Engineering Contractile 2D and 3D Human Skeletal and Cardiac Muscle Microtissues

Skeletal and cardiac muscles are crucial biological actuators with limited capacity to repair themselves after significant trauma or disease states. Engineering these complex tissues using human derived cells has potential applications to serve as more physiologically relevant and economical test beds for regenerative medicine therapies compared to animal models and costly human trials. However, before we can engineer these tissues, we must first gain a better understanding of how the structure and composition of the extracellular matrix (ECM) influences differentiation and maturation into contractile muscle in vitro. To do this, we used traditional microcontact printing techniques to determine how ECM composition and line geometry influenced differentiating human skeletal muscle in 2D, and we incorporated these differentiating human myotubes into a muscular thin film (MTF) assay previously developed to measure 2D cardiomyocyte (CM) contractility. We also engineered contractile 3D cardiac microtissues with integrated force indicators (MIFIs) by seeding embryonic stem cell derived CMs and cardiac fibroblasts in biologically derived ECM hydrogels. From the 2D work, we found that human skeletal muscle derived cells had significantly higher myotube formation on laminin (LAM) lines, and C2C12 mouse myoblasts required more specific LAM line geometries than differentiating human skeletal muscle myoblasts to form uniaxially aligned myotubes. Additionally, we found that LAM with trace amounts of perlecan significantly increased human myotube formation compared to more purified LAM solutions. We also determined that 2D human skeletal muscle was limited to 1 week of differentiation before differentiating myoblasts delaminated from patterned polydimethylsiloxane. For the 3D engineered muscle constructs, we found that cardiac MIFIs could be maintained in culture for at least 2 weeks, exerted twitch forces ~1 - 7 μN and responded as expected to excitatory pharmacological stimuli. We developed the 3D MIFI assay using CMs with the intention of applying this platform to patient specific CMs from induced pluripotent stem cells as well as to differentiating skeletal muscle. The findings we have made by engineering contractile 2D human skeletal muscle and 3D human cardiac microtissues have future applications as patient specific regenerative therapy models, test beds for pharmaceutical therapies, building blocks for engineering functional muscle replacements, and as soft robotics actuators.