The inventors have developed a microfluidic device which allows for the formation of neuromuscular junctions in a microenvironment. This device helps unravel the mechanisms leading to various neurodegenerative disorders by closely recapitulating the physiology of the neuromuscular tissue.
Neuromuscular junctions (NMJs) are the fundamental physiologic structures responsible for producing virtually all motor functions. The loss of neuromuscular junction is associated with various incapacitating or lethal disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscle atrophy (SMA). Developing in vitro assays that replicate the physiology of the neuromuscular tissue is crucial to understanding the formation of NMJs and to unravel the mechanisms leading to their degeneration. Traditional 2D culture platforms typically consist of a layer of muscle cells onto which neurons are uniformly plated. The simplistic 2D nature of the system affects neurite outgrowth and precludes direct interaction between the neurons and the muscle cells, which is essential to the understanding of NMJs.
The inventors have developed a microfluidic device which provides an in vitro platform to allow the simultaneous 3D coculture and compartmentalization of motor neurons (MNs) and skeletal muscle cells within an extracellular matrix. The device consists of two cell culture compartments, one contains the neuronal cells and the other contains the muscle cells. The two compartments are physically segregated via the buffer compartment, which contains the hydrogel comprised of collagen. When appropriate conditions are applied, the axon can extend from the neuronal cell compartment through the buffer compartment to the muscle cell compartment to form a NMJ with the muscle cells, causing the muscle cells to contract. The microfluidic chambers provide a 3D configuration similar to that of the native tissue, in which one channel serves as a surrogate for the spinal cord and the other one models the remotely innervated muscle tissue as found in the limbs. Furthermore, the inventors have added a force sensing feature to this device by fabricating flexible pillars within the muscle cell compartment. The force of muscle contractions can be monitored by measuring the deflection of the compliant pillars around which the muscle bundles are wrapped. Additionally, using optogenetics, the neural cells were genetically modified to respond to light, which allows for versatile and non-invasive in vitro control over the neurons. To further increase the physiologic relevance of the system, neuronal cells derived from patients suffering from ALS or SMA can be used to make the whole system specific for that patient.
- Mimics the spinal cord-limb separation by compartmentalizing the two cell types
- Enables direct observation of 3D axonal outgrowth and the formation of functional NMJs
- Allows quantitative measurement of force generated by the muscle cells
- Light stimulation of MNs provides great versatility over the excitation of the tissue by making it cell specific