Microfluidic Long-Term Gradient Generator with Axon Separation Prototyped by 185 nm Diffused Light Photolithography of SU-8 Photoresist.

Nobuyuki Futai,Makoto Tamura,Tomohisa Ogawa,Masato Tanaka

doi:10.3390/mi10010009

Abstract

We have developed a cast microfluidic chip for concentration gradient generation that contains a thin (~5 µm2 cross-sectional area) microchannel. The diffusion of diffused 185 nm ultraviolet (UV) light from an inexpensive low-pressure mercury lamp exposed a layer of the SU-8 photoresist from the backside and successfully patterned durable 2 µm-high microchannel mold features with smooth bell-shaped sidewalls. The thin channel had appropriate flow resistance and simultaneously satisfied both the rapid introduction of test substance and long-term maintenance of gradients. The average height and width at the half height of the channel, defined by a 2 µm-wide line mask pattern, were 2.00 ± 0.19 µm, and 2.14 ± 0.89 µm, respectively. We were able to maintain the concentration gradient of Alexa Fluor 488 fluorescent dye inside or at the exit of the thin microchannel in an H-shaped microfluidic configuration for at least 48 h. We also demonstrated the cultivation of chick embryo dorsal root ganglion neuronal cells for 96 h, and the directional elongation of axons under a nerve growth factor concentration gradient.

Highlights

IntroductionMicrofluidic systems are promising tools to maintain such concentration gradients for a longer duration than traditional devices due to their size matched to the scale of gradients, high throughput, and ease of integration of downstream assays [1,2]
Chemical or biomolecule concentration gradients in the microenvironment play a significant role in cellular behaviors, such as axon guidance, and precise targeting over long distance.Microfluidic systems are promising tools to maintain such concentration gradients for a longer duration than traditional devices due to their size matched to the scale of gradients, high throughput, and ease of integration of downstream assays [1,2]
Stability and controllability of the cross-sectional size of the thin channel casted from SU-8 positive features patterned by 185 nm/256 nm-diffused UV backside exposure

Summary

Introduction

Microfluidic systems are promising tools to maintain such concentration gradients for a longer duration than traditional devices due to their size matched to the scale of gradients, high throughput, and ease of integration of downstream assays [1,2]. Microfluidic gradient generators have been applied to other microorganisms such as bacteria [3] and roundworms [4]. As described in a review containing a case study of traditional and microfluidic chemotaxis chambers [5], traditional devices and their modification are still adopted more than microfluidic gradient generators. Microfluidic devices, including gradient generators, tend to compromise ease of production and use in exchange for their functionality and technical novelty. Simplicity in microfabrication processes and easy handling are requirements in newly developing microfluidic gradient generators

Methods

Results

Conclusion