Large-scale spontaneous self-organization and maturation of skeletal muscle tissues on ultra-compliant gelatin hydrogel substrates

Abstract

cellular self-organization is the fundamental driving force behind the complex architectures of native tissue. Yet, attempts at replicating native tissue architectures in vitro often involve complex microfabrication methods and materials. While impressive progress has been made within engineered models of striated muscle, the wide adaptation of these models is held back by the need for specific tools and knowhow. In this report, we show that C2C12 myoblasts spontaneously organize into highly aligned myotube tissues on the mm to cm scale, when cultured on sufficiently soft yet fully isotropic gelatin hydrogel substrates. interestingly, we only observed this phenomenon for hydrogels with Young's modulus of 6 kPa and below. For slightly more rigid compositions, only local micrometerscale myotube organization was observed, similar to that seen in conventional polystyrene dishes. the hydrogel-supported myotubes could be cultured for multiple weeks and matured into highly contractile phenotypes with notable upregulation of myosin heavy chain, as compared to myotubes developed in conventional petri dishes. the procedure for casting the ultra-soft gelatin hydrogels is straight forward and compatible with standardized laboratory tools. it may thus serve as a simple, yet versatile, approach to generating skeletal muscle tissue of improved physiological relevance for applied and basic research. Skeletal muscle comprises the largest tissue in the human body, and plays an essential role in locomotion as well as in metabolism and homeostasis 1,2. The diseases associated with skeletal muscle are wide ranging and includes myopathies 3 , dystrophies as well as insulin resistance in diabetes mellitus type 2 4. Within in vitro research, traditional tissue culture polystyrene (TCPS) remains the most widespread substrate for culturing and studying skeletal muscle in health and disease. The use of TCPS is common, despite the general recognition that the practically incompressible and planar TCPS dishes constitute a very poor representation of the native cellular environment. Aiming to overcome this paradox, numerous advanced in vitro models of striated muscle have emerged in recent years. These models include freestanding micro tissues 5-10 , bio-printed constructs 11,12 , 3D cultures in fibrous scaffolds 13,14 and microphysiological systems 3,15,16. These novel approaches generally display highly unidirectional muscle tissues, and native function in terms of concerted contractile shortening. Nevertheless, there are several obstacles to the widespread adaptation of these models. For instance, many custom solutions are not readily compatible with standard laboratory tools and analyses, and their fabrication may require highly specific tools and detailed expertise. In the vast majority of engineered models of striated muscle, extracellular biochemical or mechanical cues are applied for guiding cellular organization into aligned tissues, for instance in the form of nanofibrous

Customized 3D-printed stackable cell culture inserts tailored with bioactive membranes

“…S2 ). While not crucial for the barrier function explored, the shape of the cell culture area can align, for instance, muscle cells 27 .…”

“…Cells sense the stiffness of their growth matrices 21 , 22 , and the intrinsic biodegradability of hydrogels is considered a crucial feature during tissue regeneration 23 . Various natural, synthetic, and hybrid biomaterials such as Poly (ethylene glycol) (PEG) 24 , silk 25 , Matrigel 8 , decellularized tissues 26 , and gelatin 5 , 16 , 27 – 29 have been explored in the literature. An advantage of hydrogels is that they are often transparent, which is not always the case for filters.…”

Tension-driven multi-scale self-organisation in human iPSC-derived muscle fibers

“…At the cellular level, myotubes first elongate actively, then stabilise to a defined length, likely depending on the culture and matrix conditions ( Jensen et al, 2020 ). At this stage, myofiber ends start to establish stable connections with their environment, coinciding with their transition from immature to transitional myofibers.…”

Mature Myotubes Generated From Human-Induced Pluripotent Stem Cells Without Forced Gene Expression

“…Several reports have suggested that myotube culture with hydrogel covering promotes differentiation and maturation ( Maffioletti et al, 2018 ; Jensen et al, 2020 ; Urciuolo et al, 2020 ). In these reports, Matrigel diluted in the differentiation medium was used as the hydrogel material, and the myotubes indicated maturation by the expression change of various MYH gene subtypes.…”