Development of a human neuromuscular tissue-on-a-chip model on a 24-well-plate-format compartmentalized microfluidic device

Yamamoto, Kazuki; Yamaoka, Nao; Imaizumi, Yu; Nagashima, Takunori; Furutani, Taiki; Ito, Takuji; Okada, Yohei; Honda, Hiroyuki; Shimizu, Kazunori

doi:10.1039/d1lc00048a

Cited by 21 publications

(23 citation statements)

References 40 publications

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“…Other authors have reported functionality of in vitro NMJs from different cell sources [ 107 , 108 , 109 ]. This was done by evaluating muscle contraction upon excitation of MN, either by glutamate [ 110 , 111 ] or optogenetic [ 112 , 113 ] or electrophysiological [ 108 , 114 ] stimulation and by testing the specificity of transmission response by antagonists which either blocked ACh release or binding [ 16 , 107 , 115 ]. In this context, it would be informative to integrate hiPSC-derived SC into NMJ culture systems suited for such readouts in order to evaluate a possible impact on in vitro NMJ functionality.…”

Section: Discussionmentioning

confidence: 99%

hiPSC-Derived Schwann Cells Influence Myogenic Differentiation in Neuromuscular Cocultures

Hörner

Couturier

Bruch

et al. 2021

Cells

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Motoneurons, skeletal muscle fibers, and Schwann cells form synapses, termed neuromuscular junctions (NMJs). These control voluntary body movement and are affected in numerous neuromuscular diseases. Therefore, a variety of NMJ in vitro models have been explored to enable mechanistic and pharmacological studies. So far, selective integration of Schwann cells in these models has been hampered, due to technical limitations. Here we present robust protocols for derivation of Schwann cells from human induced pluripotent stem cells (hiPSC) and their coculture with hiPSC-derived motoneurons and C2C12 muscle cells. Upon differentiation with tuned BMP signaling, Schwann cells expressed marker proteins, S100b, Gap43, vimentin, and myelin protein zero. Furthermore, they displayed typical spindle-shaped morphologies with long processes, which often aligned with motoneuron axons. Inclusion of Schwann cells in coculture experiments with hiPSC-derived motoneurons and C2C12 myoblasts enhanced myotube growth and affected size and number of acetylcholine receptor plaques on myotubes. Altogether, these data argue for the availability of a consistent differentiation protocol for Schwann cells and their amenability for functional integration into neuromuscular in vitro models, fostering future studies of neuromuscular mechanisms and disease.

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Section: Discussionmentioning

confidence: 99%

hiPSC-Derived Schwann Cells Influence Myogenic Differentiation in Neuromuscular Cocultures

Hörner

Couturier

Bruch

et al. 2021

Cells

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“…Human iPSC clones (201B7 [ 17 ]) provided by Dr. Shinya Yamanaka (RIKEN BRC through the Project for Realization of Regenerative Medicine and National Bio-Resource Project of the MEXT, Japan) were cultured and differentiated into MNs, as reported previously [ 18 , 19 ]. Some hiPSC-derived MN spheroids cultured for 14 or 15 days were collected in a 15 mL tube and washed twice with PBS.…”

Section: Methodsmentioning

confidence: 99%

Alignment of Skeletal Muscle Cells Facilitates Acetylcholine Receptor Clustering and Neuromuscular Junction Formation with Co-Cultured Human iPSC-Derived Motor Neurons

Shimizu

Kassai

Kamei

et al. 2022

Cells

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In vitro neuromuscular junction (NMJ) models are powerful tools for studying neuromuscular disorders. Although linearly patterned culture surfaces have been reported to be useful for the formation of in vitro NMJ models using mouse motor neuron (MNs) and skeletal muscle (SkM) myotubes, it is unclear how the linearly patterned culture surface increases acetylcholine receptor (AChR) clustering, one of the steps in the process of NMJ formation, and whether this increases the in vitro NMJ formation efficiency of co-cultured human MNs and SkM myotubes. In this study, we investigated the effects of a linearly patterned culture surface on AChR clustering in myotubes and examined the possible mechanism of the increase in AChR clustering using gene expression analysis, as well as the effects of the patterned surface on the efficiency of NMJ formation between co-cultured human SkM myotubes and human iPSC-derived MNs. Our results suggest that better differentiation of myotubes on the patterned surface, compared to the flat surface, induced gene expression of integrin α7 and AChR ε-subunit, thereby increasing AChR clustering. Furthermore, we found that the number of NMJs between human SkM cells and MNs increased upon co-culture on the linearly patterned surface, suggesting the usefulness of the patterned surface for creating in vitro human NMJ models.

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Section: Microfluidics For Bio‐actuatorsmentioning

confidence: 99%

“…[431] Finally, microdevices with separate chambers for the precise compartmentalization of motoneurons and skeletal muscle tissues, which fit the well size of 24 well plates, have been proposed tools for fundamental research and drug development for neuromuscular disorders (Figure 13I). [432] Of note, microfluidic platforms for neuromuscular junction modeling can also include optically excitable cells. Uzel et al differentiated motor neurons from mouse embryonic stem cells and co-cultured them with myoblast-derived muscle strips within a 3D hydrogel (Figure 14).…”

Section: Functional Control Of Muscle Tissue In Microfluidic Devicesmentioning

confidence: 99%

Microfluidic Tissue Engineering and Bio‐Actuation

et al. 2022

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Bio‐hybrid technologies aim to replicate the unique capabilities of biological systems that could surpass advanced artificial technologies. Soft bio‐hybrid robots consist of synthetic and living materials and have the potential to self‐assemble, regenerate, work autonomously, and interact safely with other species and the environment. Cells require a sufficient exchange of nutrients and gases, which is guaranteed by convection and diffusive transport through liquid media. The functional development and long‐term survival of biological tissues in vitro can be improved by dynamic flow culture, but only microfluidic flow control can develop tissue with fine structuring and regulation at the microscale. Full control of tissue growth at the microscale will eventually lead to functional macroscale constructs, which are needed as the biological component of soft bio‐hybrid technologies. This review summarizes recent progress in microfluidic techniques to engineer biological tissues, focusing on the use of muscle cells for robotic bio‐actuation. Moreover, the instances in which bio‐actuation technologies greatly benefit from fusion with microfluidics are highlighted, which include: the microfabrication of matrices, biomimicry of cell microenvironments, tissue maturation, perfusion, and vascularization.

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Development of a human neuromuscular tissue-on-a-chip model on a 24-well-plate-format compartmentalized microfluidic device

Abstract: Engineered three-dimensional models of neuromuscular tissues are promising for use in mimicking their disorder states in vitro. Although several models have been developed, it is still challenging to mimic the...

Cited by 21 publications

References 40 publications

hiPSC-Derived Schwann Cells Influence Myogenic Differentiation in Neuromuscular Cocultures

hiPSC-Derived Schwann Cells Influence Myogenic Differentiation in Neuromuscular Cocultures

Alignment of Skeletal Muscle Cells Facilitates Acetylcholine Receptor Clustering and Neuromuscular Junction Formation with Co-Cultured Human iPSC-Derived Motor Neurons

Microfluidic Tissue Engineering and Bio‐Actuation

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