Method Article

Generating Neuromuscular Junctions Using a Microfluidic Device

July 8th, 2025

In This Article

Abstract

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Source: Stoklund Dittlau, K. et al., Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices. J. Vis. Exp. (2021)

The video demonstrates the formation of neuromuscular junctions using a microfluidic device. Neural precursor cells and muscle progenitor cells are seeded into different wells within the device and left to mature into motor neurons and myotubes, respectively. The creation of a volume and chemical gradient leads to the development of active neuromuscular junctions.

Protocol

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All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

All reagents and equipment used in this protocol are listed in the Table of Materials and should be used sterile. Media should be heated to room temperature (RT) before use unless otherwise specified. For an overview of the co-culture protocol, please see Figure 1.

1. Differentiation of motor neuron progenitors from induced pluripotent stem cells (iPSCs)

  1. Follow the motor neuron differentiation protocol, adapted from a previous study, until....

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Results

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Neural differentiation diagram with cell plating over time, showing chemotactic gradient and analysis.
Figure 1: Schematic overview of the motor unit protocol in microfluidic devices. Differentiation timeline and co-culture overview from day 0 to day 28 according to the timeline of the motor neuron differentiation protocol. Motor neuron differentiation.......

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Disclosures

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No conflicts of interest declared.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Acetic AcidCHEM-Lab NVCL00.0116.1000Coating component. H226, H314. P280
Agrin (recombinant human protein)R&D systems6624-AG-050Media supplement
Ascorbic acidSigmaA4403Media component
βIII-tubulin (Tubulin)Abcamab7751Antibody (1:500)
β-mercaptoethanolThermo Fisher Scientific31350010Media component. H317. P280.
B-27 without vitamin AThermo Fisher Scientific12587-010Media component
BDNF (brain-derived neurotrophic factor)Peprotech450-02BGrowth factor
bFGF (recombinant human basic fibroblast growth factor)Peprotech100-18BGrowth factor
Collagen from calfskinThermo Fisher Scientific17104019Coating component
CNTF (ciliary neurotrophic factor)Peprotech450-13BGrowth factor
DAPTTocris Bioscience2634Media supplement
DMEM/F12Thermo Fisher Scientific11330032Media component
DMSOSigmaD2650-100MLCryopreservation component. H315, H319, H335. P280.
Dulbecco's phosphate-buffered saline (DPBS)Thermo Fisher Scientific14190250no calcium, no magnesium
EthanolVWR20.821.296Sterilization. H225. P280
Fetal bovine serumThermo Fisher Scientific10270106Media component
Fluorescence Mounting MediumDakoS3023Immunocytochemistry component
GDNF (glial cell line-derived neurotrophic factor)Peprotech450-10BGrowth factor
Horse serumThermo Fisher Scientific16050122Media component
IMDMThermo Fisher Scientific12440053Media component
Insulin transferrin seleniumThermo Fisher Scientific41400045Media component
Knockout serum replacementThermo Fisher Scientific10828-028Cryopreservation component
Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membraneSigmaL2020-1MGCoating component and media supplement
L-glutamineThermo Fisher Scientific25030-024Media component
N-2 supplementThermo Fisher Scientific17502-048Media component
Neurobasal mediumThermo Fisher Scientific21103049Coating and media component
Non-essential amino acidsThermo Fisher Scientific11140050Media component
Parafilm MSigmaP7793-1EAStoring equipment
Penicillin/Streptomycin (5000 U/mL)Thermo Fisher Scientific15070063Media component
Petri dish (3 cm)nunc153066Diameter: 3 cm
Petri dish (10 cm)Sarstedt833.902Diameter: 10 cm
Plate (6-well)Cellstar Greiner bio-one657160Culture plate
Poly-L-ornithine (PLO)SigmaP3655-100MGCoating component
Retinoic acidSigmaR2625Media supplement. H302, H315, H360FD, H410. P280.
Revita cell supplementThermo Fisher ScientificA2644501ROCK inhibitor solution
Smoothened agonistMerch Millipore566660Media supplement
Sodium pyruvateLife Technologies11360-070Media component
T75 flaskSigmaCLS3276Culture plate
TrypLE expressThermo Fisher Scientific12605010MAB dissociation solution
Tubocyrarine hydrochloride pentahydrateSigmaT2379-100GAcetylcholine receptor blocker. H301. P280.
XonaChips pre-assembled microfluidic deviceXona MicrofluidicsXC150Microgroove length: 150 μm

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Tags

Neuromuscular JunctionsMicrofluidic DeviceNeural Progenitor CellsMuscle Progenitor CellsMotor NeuronsMyotubesMicrogroovesCell SeedingGrowth MediumDifferentiation Medium

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