Engineered Human Contractile Myofiber Sheets as a Platform for Studies of Skeletal Muscle Physiology

Abstract

Skeletal muscle physiology and the mechanisms of muscle diseases can be effectively studied by an in-vitro tissue model produced by muscle tissue engineering. Engineered human cell-based tissues are required more than ever because of the advantages they bring as tissue models in research studies. This study reports on a production method of a human skeletal myofiber sheet that demonstrates biomimetic properties including the aligned structure of myofibers, basement membrane-like structure of the extracellular matrix, and unidirectional contractile ability. The contractile ability and drug responsibility shown in this study indicate that this engineered muscle tissue has potential as a human cell-based tissue model for clinically relevant in-vitro studies in muscle physiology and drug discovery. Moreover, this engineered tissue can be used to better understand the relationships between mechanical stress and myogenesis, including muscle growth and regeneration. In this study, periodic exercise induced by continuous electrical pulse stimulation enhanced the contractile ability of the engineered myofibers and the secretion of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) from the exercising myofibers. Since the physiology of skeletal muscle is directly related to mechanical stress, these features point to application as a tissue model and platform for future biological studies of skeletal muscle including muscle metabolism, muscle atrophy and muscle regeneration.

A 96-well culture platform enables longitudinal analyses of engineered human skeletal muscle microtissue strength

“…In addition, engineered 3D skeletal muscle models mimic native muscle architecture 21 , provide structural integrity for long-term culture of myotubes in vitro, and enable contractile force measurements 22 . Recent articles report successful development of 3D culture models of human skeletal muscle 9,[23][24][25][26][27][28][29][30][31][32][33] . In these studies, active force is quantified on tissues removed from the supporting culture device to implement a force transducer, which is precise, but invasive.…”

Coupling 3D Printing and Novel Replica Molding for In House Fabrication of Skeletal Muscle Tissue Engineering Devices

“…The structures realized with the Direct Peeling method can be produced with other fabrication techniques, such as milling [ 19 ] or injection molding, [ 27,28 ] thanks to their millimetric dimensions. However, 3D printing offers higher versatility, as the fabrication process can be fully tuned in house from design to manufacturing to fit different needs and possible new necessities naturally arising during research.…”

“…For adequate lodging of 3D contractile tissues, flexible pillars are preferred to promote maturation and to provide tendon‐like attachment points for contraction. [ 17 ] Compared to alternative methods, such as micropatterned surfaces, [ 18,19 ] flexible pillars offer mechanical and functional advantages in the tracking of their displacement as a consequence of a tissue’s contraction. [ 17 ] Downscaling the size of engineered tissues is also possible by producing pillars in a T‐shape: “caps” on top of each pillar help the retention of smaller tissues under tension.…”

“…To avoid multistep photolithography, molds have been generated using single‐step lithography [ 8–10 ] or milled Teflon. [ 19 ] This was followed by manually gluing a thin square of PDMS on top of each pillar for obtaining the T‐shape, making this method time‐consuming and not prone to high throughput. A multiple step aluminum mold has recently been used to fabricate hammer‐like PDMS pillars.…”

Global and local tension measurements in biomimetic skeletal muscle tissues reveals early mechanical homeostasis

“…In contrast, modern reconstituted 3D in vitro skeletal muscle systems are demonstrated to be an efficient tool for rapid and reliable drug screening [2,54] and allow for testing of personalized treatments on cells harvested from individual patients. Besides these medical advantages, such functional muscle tissues were reported to successfully mimic native muscle tissue with long-term structural integrity [23,27] and allow new insights and fundamental knowledge of muscle tissue development, force generation during contraction as well as the phases of disease onset and progression [2,11,19,24,25,31,32,41,48,51]. In culture platforms allowing for in situ force measurements, 3D skeletal muscle tissues self organize between mm sized posts around which muscle precursor cells are seeded together with an extracellular matrix.…”