Long-term microfluidic cultures of myotube microarrays for high-throughput focal stimulation.

We have developed a microfluidic cell culture method that allows for the formation of linear isolated myotubes organized in a parallel microarray. Attachment and spreading of cells are confined within microtracks of cell-adherent proteins separated by a protein-repellent coating. Signaling molecules or other molecules of interest can be focally delivered to the myotubes using heterogeneous microfluidic streams.

Objective quantification of acetylcholine receptor aggregation using fast Fourier transforms.

A new approach for objectively analyzing the aggregation of acetylcholine receptors (AChRs) through power spectrum analysis derived from fast Fourier transform (FFT) of images has been developed. Presently, detection of AChR aggregates at neuromuscular junctions is not easily accomplished. Though the formation of AChR clusters results in periodic gray-level variations that differ with time, no study reporting their correlation with frequency information in the Fourier domain for aggregates' detection purposes exists.

Long-term micropatterned cell cultures in heterogeneous microfluidic environments.

Microfluidic poly(dimethylsiloxane) (PDMS) devices were constructed and used as long-term cell culture platforms for skeletal muscle cell differentiation and for dynamic application of chemical stimuli to the cells. The devices featured two orthogonal fluidic networks: one for long-term cell perfusion at minimal rates and the other one for short-term selective cell treatment and stimulation with biologically relevant molecules. The cells were micropatterned within the microfluidic channels using surface modification techniques, cultured under continuous flow, and allowed to fuse into polynucleated myotubes (a major milestone in muscle cell differentiation).

A nanofabricated planar aperture as a mimic of the nerve-muscle contact during synaptogenesis.

The release of synaptogenic factors by the nerve terminal plays a central role in the aggregation of neurotransmitter receptors at the postsynaptic membrane, a precisely timed and localized process that is essential for the correct formation and functioning of the synapse. This process has been difficult to re-capitulate in cell culture because present cell stimulation methods do not have sufficient spatiotemporal control of the delivery of soluble signals. We cultured myotubes atop nanofabricated planar apertures (2-8 microm diameter) to focally stimulate the muscle cell membrane with neural agrin, a synaptogenic factor released by motor neurons during development.

Differentiation-on-a-chip: a microfluidic platform for long-term cell culture studies.

Here we demonstrate a microfluidic perfusion system suitable for a long-term (>2 week) culture of muscle cells spanning the whole process of differentiation from myoblasts to myotubes. Cell-adhesive surface microdomains alternating with a robust cell-repellent coating mimic in vivo spatial cues for muscle cell assembly and allow for confining the fusion of myoblasts into aligned, isolated multinucleated myotubes. The microfluidic system provides accurate control of the perfusion rates and biochemical composition of the environment surrounding the cells.