Characterization of Gelatin Hydrogels Cross-Linked with Microbial Transglutaminase as Engineered Skeletal Muscle Substrates

Abstract

Engineered in vitro models of skeletal muscle are essential for efficiently screening drug safety and efficacy. However, conventional culture substrates poorly replicate physical features of native muscle and do not support long-term culture, which limits tissue maturity. Micromolded gelatin hydrogels cross-linked with microbial transglutaminase (gelatin-MTG hydrogels) have previously been shown to induce C21C2 myotube alignment and improve culture longevity. However, several properties of gelatin-MTG hydrogels have not been systematically characterized, such as changes in elastic modulus during incubation in culture-like conditions and their ability to support sarcomere maturation. In this study, various gelatin-MTG hydrogels were fabricated and incubated in ambient or culture-like conditions. Elastic modulus, mass, and transmittance were measured over a one- or two-week period. Compared to hydrogels in phosphate buffered saline (PBS) or ambient air, hydrogels in Dulbecco’s Modified Eagle Medium (DMEM) and 5% CO2 demonstrated the most stable elastic modulus. A subset of gelatin-MTG hydrogels was micromolded and seeded with C2C12 or primary chick myoblasts, which aligned and fused into multinucleated myotubes with relatively mature sarcomeres. These data are important for fabricating gelatin-MTG hydrogels with predictable and stable mechanical properties and highlight their advantages as culture substrates for engineering relatively mature and stable muscle tissues.

The Role of Embryonic Chick Muscle Cell Culture in the Study of Skeletal Myogenesis

“…It is important to mention that chick primary muscle cell cultures have been used in the last 70 years as a two-dimensional (2D) layer of cells, and a recent study reported a promising work using three-dimensional (3D) chick skeletal muscle cell cultures to promote muscle differentiation ( Gupta et al, 2021 ). These authors harvested thigh muscle cells from 10-day-old chick embryos, seeded them onto gelatin hydrogels and observed that myoblasts fused into aligned, multinucleated myotubes with robust sarcomere formation in long-term cultures.…”

Current Strategies for the Regeneration of Skeletal Muscle Tissue

“…Gelatin is a denatured low-cost form of collagen that can be cross-linked into tunable constructs with elastic moduli similar to native muscle. Gelatin hydrogels crosslinked with microbial transglutaminase (MTG), an FDA-approved enzyme, improved the long-term culture and maturation of myotubes [ 143 ]. Bettadapur et al were able to maintain differentiated skeletal myotubes from C2C12 mouse skeletal myoblasts aligned for three weeks [ 144 ].…”

The Role of Soft Robotic Micromachines in the Future of Medical Devices and Personalized Medicine

“…In Figure 8 (inset), we show an example of an ECM-based gelatin actuator crosslinked with polycitrate. A hybrid gelatin-polycitrate hydrogel, increases the tensile strength and Young's modulus [162] of the resulting material. The mechanism behind this is hydrophilic interactions between polar groups on the polycitrate and gelatin molecules, as well as entanglement with the long polycitrate chains [162].…”