Differentiation of Bioengineered Skeletal Muscle within a 3D Printed Perfusion Bioreactor Reduces Atrophic and Inflammatory Gene Expression

Bioengineered skeletal muscle tissues benefit from dynamic culture environments which facilitate the appropriate provision of nutrients and removal of cellular waste products. Biologically compatible perfusion systems hold the potential to enhance the physiological biomimicry of in vitro tissues via dynamic culture, in addition to providing technological advances in analytical testing and live cellular imaging for analysis of cellular development. To meet such diverse requirements, perfusion systems require the capacity and adaptability to incorporate multiple cell laden constructs of both monolayer and bioengineered tissues. This work reports perfusion systems produced using additive manufacturing technology for the in situ phenotypic development of myogenic precursor cells in monolayer and bioengineered tissue. Biocompatibility of systems 3D printed using stereolithography (SL), laser sintering (LS), and PolyJet outlined preferential morphological development within both SL and LS devices. When exposed to intermittent perfusion in the monolayer, delayed yet physiologically representative cellular proliferation, MyoD and myogenin transcription of C₂C₁₂ cells was evident. Long-term (8 days) intermittent perfusion of monolayer cultures outlined viable morphological and genetic in situ differentiation for the live cellular imaging of myogenic development. Continuous perfusion cultures (13 days) of bioengineered skeletal muscle tissues outlined in situ myogenic differentiation, forming mature multinucleated myotubes. Here, reductions in IL-1β and TNF-α inflammatory cytokines, myostatin, and MuRF-1 atrophic mRNA expression were observed. Comparable myosin heavy chain (MyHC) isoform transcription profiles were evident between conditions; however, total mRNA expression was reduced in perfusion conditions. Decreased transcription of MuRF1 and subsequent reduced ubiquitination of the MyHC protein allude to a decreased requirement for transcription of MyHC isoform transcripts. Together, these data appear to indicate that 3D printed perfusion systems elicit enhanced stability of the culture environment, resulting in a reduced basal requirement for MyHC gene expression within bioengineered skeletal muscle tissue.