Prolonged Culture of Aligned Skeletal Myotubes on Micromolded Gelatin Hydrogels

Abstract

In vitro models of skeletal muscle are critically needed to elucidate disease mechanisms, identify therapeutic targets, and test drugs pre-clinically. However, culturing skeletal muscle has been challenging due to myotube delamination from synthetic culture substrates approximately one week after initiating differentiation from myoblasts. In this study, we successfully maintained aligned skeletal myotubes differentiated from C2C12 mouse skeletal myoblasts for three weeks by utilizing micromolded (μmolded) gelatin hydrogels as culture substrates, which we thoroughly characterized using atomic force microscopy (AFM). Compared to polydimethylsiloxane (PDMS) microcontact printed (μprinted) with fibronectin (FN), cell adhesion on gelatin hydrogel constructs was significantly higher one week and three weeks after initiating differentiation. Delamination from FN-μprinted PDMS precluded robust detection of myotubes. Compared to a softer blend of PDMS μprinted with FN, myogenic index, myotube width, and myotube length on μmolded gelatin hydrogels was similar one week after initiating differentiation. However, three weeks after initiating differentiation, these parameters were significantly higher on μmolded gelatin hydrogels compared to FN-μprinted soft PDMS constructs. Similar results were observed on isotropic versions of each substrate, suggesting that these findings are independent of substrate patterning. Our platform enables novel studies into skeletal muscle development and disease and chronic drug testing in vitro.

Gelatin–genipin‐based biomaterials for skeletal muscle tissue engineering

“…Although future studies will be required to enhance and optimize culture conditions, our present results provide a proof of concept of the feasibility of the use of micropatterned gelatin–GP biomaterial to generate in vitro cultures of unidirectionally aligned contracting primary myotubes where electrophysiological studies can be performed. A recent study showed that micropatterned gelatin hydrogels, realized with different cross‐linker and smaller strip size, are more effective in driving the growth and orientation of C2C12 myotubes in in vitro long‐term cultures, when compared to the commonly used extracellular microcontact printed PDMS . Although we did not analyze our cells after 3 weeks in culture, we could assume that our system may be as effective as this, and find application for in vitro studies on muscle development and disease, and on chronic drug testing.…”

“…A recent study showed that micropatterned gelatin hydrogels, realized with different cross-linker and smaller strip size, are more effective in driving the growth and orientation of C2C12 myotubes in in vitro long-term cultures, when compared to the commonly used extracellular microcontact printed PDMS. 59 Although we did not analyze our cells after 3 weeks in culture, we could assume that our system may be as effective as this, and find application for in vitro studies on muscle development and disease, and on chronic drug testing.…”

Mechanochemical Patternable ECM‐Mimetic Hydrogels for Programmed Cell Orientation

“…Pre‐defining topographical features at the time of hydrogel crosslinking, via pre‐formed template micro‐molding, could constitute fitting alternatives that can conjugate topography and ECM composition. [ 89,101,102 ] However, these strategies are limited by confining the patterning process within the manufacturing timeframe. Hence, approaches that can enable on‐demand patternability are being pursued, such as spatially controlled hydrogel photodegradation, but are still restricted to either fully synthetic PEG‐based networks or more recently, to hybrid PEG/gelatin combinations.…”

Combined substrate micropatterning and FFT analysis reveals myotube size control and alignment by contact guidance

“…Hsiao et al (2009) reported a DNA oligonucleotide based micropatterned glass substrate for selective adherence and subsequent differentiation of primary myoblasts. Bajaj et al (2011) Antigny, Koenig, Bernheim, & Frieden, 2013;Bettadapur et al, 2016;Shahini et al, 2018). PDMS membranes were utilised for micropatterning C2C12 cells by Huang, Lee, and Li (2010) to study myoblast alignment.…”

“…Zatti et al (2012) went a step further by micropatterning C2C12 cells on hydrogels with tissue like stiffness and elucidating the importance of secretory factors in myogenesis. Furthermore, several other studies have utilized morphometric based characterization parameters, for example, myotube width, fusion index, and nuclear distribution for assessing differentiation and myotube formation (Agley, Velloso, Lazarus, & Harridge, 2012;Antigny, Koenig, Bernheim, & Frieden, 2013;Bettadapur et al, 2016;Shahini et al, 2018). In addition, techniques like 2D FFT analysis and orientation order parameter (OOP) have been applied for assessing alignment in muscle tissues (Bajaj, Rivera, Marchwiany, Solovyeva, & Bashir, 2014;Drew, Eagleson, Baldo Jr., Parker, & Grosberg, 2015;Feinberg et al, 2012).…”