A novel 3D bilayer hydrogel tri-culture system for studying functional motor units

Yu Lung Lin, Jennifer Nhieu, Thomas Lerdall, Liming Milbauer, Chin Wen Wei, Dong Jun Lee, Sang Hyun Oh, Stanley Thayer, Li Na Wei

Research output: Contribution to journalArticlepeer-review


Background: A motor unit (MU) is formed by a single alpha motor neuron (MN) and the muscle fibers it innervates. The MU is essential for all voluntary movements. Functional deficits in the MU result in neuromuscular disorders (NMDs). The pathological mechanisms underlying most NMDs remain poorly understood, in part due to the lack of in vitro models that can comprehensively recapitulate multistage intercellular interactions and physiological function of the MU. Results: We have designed a novel three-dimensional (3D) bilayer hydrogel tri-culture system where architecturally organized MUs can form in vitro. A sequential co-culture procedure using the three cell types of a MU, MN, myoblast, and Schwann cell was designed to construct a co-differentiating tri-culture on a bilayer hydrogel matrix. We utilized a µ-molded hydrogel with an additional Matrigel layer to form the bilayer hydrogel device. The µ-molded hydrogel layer provides the topological cues for myoblast differentiation. The Matrigel layer, with embedded Schwann cells, not only separates the MNs from myoblasts but also provides a proper micro-environment for MU development. The completed model shows key MU features including an organized MU structure, myelinated nerves, aligned myotubes innervated on clustered neuromuscular junctions (NMJs), MN-driven myotube contractions, and increases in cytosolic Ca2+ upon stimulation. Conclusions: This organized and functional in vitro MU model provides an opportunity to study pathological events involved in NMDs and peripheral neuropathies, and can serve as a platform for physiological and pharmacological studies such as modeling and drug screening. Technically, the rational of this 3D bilayer hydrogel co-culture system exploits multiple distinct properties of hydrogels, facilitating effective and efficient co-culturing of diverse cell types for tissue engineering.

Original languageEnglish
Article number168
JournalCell and Bioscience
Issue number1
Publication statusPublished - Dec 2023


  • Co-culture
  • Hydrogel
  • Motor neuron
  • Motor neuron disorders
  • Motor unit
  • Muscle
  • Neuromuscular
  • Schwann cell

ASJC Scopus subject areas

  • General Biochemistry,Genetics and Molecular Biology


Dive into the research topics of 'A novel 3D bilayer hydrogel tri-culture system for studying functional motor units'. Together they form a unique fingerprint.

Cite this