Lin Yu-Lung, Nhieu Jennifer, Lerdall Thomas, Milbauer Liming, Wei Chin-Wen, Lee Dong Jun, Oh Sang-Hyun, Thayer Stanley, Wei Li-Na
Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN, 55455, USA.
The Ph.D. Program for Translational Medicine, College of Medical Sciences and Technology, Taipei Medical University, Taipei, Taiwan.
Cell Biosci. 2023 Sep 12;13(1):168. doi: 10.1186/s13578-023-01115-2.
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.
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 Ca upon stimulation.
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.
一个运动单位（MU）由单个α运动神经元（MN）及其支配的肌纤维组成。运动单位对所有自主运动至关重要。运动单位的功能缺陷会导致神经肌肉疾病（NMDs）。大多数神经肌肉疾病的病理机制仍知之甚少，部分原因是缺乏能够全面重现运动单位多阶段细胞间相互作用和生理功能的体外模型。
我们设计了一种新型的三维（3D）双层水凝胶三培养系统，在体外可以形成结构有序的运动单位。设计了一种使用运动单位的三种细胞类型（运动神经元、成肌细胞和雪旺细胞）的顺序共培养程序，以在双层水凝胶基质上构建共同分化的三培养物。我们利用带有额外基质胶层的微模塑水凝胶形成双层水凝胶装置。微模塑水凝胶层为成肌细胞分化提供拓扑线索。嵌入雪旺细胞的基质胶层不仅将运动神经元与成肌细胞分开，还为运动单位的发育提供了合适的微环境。完整的模型显示了运动单位的关键特征，包括有序的运动单位结构、有髓神经、在成簇的神经肌肉接头（NMJs）上排列的肌管、运动神经元驱动的肌管收缩以及刺激后胞质钙的增加。
这种有序且功能化的体外运动单位模型为研究神经肌肉疾病和周围神经病变中涉及的病理事件提供了机会，并可作为生理和药理研究的平台，如建模和药物筛选。从技术上讲，这种3D双层水凝胶共培养系统的原理利用了水凝胶的多种不同特性，有助于多种不同细胞类型进行有效且高效的共培养用于组织工程。
