Poster BB18 - Neuromuscular Junction Modeling Using a Tissue Culture Device

Introduction: The neuromuscular junction (NMJ) is a synapse between a motor neuron and a muscle fiber that allows for voluntary movement. It is difficult to study the effects of various treatments on NMJs due to challenges presented by working with in vivo models. In this work, we develop a 3D-printed device to co-culture NSC-34 and C2C12 cells. The goal of this study is to develop an in vitro culture model of the NMJ to better understand the conditions for its degeneration and regeneration.

Materials and

Methods: We have established optimal cell culture conditions for motor neurons (NSC-34) and myoblasts (C2C12). NSC-34 is a hybrid cell line produced by the fusion of embryonic mouse spinal cord and mouse neuroblastoma. C2C12 is an immortalized mouse myoblast cell line which differentiates into myotubes. NSC-34 cells were cultured on 100 µg/ml extracellular matrix-coated (collagen, laminin, hyaluronic acid, and gelatin) 48-well plates at a density of 20,000 cells/well and cultured for 3 days in differentiation media. In a separate experiment, NSC-34 cells were cultured in differentiation media supplemented with glial cell line-derived neurotrophic factor (GDNF) for 4 days. GDNF is a growth factor known to enhance neurite outgrowth. Brightfield images were taken at 20x magnification, and neurite length was determined using ImageJ. C2C12 cells were cultured in 48-well plates at a density of 200,000 cells/well in differentiation media supplemented with Agrin (100ng/mL) for 5 days. Agrin is a proteoglycan that enhances acetylcholine receptor (AchR) clustering. Alpha bungarotoxin and desmin fluorescent staining was performed, and 10x images were taken. We have designed and 3D printed a tissue culture device of polycaprolactone that has two compartments for culturing NSC-34 and C2C12 cells connected by parallel microchannels (Figure 1).

Results, Conclusions, and Discussions: Our results revealed that NSC-34 cells cultured on gelatin (100 µg/ml) coated substrates significantly enhances neurite extensions in the size range of 50-100 microns compared to extracellular matrix proteins, such as collagen (p=0.0049), laminin (p=0.0098), and tissue culture plastic (p=0.0018) (Figure 2). NSC-34 cells cultured with GDNF (100ng/mL) had a significantly higher percentage of 30-45 microns sized neurite extensions (p=0.0011) (Figure 3). The C2C12 myoblasts readily formed myotubes following serum deprivation, and stimulation with Agrin (100 ng/ml) enhanced AchR clustering (Figure 4).
Our studies show that gelatin is an excellent substrate for motor neurite differentiation and outgrowth. GDNF supplementation results in longer neurite outgrowth, and Agrin supplementation results in formation of AchRs. Based on these results, these culture conditions will be utilized on the 3D printed device in order to observe the formation of NMJs.
Ongoing work involves culturing these cells in the device compartments and monitoring neurite outgrowth through the microchannels. Once this device has been optimized, other small biomolecules will be supplemented to the cell culture and their effect will be observed. NMJs will be identified by staining for bungarotoxin, neurofilament, and desmin which will allow visualization of neurites anchored onto AchRs on myotubes.

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