Abstract
Tension and mechanical properties of muscle tissue are tightly related to proper skeletal muscle function, which makes experimental access to the biomechanics of muscle tissue formation a key requirement to advance our understanding of muscle function and development. Recently developed elastic in vitro culture chambers allow for raising 3D muscle tissue under controlled conditions and to measure global tissue force generation. However, these chambers are inherently incompatible with high-resolution microscopy limiting their usability to global force measurements, and preventing the exploitation of modern fluorescence based investigation methods for live and dynamic measurements. Here, we present a new chamber design pairing global force measurements, quantified from post-deflection, with local tension measurements obtained from elastic hydrogel beads embedded in muscle tissue. High-resolution 3D video microscopy of engineered muscle formation, enabled by the new chamber, shows an early mechanical tissue homeostasis that remains stable in spite of continued myotube maturation.
Highlights
Skeletal muscle is one of the most abundant tissues in the human body and is crucial for essential functions such as limb movement, thermogenesis, and maintaining posture (Periasamy et al, 2017; Lauretani et al, 2003; Buckingham, 2001)
Molecular biology has contributed a phlethora of fluorescence microscopy-based tools to label molecules (Heilemann et al, 2008; Walker et al, 2015; Bourgeois et al, 2012), modify signaling cascades (Fenno et al, 2011) and measure molecular interactions (Algar et al, 2019) as well as mechanical tension (Lee et al, 2019; Grashoff et al, 2010; Ringer et al, 2017), the field of muscle tissue research is partially hampered in exploiting these tools due in part to the limited access to high end and super resolution fluorescence
The bottom component is milled from a PMMA block to contain eight individual oval-shaped cell culture chambers (Figure 1A, bottom; Figure 1—figure supplement 1). This bottom part is glued to a standard microscopy coverslip, which enables high- as well as super resolution inverted fluorescence microscopy compatible with oil or water immersion objectives
Summary
Skeletal muscle is one of the most abundant tissues in the human body and is crucial for essential functions such as limb movement, thermogenesis, and maintaining posture (Periasamy et al, 2017; Lauretani et al, 2003; Buckingham, 2001). This characterization has been largely achieved on the global tissue level, the local mechanical properties during muscle development and maturation remain inaccessible This is largely due to the limited optical access of muscle tissue which prevents the use of modern fluorescence microscopy-based methods. Non-destructive experimental approaches to investigate spatial and temporal forces on a cellular level are limited and characterization of local cell niches within a tissue remains a challenge To overcome this problem, Campas et al introduced biocompatible oil microdroplets to evaluate cellgenerated forces in living tissue for the first time (Campas et al, 2014) and very recently, deformable (Polyacrylamide) PAA beads were used to determine local tension on a cell scale within cancer spheroids, zebrafish embryos and during phagocytosis (Dolega et al, 2017; Lee et al, 2019; Traber et al, 2019; Vorselen et al, 2020). By enabling real-time high-resolution microscopy on 3D in vitro muscle tissues for the first time, we envision our novel reconstituted muscle tissue device will be an advantage to the skeletal muscle research community by enabling the dynamic study of (sub-)cellular events
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