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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session A

Poster H14 - 2D Micropatterned Membrane for Studying Mechanotransduction in Striated Muscle

Thursday, October 24, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Mutations in the LMNA gene, which encodes the nuclear envelope proteins lamin A/C, lead to a spectrum of diseases collectively known as laminopathies. Specific mutations can cause defects in fat storage, peripheral nerves, or in muscles, including muscular dystrophy and dilated cardiomyopathy. While lamin A/C is expressed in nearly every tissue, the majority of lamin mutations affect striated muscle, which suggests that the mutation impacts mechanosensitive pathways. However, studying these mutations is challenging as in vitro models of skeletal and cardiac muscle tissue often diverge significantly from mature muscle cells found in vivo. Striated muscle has a highly ordered structure, and muscle cells grown in tissue culture fail to to reach maturity without proper conditioning. Thus, there is a pressing need to develop better in vitro models to recreate the in vivo structure to effectively study laminopathy muscle defects and the underlying mechanisms. Previous work has shown that a combination of substrate patterning and specific agonists of signaling pathways can help create more mature muscle fibers, motivating the work here.

Materials and

Methods:  Our goal was to create a cell culture substrate with nanostructures, 800 nm wide ridges that are 1000 nm tall, separated by 800 nm wide gaps, to resemble in vivo tissue architecture and promote muscle cell differentiation and alignment. To create these nanoscale structures, I fabricated silicon wafers to serve as mold to cast polydimethylsiloxane (PDMS) substrates. First I spin-coated Oir620-7i photoresist onto a 4 inch wafer. Then I exposed the photoresist to UV light using the GCA AutoStepper to imprint my pattern into the photoresist layer. Then I developed the pattern using 726 MIF to fully create my pattern in the photoresist. I performed a silicon etch with the Unaxis 770 Deep Silicon Etcher which removes silicon in the regions not covered by photoresist hence etching the pattern one micron into the wafer (Figure 1). After etching, I stripped the remaining photoresist and applied a hydrophobic teflon coating to make the wafer suitable for creating a mold to cast thin sheets of PDMS with the nanostructures.

After obtaining the PDMS sheets, we plated primary mouse myoblasts onto the PDMS nanostructured surfaces coated with matrigel and induced differentiation of myoblasts into myotubes starting one day after plating. We used an Airyscan LSM900 confocal microscope to observe the cells and assess their alignment and maturity based on their sarcomeric structures. In future work, I will apply uniaxial stretch to these membranes and compare nuclear deformation between wild type and Lmna knockout myotubules.

Results, Conclusions, and Discussions: Lmna knockout myotubes have defects in mechanotransduction but the exact mechanisms for those defects are still unknown. In this study, I developed a tool to create a 2D patterned structure for live imaging and stretch testing of skeletal and cardiac myotubes in vitro. Using photolithographic techniques, I developed a method to create thin nanopatterned, flexible PDMS membranes, which can be used to study mechanotransduction systems in real time by applying static or cyclical tension to cells seeded on these membranes. The wafer I created has an array of 1 cm by 1 cm squares with ridges running along the length of each square. I have used the Zygo Optical Profilometer to confirm the dimensions of the ridges and the etch depth. Figure 2 shows an image of the completed wafer. I am currently evaluating a prototype of the nanopatterned membrane to determine its ability to enable myotubules to align along the pattern in an ordered fashion. Once the experimental platform is established and validated, I will use it to study the responses of Lmna knockout myotubes and cardiomyocytes to mechanical stimuli. Ultimately, we plan to use this platform to elucidate the molecular mechanisms responsible for the muscular defects in laminopathies. By discovering the underlying mechanisms causing laminopathies we can more effectively work to develop treatments to help people suffering from these conditions.

Acknowledgements (Optional):  This material is based upon work supported by the National Science Foundation under Grant No. NNCI-2025233. Any opinions, findings, conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.