Microfluidic Device for the 3D and Compartmentalized Coculture of Neuronal and Muscle Cells, with Functional Force Readout

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Microfluidic design and assembly. (A) The microfluidic design features three parallel gel regions accessible by six gel filling ports and flanked by two medium channels connected to four medium reservoirs. A surrounding vacuum channel allows for temporary bonding. Scale bar, 2 mm. (B) The platform is composed of a top microfluidic layer assembled on top of a PDMS membrane featuring two sets of two capped pillars (inset), itself bonded to a coverslip. (C) Schematic displaying the final coculture arrangement: embedded in a hydrogel, muscle bundles that are wrapped around and exerted force to the pillars are innervated by neurospheres located in the opposite gel chamber separated by a 1-mm-wide gel region.Muscle differentiation and neuromuscular tissue formation. (A) Immunostaining of the myogenic marker a-actinin (green) and DAPI (blue), demonstrating proper muscle differentiation and formation of sarcomeric striations (examples of striations are indicated by arrowheads in the inset). Scale bars, 50 mm. (B) Muscle bundle width relative to the width at day 0. (C) Passive force generation over the course of 16 days.(D) Representative image of a neuron-muscle coculture in the microfluidic device on day 1 of coculture. Scale bar, 500 mm. (E) Neurite extension over 4 days of coculture. Scale bar, 250 mm. (F) Absolute maximum neurite outgrowth in millimeters over the first 4 days of culture. For comparison, the red dashed line represents the average initial distance between neurospheres and muscle bundles (the shaded area in between the dotted line indicates the SEM). (G) Percentage of muscle bundles contacted by at least one neurite over the course of 4 days. (H) Confocal 3D reconstruction of neurites in the bridge gel region after 1 day of coculture. Scale bar, 200 mm. All error bars, SEM.Activation of NMJs within the microfluidic device. (A) Application of glutamate to the medium results in a delayed stimulation of the muscle, leading to the initiation of muscle twitching with force (left y axis) at an increasing frequency (right y axis) as glutamate diffuses within the neurospheres. (B) Force generated by the muscle bundle upon illumination of the ChR2H134R-HBG3-MN neurospheres on day 15. Application of aBTX inhibited the contractions. (C) Colocalization of incoming motor axons and clusters of AChR indicative of the presence of NMJ. Scale bar, 100 mm.(D) Kymographs of the pillar displacement on day 16 for three stimulation light intensities. (E) Muscle-twitching frequencies as a function of light intensity. *P <0.05, **P <0.001,***P < 0.0005.
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Inventors
Professor Roger Kamm
Department of Mechanical Engineering, MIT
External Link (web.mit.edu)
Sebastian Uzel
Department of Mechanical Engineering, MIT
Managed By
Michelle Hunt
MIT Technology Licensing Officer
Patent Protection

Microfluidic Device for the Three Dimensional and Compartmentalized Coculture of Neuronal and Muscle Cells, with Functional Force Readout

Provisional Patent Application Filed
Publications
Microfluidic Device for the Formation of Optically Excitable, Three-Dimensional, Compartmentalized Motor Units
Science Advances , August 3, 2016, vol. 2, no. 8

Applications

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.

Problems Addressed

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.

Technology

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.

Advantages

  • 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