Name: Microfluidic Chip for Axonal Injury Models Construction and Enabling Multi-Omics Analysis (Video) | JoVE Journal
Uploaded: 2025-10-14T12:30:00.000+00:00
Duration: 11 min
Description: Explore microfluidic technology for multi-omics analysis of neuronal metabolic dynamics following axonal injury. This method enables transcriptomics and metabolic flux studies in cortical neurons.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was supported by the Beijing Natural Science Foundation (Grant Nos. L222080, F251031, Z240011, 7192103) and the National Natural Science Foundation of China (Grant Nos. 82271513)

<ol><li>Namba, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pioneering+axons+regulate+neuronal+polarization+in+the+developing+cerebral+cortex%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Pioneering axons regulate neuronal polarization in the developing cerebral cortex.</a> Neuron. 81 (4), 814-829 (2014).</li>
<li>Donato, A., Kagias, K., Zhang, Y., Hilliard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuronal+sub-compartmentalization%3A+a+strategy+to+optimize+neuronal+function%5BTitle%5D)+AND+%22Biol+Rev+Camb+Philos+Soc%22%5BJournal%5D)">Neuronal sub-compartmentalization: a strategy to optimize neuronal function.</a> Biol Rev Camb Philos Soc. 94 (3), 1023-1037 (2019).</li>
<li>Anderson, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Required+growth+facilitators+propel+axon+regeneration+across+complete+spinal+cord+injury%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Required growth facilitators propel axon regeneration across complete spinal cord injury.</a> Nature. 561 (7723), 396-400 (2018).</li>
<li>Hilton, B. J., Bradke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Can+injured+adult+CNS+axons+regenerate+by+recapitulating+development%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Can injured adult CNS axons regenerate by recapitulating development.</a> Development. 144 (19), 3417-3429 (2017).</li>
<li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia-organized+scar-free+spinal+cord+repair+in+neonatal+mice%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Microglia-organized scar-free spinal cord repair in neonatal mice.</a> Nature. 587 (7835), 613-618 (2020).</li>
<li>Hilton, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+active+vesicle+priming+machinery+suppresses+axon+regeneration+upon+adult+CNS+injury%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">An active vesicle priming machinery suppresses axon regeneration upon adult CNS injury.</a> Neuron. 110 (1), 51-69 (2022).</li>
<li>Alizadeh, A., Dyck, S. M., Karimi Abdolrezaee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Traumatic+spinal+cord+injury%3A+An+overview+of+pathophysiology%2C+models+and+acute+injury+mechanisms%5BTitle%5D)+AND+%22Front+Neurol%22%5BJournal%5D)">Traumatic spinal cord injury: An overview of pathophysiology, models and acute injury mechanisms.</a> Front Neurol. 10, 441408(2019).</li>
<li>Anjum, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spinal+cord+injury%3A+Pathophysiology%2C+multimolecular+interactions%2C+and+underlying+recovery+mechanisms%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Spinal cord injury: Pathophysiology, multimolecular interactions, and underlying recovery mechanisms.</a> Int J Mol Sci. 21 (20), 7533(2020).</li>
<li>Zhao, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidic+model+to+mimic+initial+event+of+neovascularization%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Microfluidic model to mimic initial event of neovascularization.</a> J Vis Exp. (170), e62003(2021).</li>
<li>Li, N., Sip, C., Folch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidic+chips+controlled+with+elastomeric+microvalve+arrays%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Microfluidic chips controlled with elastomeric microvalve arrays.</a> J Vis Exp. (8), e296(2007).</li>
<li>Chowdhury, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+customizable+chamber+for+measuring+cell+migration%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">A customizable chamber for measuring cell migration.</a> J Vis Exp. (121), e55264(2017).</li>
<li>De Ninno, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidic+co-culture+models+for+dissecting+the+immune+response+in+in+vitro+tumor+microenvironments%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Microfluidic co-culture models for dissecting the immune response in in vitro tumor microenvironments.</a> J Vis Exp. (170), e61895(2021).</li>
<li>Shahryari, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pre-clinical+orthotopic+murine+model+of+human+prostate+cancer%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Pre-clinical orthotopic murine model of human prostate cancer.</a> J Vis Exp. (114), e54125(2016).</li>
<li>Kim, J., Hegde, M., Jayaraman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidic+co-culture+of+epithelial+cells+and+bacteria+for+investigating+soluble+signal-mediated+interactions%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Microfluidic co-culture of epithelial cells and bacteria for investigating soluble signal-mediated interactions.</a> J Vis Exp. (38), e1749(2010).</li>
<li>Taylor, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+microfluidic+culture+platform+for+CNS+axonal+injury%2C+regeneration+and+transport%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">A microfluidic culture platform for CNS axonal injury, regeneration and transport.</a> Nat Methods. 2 (8), 599-605 (2005).</li>
<li>Liu, R., Zhou, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Harmine+promotes+axon+regeneration+through+enhancing+glucose+metabolism%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Harmine promotes axon regeneration through enhancing glucose metabolism.</a> J Biol Chem. 301 (3), 108254(2025).</li>
<li>Shrirao, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidic+platforms+for+the+study+of+neuronal+injury+in+vitro%5BTitle%5D)+AND+%22Biotechnol.+Bioeng%22%5BJournal%5D)">Microfluidic platforms for the study of neuronal injury in vitro.</a> Biotechnol. Bioeng. 115 (4), 815-830 (2018).</li>
<li>Van Laar, V. S., Arnold, B., Berman, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Primary+embryonic+rat+cortical+neuronal+culture+and+chronic+rotenone+treatment+in+microfluidic+culture+devices%5BTitle%5D)+AND+%22Bio+Protoc%22%5BJournal%5D)">Primary embryonic rat cortical neuronal culture and chronic rotenone treatment in microfluidic culture devices.</a> Bio Protoc. 9 (6), e3192(2019).</li>
<li>Huang, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reprogramming+an+energetic+AKT-PAK5+axis+boosts+axon+energy+supply+and+facilitates+neuron+survival+and+regeneration+after+injury+and+ischemia%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">Reprogramming an energetic AKT-PAK5 axis boosts axon energy supply and facilitates neuron survival and regeneration after injury and ischemia.</a> Curr Biol. 31 (14), 3098-3114 (2021).</li>
<li>Zhou, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Facilitation+of+axon+regeneration+by+enhancing+mitochondrial+transport+and+rescuing+energy+deficits%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Facilitation of axon regeneration by enhancing mitochondrial transport and rescuing energy deficits.</a> J Cell Biol. 214 (1), 103-119 (2016).</li>
<li>Cartoni, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mammalian-specific+protein+armcx1+regulates+mitochondrial+transport+during+axon+regeneration%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">The mammalian-specific protein armcx1 regulates mitochondrial transport during axon regeneration.</a> Neuron. 92 (6), 1294-1307 (2016).</li>
<li>Han, S. M., Baig, H. S., Hammarlund, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondria+localize+to+injured+axons+to+support+regeneration%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Mitochondria localize to injured axons to support regeneration.</a> Neuron. 92 (6), 1308-1323 (2016).</li>
<li>Yang, L. Q., Chen, M., Ren, D. L., Hu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dual+oxidase+mutant+retards+mauthner-cell+axon+regeneration+at+an+early+stage+via+modulating+mitochondrial+dynamics+in+zebrafish%5BTitle%5D)+AND+%22Neurosci+Bull%22%5BJournal%5D)">Dual oxidase mutant retards mauthner-cell axon regeneration at an early stage via modulating mitochondrial dynamics in zebrafish.</a> Neurosci Bull. 36, 1500-1512 (2020).</li>
<li>Yuan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+and+in+vivo+stable+isotope+labelling+of+central+carbon+metabolism+and+related+pathways+with+analysis+by+LC-MS%2FMS%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Ex vivo and in vivo stable isotope labelling of central carbon metabolism and related pathways with analysis by LC-MS/MS.</a> Nat Protoc. 14 (2), 313-330 (2019).</li>
<li>Mercimek, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+effects+of+3D+printed+mold+material%2C+curing+temperature%2C+and+duration+on+polydimethylsiloxane+%28PDMS%29+curing+characteristics+for+lab-on-a-chip+applications%5BTitle%5D)+AND+%22Micromachines+%28Basel%29%22%5BJournal%5D)">On the effects of 3D printed mold material, curing temperature, and duration on polydimethylsiloxane (PDMS) curing characteristics for lab-on-a-chip applications.</a> Micromachines (Basel). 16 (6), 684(2025).</li>
<li>Derosa, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Convergent+pathways+in+idiopathic+autism+revealed+by+time+course+transcriptomic+analysis+of+patient-derived+neurons%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Convergent pathways in idiopathic autism revealed by time course transcriptomic analysis of patient-derived neurons.</a> Sci Rep. 8 (1), 8423(2018).</li>
<li>Cheng, X. T., Huang, N., Sheng, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Programming+axonal+mitochondrial+maintenance+and+bioenergetics+in+neurodegeneration+and+regeneration%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Programming axonal mitochondrial maintenance and bioenergetics in neurodegeneration and regeneration.</a> Neuron. 110 (12), 1899-1923 (2022).</li>
<li>Huang, N., Sheng, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidic+devices+as+model+platforms+of+CNS+injury-ischemia+to+study+axonal+regeneration+by+regulating+mitochondrial+transport+and+bioenergetic+metabolism%5BTitle%5D)+AND+%22Cell+Regen%22%5BJournal%5D)">Microfluidic devices as model platforms of CNS injury-ischemia to study axonal regeneration by regulating mitochondrial transport and bioenergetic metabolism.</a> Cell Regen. 11 (1), 33(2022).</li>
</ol>

The authors have nothing to disclose.

The establishment of advanced in vitro models for studying axonal injury and regeneration is essential for uncovering the molecular and metabolic mechanisms involved in neuronal repair16,19. In this study, we present a standardized workflow that integrates microfluidic devices, large-scale neuronal cultures, and precise axonal injury protocols, enabling robust multi-omics analysis of injured cortical neurons.
The success of th...

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 traumatic brain injuries7,8. Thus, developing strategies to enhance CNS repair and elucidating the mechanisms underlying axonal regeneration are crucial.
Microfluidic devices, functioning as integrated, cost-effective, and high-throughput in vitro culture systems, exhibited remarkable advantages in cell and neuron research in recent years9,10. Compared with traditional methods, microfluidic technology enables precise regulation of fluid shear stress, concentration gradients, and spatial structures, thereby closely simulating authentic physiological and pathological microenvironments to promote cell growth, migration, differentiation, and interactions11,12,13,14. Microfluidics-based in vitro axonal injury models enable compartmentalized culture of neuronal cell bodies and processes through spatial polarization, providing an indispensable tool for exploring the intrinsic regenerative capacity of neurons and their metabolic adaptations following injury15,16,17,18. It provides a critical window into how neurons respond to injury, regulate metabolic processes19, and promote axonal regeneration20. However, existing microfluidic platforms for axonal injury and metabolic research still face challenges, including low neuronal cell yields, reduced accuracy in metabolic measurements, and a lack of standardized operational procedures.
In response to these limitations, this study developed a novel large-scale microfluidic platform that enables standardized, high-throughput induction of axonal injury and provides sufficient cell material for compartmentalized multi-omics analyses. This integrated design directly confronts the critical unmet need in neural injury research. Specifically, the microfluidic chip we designed features an alternating arrangement of somal and axonal chambers connected by microgrooves, with axonal transection achieved through controlled vacuum aspiration. This approach is ideal for studies that require reproducible, high-throughput axonal injury models and precise metabolic profiling, such as investigating the metabolic mechanisms underlying axonal regeneration. Compared to traditional methods, this technique offers higher throughput, greater metabolic precision, and the capability to establish standardized injury models. For example, previous studies have demonstrated the importance of mitochondrial transport in axonal regeneration20,21,22,23, whereas our platform further reveals the role of glucose metabolism in axonal regeneration following injury. This protocol establishes a technical system for large-scale axotomy and metabolic analysis that is both visually verifiable and operationally standardized.

<table><tbody><tr><td>Neurobasal-A Medium</td><td>Gibco</td><td>10888022</td><td>Culture medium</td></tr><tr><td>Alexa Fluor 488 goat anti-mouse</td><td>Invitrogen</td><td>A-21042</td><td>Immunofluorescence</td></tr><tr><td>Analytical balance</td><td>Shanghai Shunyu Hengping Scientific Instrument Co., Ltd.</td><td>FA1004</td><td>Precise weighing of reagents/samples</td></tr><tr><td>B27 (50x)</td><td>Gibco</td><td>17504044</td><td>Culture medium</td></tr><tr><td>BCA protein assay</td><td>Boster</td><td>AR1189</td><td>Protein concentrations</td></tr><tr><td>Bioanalyzer</td><td>Agilent</td><td>https://www.agilent.com/en/product/automated-electrophoresis/bioanalyzer-systems/bioanalyzer-instrument</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Genview</td><td>FA016</td><td>Immunofluorescence</td></tr><tr><td>Centrifugal stirrer mixer</td><td>THINK</td><td>ARE-310</td><td>Mix PDMS and degasification</td></tr><tr><td>CLARIOstarPlus-ACU analyzer</td><td>BMG LABTECH</td><td>CLARIOstar Plus</td><td>protein concentrations</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo Fisher Scientific (Asheville) LLC</td><td>3111</td><td>Cell culture incubation at 37 &amp;deg;C, 5% CO?</td></tr><tr><td>Corrosion-resistant Diaphragm Vacuum Pump</td><td>Shanghai Lichen Bangxi Instrument Technology Co., Ltd.</td><td>LC-85DLC</td><td>Aspirate waste liquid and provide suction for axotomy</td></tr><tr><td>D-GLUCOSE (U-&lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;6&lt;/sub&gt;, 99%)</td><td>Cambridge Isotope Laboratories</td><td>CLM-1396</td><td>Metabolic flux</td></tr><tr><td>DNase I</td><td>Roche</td><td>11284932001</td><td>Neuronal culture</td></tr><tr><td>Dr.TOM2 platform&amp;nbsp;</td><td>BGI Genomics</td><td></td><td>Perform and analyze RNA-seq&amp;nbsp;</td></tr><tr><td>Electric hot-air drying oven</td><td>BIOM Huahong Bonn / Huaying Bosi</td><td>DHG-9070A</td><td>Curing PDMS</td></tr><tr><td>Electronic Balance</td><td>Shanghai Huachao Industrial Co., Ltd.</td><td>HC313</td><td>Routine weighing of materials</td></tr><tr><td>Fluoromount Aqueous Mounting Medium</td><td>Sigma-Aldrich</td><td>F4680</td><td>Immunofluorescence</td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>35050061</td><td>Culture medium</td></tr><tr><td>Glycine</td><td>Solarbio</td><td>G8200</td><td>Immunofluorescence</td></tr><tr><td>HBSS</td><td>Gibco</td><td>14025092</td><td>Neuronal culture</td></tr><tr><td>HBSS, no calcium, no magnesium</td><td>Gibco</td><td>14175095</td><td>Neuronal culture</td></tr><tr><td>High-pressure steam sterilizer</td><td>STIK Instrument (Shanghai) Co., Ltd.</td><td>HMJ-54A</td><td>Sterilization of lab equipment and media</td></tr><tr><td>Hypoxic chamber</td><td>STEMCELL Technologies</td><td>27310</td><td>Hypoxic treatment</td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td>Used to analyze microscopy images</td></tr><tr><td>Leica Microscope</td><td>Leica Microsystems CMS GmbH</td><td>DMIL LED Fluo</td><td>Live and fluorescence observation</td></tr><tr><td>Live cell imaging solution</td><td>Invitrogen</td><td>A14291DJ</td><td>Live cell imaging</td></tr><tr><td>Medical low-temperature storage box (-80 &amp;deg;C)</td><td>Qingdao Haier Biomedical Co., Ltd.</td><td>DW-86L388J</td><td>Long-term sample storage at -80 &amp;deg;C</td></tr><tr><td>Mini desktop vacuum pump</td><td>Haimen Qilin-Bell Instrument Manufacturing Co., Ltd.</td><td>GL-805</td><td>Small-scale vacuum applications</td></tr><tr><td>MitoTracker Orange CMTMRos</td><td>Invitrogen</td><td>M7510</td><td>Mitochondrial membrane potential staining</td></tr><tr><td>MRMPROBS program</td><td></td><td>Version 2.60</td><td>To extract raw mass spectrometry data&amp;nbsp;</td></tr><tr><td>Neurobasal Medium</td><td>Gibco</td><td>21103049</td><td>Culture medium</td></tr><tr><td>Neurobasal-A without glucose</td><td>Gibco</td><td>A2477501</td><td>Culture medium</td></tr><tr><td>Papain</td><td>Worthington</td><td>LS003127</td><td>Neuronal culture</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>Cell fixation</td></tr><tr><td>PDMS</td><td>Dow Corning</td><td>DC184</td><td>Device fabrication</td></tr><tr><td>PDS Kit Inhibitor Vial</td><td>Worthington</td><td>LK003182</td><td>Neuronal culture</td></tr><tr><td>Penicillin Streptomycin</td><td>Gibco</td><td>15140122</td><td>Culture medium</td></tr><tr><td>Phosphate Buffered Saline</td><td>Biosharp</td><td>BL302A</td><td>Dilute the substances and clean the cell containers</td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>ODC-32G-2</td><td>Activation</td></tr><tr><td>Poly-D-lysine</td><td>Sigma-Aldrich</td><td>P6407</td><td>Neuronal culture</td></tr><tr><td>Prism&amp;nbsp;</td><td>GraphPad</td><td>Version 8.4.3</td><td>Statistical analyses</td></tr><tr><td>Refrigerated centrifuge</td><td>Eppendorf AG (Germany)</td><td>5430R</td><td>Sample separation at controlled temperatures</td></tr><tr><td>RNArep Pure Cell/Bacterial Total RNA Extraction Kit</td><td>Tiangen</td><td>dp430</td><td>Transcriptomics</td></tr><tr><td>Rotameter</td><td>Dwyer</td><td>RMA-23-SSV</td><td>Control the low oxygen flow</td></tr><tr><td>Stereo microscope</td><td>Olympus Corporation</td><td>SZ2-ILST</td><td>Sample observation and dissection</td></tr><tr><td>Stuart Orbital Shaker</td><td>Kylin-Bell</td><td>TS-200</td><td>Mixing cell cultures/solutions at adjustable speeds</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td>Immunofluorescence</td></tr><tr><td>ULVAC Rotary Vane Vacuum Pump</td><td>ULVAC (Ningbo) Co., Ltd.</td><td>GLD-N202</td><td>Creating vacuum for filtration/drying</td></tr><tr><td>&amp;beta;III-tubulin</td><td>Promega</td><td>G7121</td><td>Immunofluorescence</td></tr></tbody></table>

microfluidic chip for axonal injury models construction and enabling multi-omics analysis

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

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.

To establish a comprehensive research platform for studying the metabolic regulation mechanisms in cortical neurons, we first cultured cortical neurons in a conventional microfluidic device, precisely controlling axon growth along microchannels (Figure 2A). With an appropriate design, the microchannels were constructed with a low height (5 µm) and a larger width (10 µm), allowing each channel to host more axons without allowing cell bodies to enter. Immunoflu...

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.

Neurons polarize to form dendrites and axons, enabling intercellular communication. Axonal injury disrupts these connections and transmits damage signals ...

We have developed large-scale microfluidic chips to enable multiomics analysis, with the aim of for illustrating the metabolic mechanism of neurons following axonal injury. We mainly utilize microfluidic technology, metabolic flux analysis, and the transcriptomics to advance the research on the metabolic mechanisms of neurons during axonal injury and regeneration. We found that there are metabolic differences among cortical neurons at different developmental disease, and that young neurons undergo metabolic remodeling after external injury.The core advantage of our protocol lies in the larger-scale microfluidic platform design and operational standards, which enable accurate multiomics and size of millions of cells. To begin, transfer the PDMS base and curing agent into a centrifuge tube. Place the centrifuge tube containing the mixture into a centrifugal stirrer mixer.Centrifuge at 2, 000 G for four minutes twice to mix and to degas. Pour 13 to 15 grams of the degassed PDMS into the microfluidic mold, ensuring the bottom of the mold is completely covered. Now, place the mold in a vacuum desiccator, then use a vacuum pump to evacuate air for five to 10 minutes to thoroughly remove bubbles from the PDMS.Using a rubber bulb, gently tap the surface of the PDMS to break any remaining surface bubbles. Transfer the mold to a convection oven and bake at 80 degrees Celsius for 100 minutes to cure the polydimethylsiloxane. Once the PDMS is fully cured, gently slide the tip of a scalpel under the edge of the PDMS to carefully lift and separate it from the mold.Using a biopsy punch with a diameter of 2.0 to 2.5 millimeters, create holes in the PDMS for culture medium infusion and cell loading. Remove any surface impurities from the PDMS using adhesive tape, then place it in a clean glass dish and wrap it with aluminum foil for storage. Prior to the experiment, autoclave the microfluidic device at 121 degrees Celsius and 101 kilopascals for five minutes to ensure sterility.Place a cover slip intended for conventional microfluidic devices in a 35 millimeter culture dish. Add two milliliters of 0.1 milligram per milliliter poly-D-lysine solution to the dish. Incubate the dish at 37 degrees Celsius for six hours or overnight.After incubation, carefully retrieve the poly-D-lysine solution for reuse or proper disposal. Now, add two milliliters of sterile ultrapure water to the dish. Rotate it 50 times clockwise, followed by 50 times counterclockwise.Aspirate the water using a vacuum pump connected to a sterile pipette tip, collecting waste in a liquid waste container. After all washes are complete, allow the cover slips to air dry naturally in a biosafety cabinet before use. Using sterile fine tip forceps, place the autoclaved microfluidic chip at the center of the glass surface with microchannels facing downward.Gently press the chip onto the glass surface using a 200 microliter pipette tip to ensure complete adhesion. Next, mark the left chamber with a dot using a permanent marker to designate the soma compartment. Aspirate 10 microliters of neuronal suspension with a 10 microliter pipette, then slowly dispense the suspension into the loading port above the marked soma compartment.Confirm proper fluid flow into the lower reservoir of the left chamber. Transfer the culture dish to a humidified incubator set at 37 degrees Celsius and 5%carbon dioxide for 20 minutes. After incubation, place the dish under a 20X microscope to confirm media profusion from the soma compartment to the axonal compartment through the microchannels.Now, pipette 150 microliters of neuronal basal medium to the upper port of the axonal compartment. Similarly, add 150 microliters of complete neuronal medium to the upper port of the soma compartment. Next, pour 20 milliliters of ultrapure water into a 150 millimeter culture dish to create a humidity chamber.Place the 35 millimeter culture dish inside the large dish, then transfer the entire culture system to the incubator and maintain for the required duration. Use a 10 centimeter wide Petri dish for placement of the large-scale microfluidic device. Choose either full channel or interval channel seeding based on experimental needs, as each method requires different axonal injury protocols.After seeding is completed, add five milliliters of complete neuron culture medium to the Petri dish to maintain neuron growth. Transfer an appropriate amount of neuronal basal medium into a new 15 milliliter centrifuge tube for later use. Place a microfluidic device containing cortical neurons on a clean bench.Using lint-free paper, dry the bottom of the 35 millimeter culture dish, then use a 200 microliter pipette to aspirate the medium from the two right side holes of the microfluidic device. Next, connect a vacuum pump tubing to a sterile, filter-free 200 microliter pipette tip. Turn on the vacuum pump at a suction rate of 60 liters per minute and aim the tip at the connection point between the lower hole of the axon terminal chamber and the chamber to aspirate the old medium, causing axon breakage due to negative pressure.Replace the pipette tip and draw 150 microliters of neuronal basal medium, slowly adding it through the lower hole of the right chamber. After the performing addition and aspiration four times, replace with fresh complete neuronal medium, then aspirate the old medium from the cell body side hole and add fresh complete neuronal medium containing drugs if needed. Place the microfluidic device along with the culture dish in a 150 millimeter culture dish and continue culturing for the required time, such as 24 hours.For manual axonal injury, seed neurons into all chambers of the large-scale microfluidic device. Incubate the device at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide for three days. On day seven in vitro, remove the culture dish containing the microfluidic device from the incubator and place it on a sterile stage.Prepare a microscope with 20X magnification and sterilize the work area using 75%ethanol. Hold a 10 microliter sterile filtered pipette tip and identify axon bundles in the original microchannels under microscope guidance. Perform longitudinal scratches along each axon bundle using the pipette tip.Observe the axon breakage under the microscope in real time. The microchannels of a standard microfluidic device permit the growth of axons, but not soma, as confirmed by beta three tubulin staining at seven days in vitro. Neuronal density showed no significant difference following axonal injury, indicating no observable neuronal death.A large-scale microfluidic device with a three-dimensional expandable design was developed for multiomics analyses. Both unpunched and perforated PDMS replicas adhered securely to 10 centimeter dishes or PC boards. Axonal damage induced by pipette tip scratching led to clearly severed axons with disrupted morphology, in contrast to intact axons prior to injury.Axonal regeneration was observed at both six hours and 24 hours following injury, while uninjured axons remained stable. Neuronal protein concentration rose significantly from 1420.4 micrograms per milliliter at baseline to 1748.9 per milliliter at six hours, and 1823.7 micrograms per milliliter at 24 hours post-injury. RNA yields from control and axon injured groups were nearly identical.RNA sequencing revealed 595 injury-specific genes and 609 control-specific genes, with 17, 471 genes shared between groups. KEGG pathway analysis identified significant upregulation of oxidative phosphorylation, reactive oxygen species response, autophagy, and TCA cycle pathways post-injury.

microfluidic-chip-for-axonal-injury-models-construction-enabling

Research

JoVE Journal

Neuroscience

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

This video describes the fabrication and use of a microfluidic device to culture central nervous system (CNS) neurons.  This device is compatible with ...

BioMEMS: Forging New Collaborations Between Biologists and Engineers

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

Preparation of Neuronal Co-cultures with Single Cell Precision

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

This protocol describes the use of plastic chips to culture and compartmentalize primary murine neurons. These chips are preassembled, user-friendly, and compatible with high-resolution, live, and fluorescence imaging. This protocol describes how to plate rat hippocampal neurons within these chips and perform fluidic isolation, axotomy and immunostaining.

Use of Pre-Assembled Plastic Microfluidic Chips for Compartmentalizing Primary Murine Neurons

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

This protocol demonstrates the use of compartmentalized microfluidic chips, injection molded in a cyclic olefin copolymer to cultured neurons differentiated from human stem cells. These chips are preassembled and easier-to-use than traditional compartmentalized poly(dimethylsiloxane) devices. Multiple common experimental paradigms are described here, including viral labeling, fluidic isolation, axotomy, and immunostaining.

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips

Here, we present a protocol for preparing and culturing a blood brain barrier metastatic tumor micro-environment and then quantifying its state using confocal imaging and artificial intelligence (machine learning).

Quantifying the Brain Metastatic Tumor Micro-Environment using an Organ-On-A Chip 3D Model, Machine Learning, and Confocal Tomography

A protocol for the culture and manipulation of mouse embryonic tissue on a microfluidic chip is provided. By applying pulses of pathway modulators, this system can be used to externally control signaling oscillations for the functional investigation of mouse somitogenesis.

A Microfluidics Approach for the Functional Investigation of Signaling Oscillations Governing Somitogenesis

Explore microfluidic technology for multi-omics analysis of neuronal metabolic dynamics following axonal injury. This method enables transcriptomics and metabolic flux studies in cortical neurons.

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

Explore More Videos