Gel Alignment in 3D Microfluidic Neuronal Culture Systems

Abstract

Axon plays an important role in the brain mechanisms as electric signal transmission, axonal transport, and local protein synthesis. By developing of valuable microfluidic systems, researchers easily isolate, observe, and test neurites development. In 2D culture, a microfluidic culture platform is a strong tool to probe axons independently from cell bodies, and micro-patterning method pattern axonal guidance molecules to selectively control axonal growth. However, conventional 2D environments disadvantage is not to closely mimic physiological axon growth. To understand fully how axons as well as neural networks form and operate, some 3D cell-culture models have been recently applied widely. In vitro, a lack of an appropriate method which guides axons in 3D environment prevented investigation of axonal biology and neural networks. Here, we describe a novel method that guides axon growth by controlling 3D gel alignment. In this method, we design a microfluidic device that maintains 3D gel environment (Matrigel) in channel. Before complete polymerization, the random structure of Matrigel is aligned by the directional diffusion and pressure of medium from top to bottom. Then, neurospheres are cultured next to the aligned gel. Neural cells are differentiated from neurospheres inside the 3D scaffold. Because the cell motility depends on the scaffolds structure, neural cells as well as their dendrites migrate and invade directionally inside the aligned gel. Neurosphere-derived cells express the fluorescent of tubulin β III-positive (Tuj-III) neurons show the data of axon alignment (80% axons grow straight, less than 30 degree). Besides, depending on the concentration of Matrigel, the alignment of axons will be formed completely or partially. For neural network formation, synapse signals can be detected on the designated areas between the directional axons and local dendrites The axon guidance in 3D environment provides the understanding of directional controlling axon growth, supporting the formation neural network in 3D and solving the 2D culture limitation by utilizing extracellular matrix gel on axon migration. In addition to its compatibility with optics system, the platform is useful for guiding axon with light, forming neural network, making axon injury, and observing regeneration in 3D environment.