Microfluidic Chip for Axonal Injury Models Construction and Enabling Multi-Omics Analysis

Bing Zhou; Ruixuan Liu; Jiaxin Sun; Maoliang Lu; Qianwen Zhang

doi:10.3791/68915

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Method Article

Microfluidic Chip for Axonal Injury Models Construction and Enabling Multi-Omics Analysis

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DOI:

10.3791/68915

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October 14th, 2025

Bing Zhou¹^,² , Ruixuan Liu¹^,³ , Jiaxin Sun¹^,³ , Maoliang Lu⁴ , Qianwen Zhang⁵

¹Beijing Advanced Innovation Center for Big Data-Based Precision Medicine, Beihang University, ²Interdisciplinary Innovation Institute of Medicine and Engineering, Beihang University, ³School of Biological Science and Medical Engineering, Beihang University, ⁴School of Software, Beihang University, ⁵School of Beijing, Beihang University

Summary

This protocol describes a microfluidic system modeling neuronal metabolic dynamics post-axonal injury, enabling imaging, multi-omics analysis, and mechanistic studies of intrinsic metabolic remodeling.

Abstract

Neurons polarize to form dendrites and axons, enabling intercellular communication. Axonal injury disrupts these connections and transmits damage signals to the soma, often leading to neuronal degeneration. Thus, maintaining axonal homeostasis is essential for promoting local axon regeneration and protecting against neurodegeneration. This process relies on cellular metabolism to supply energy and biosynthetic precursors and is sustained by mechanisms that regulate metabolic balance and eliminate by-products. However, neuronal metabolism is compartmentalized between the soma and axon and is further influenced in vivo by the surrounding microenvironment, such as astrocyte-derived metabolic activity (e.g., the astrocyte-neuron lactate shuttle). These factors complicate the investigation of neurons' intrinsic metabolic mechanisms. To address these challenges, here we developed a microfluidic platform for culturing primary cortical neurons in vitro that preserves key metabolic characteristics observed in vivo, including physiological glycolytic flux and mitochondrial respiration. This system provides a simplified model for investigating intrinsic metabolic remodeling in neurons after axonal injury. Conventional microfluidic chips support in vitro axonal injury models and are compatible with live-cell imaging, immunofluorescence staining, and hypoxia treatment. To accommodate large-scale transcriptomic and metabolomic analyses involving millions of cells, we further designed and fabricated high-throughput microfluidic chips with optimized operational protocols. The device features alternately arranged soma and axon chambers connected by microchannels, and axonal injury is induced by vacuum aspiration of fluid from the axon compartment. This platform enables rapid assessment of metabolite and enzyme dynamics, improving the accuracy and reproducibility of multi-omics investigations.

Introduction

Neurons establish two distinct types of protrusions with specialized functions, axons and dendrites, via polarization during development¹^,². Nerve regeneration refers to the process of axonal regrowth after neural injury³, with a research focus on central nervous system (CNS) repair. Unlike the peripheral nervous system, the CNS has limited regenerative capacity⁴^,⁵^,⁶, often resulting in persistent functional impairments or permanent disabilities in patients with spinal cord injuries or traumati....