Formation and optogenetic control of engineered 3D skeletal muscle bioactuators

Abstract

Densely arrayed skeletal myotubes are activated individually and as a group using precise optical stimulation with high spatiotemporal resolution. Skeletal muscle myoblasts are genetically encoded to express light-activated cation channel, Channelrhodopsin-2, which allows for spatiotemporal coordination of the multitude of skeletal myotubes that contract in response to pulsed blue light. Furthermore, ensembles of mature functional 3D muscle microtissues have been formed from the optogenetically encoded myoblasts using a high-throughput device. The device, called “skeletal muscle on a chip”, not only provides the myoblasts with controlled stress and constraints necessary for muscle alignment, fusion and maturation, but also facilitates to measure forces and characterize the muscle tissue. We measured the specific static and dynamic stresses generated by the microtissues, and characterized the morphology and alignment of the myotubes within the constructs. The device allows for testing the effect of a wide range of parameters (cell source, matrix composition, microtissue geometry, auxotonic load, growth factors, and exercise) on the maturation, structure, and function of the engineered muscle tissues in a combinatorial manner. Our studies integrate tools from optogenetics and microelectromechanical systems (MEMS) technology with skeletal muscle tissue engineering to open up opportunities to generate soft robots actuated by multitude of spatiotemporally coordinated 3D skeletal muscle microtissues.

Optogenetic skeletal muscle-powered adaptive biological machines

“…Ion channels that respond to light stimuli have been implemented in a variety of applications that require cellular manipulation. Although the seminal studies in this field were performed on neurons, recent studies have demonstrated optogenetic approaches to control other cell types, including skeletal muscle (12). To implement optogenetic control of our muscle actuators, we used an existing lentiviral transduction protocol to engineer C2C12 murine myoblast cells with a mutated variant of the blue light-sensitive ion channel, Channelrhodopsin-2 (ChR2), namely ChR2(H134R) (Fig.…”

“…Nearly all machines require actuators, modules that convert energy into motion, to produce a measurable output in response to input stimuli. Efforts to manufacture bio-integrated actuators have targeted a range of cell types (5), including flagellated bacteria (6), cardiac muscle (7)(8)(9), and skeletal muscle (10)(11)(12). We previously demonstrated a millimeter-scale soft robotic device, or biobot, that uses the contractile force produced by electrically paced skeletal muscle to drive locomotion across a substrate (10).…”

Biomimicry, Biofabrication, and Biohybrid Systems: The Emergence and Evolution of Biological Design

“…The bioactuator was shown to generate passive tension forces comparable to other demonstrations of tissue engineered skeletal muscle. [118,119] The passive tension force generated could be increased by imposing a static mechanical stretch stimulus during differentiation, and by the addition of biochemical factors, such as human insulin-like growth factor 1 (IGF-1) to the differentiation medium. This was an important confirmation that the adaptive behavior inherent to natural biological systems could be mimicked in biohybrid systems cultured in vitro.…”

3D culture models of tissues under tension

“…In particular, progress has been made in developing a model for skeletal muscle (Juhas et al, 2014;Sakar et al, 2012;Uzel et al, 2014), which could be useful for understanding and modeling dystrophic muscle diseases. Constrained microtissues also have been applied to model airway smooth muscle (West et al, 2013), aortic valve tissue (Kural and Billiar, 2016) and lung (Chen et al, 2016b).…”