explant cultures[1] and dissociated primary neurons[2]. A hypothetical advancing growth cone encounters an evolving gradient and is exposed to the gradient at varying angles as the growth progresses (Figure 1c), which makes analysis difficult. In a microfluidic device, the same advancing growth cone faces a stable concentration field without changing the angle of incidence of the gradient (Figure 1d). Gradients of biochemical molecules generated by microfluidic devices have been used extensively to study cell guidance, including neutrophil migration[7,8,9], breast cancer cell metastases[10], bacterial chemotaxis[11], stem cell differentiation[12,13], and axon growth and guidance[14,15,16,17]. Microdevice-based approaches, which are used for investigating axon guidance, have recently been reviewed comprehensively[18,19]

Methods

Results

Discussion

Conclusion