Differentiation of mammalian skeletal muscle cells cultured on microcarrier beads in a rotating cell culture system

Abstract

The growth and repair of adult skeletal muscle are due in part to activation of muscle precursor cells, commonly known as satellite cells or myoblasts. These cells are responsive to a variety of environmental cues, including mechanical stimuli. The overall goal of the research is to examine the role of mechanical signalling mechanisms in muscle growth and plasticity through utilisation of cell culture systems where other potential signalling pathways (i.e. chemical and electrical stimuli) are controlled. To explore the effects of decreased mechanical loading on muscle differentiation, mammalian myoblasts are cultured in a bioreactor (rotating cell culture system), a model that has been utilised to simulate microgravity. C2C12 murine myoblasts are cultured on microcarrier beads in a bioreactor and followed throughout differentiation as they form a network of multinucleated myotubes. In comparison with three-dimensional control cultures that consist of myoblasts cultured on microcarrier beads in teflon bags, myoblasts cultured in the bioreactor exhibit an attenuation in differentiation. This is demonstrated by reduced immunohistochemical staining for myogenin and alpha-actinin. Western analysis shows a decrease, in bioreactor cultures compared with control cultures, in levels of the contractile proteins myosin (47% decrease, p < 0.01) and tropomyosin (63% decrease, p < 0.01). Hydrodynamic measurements indicate that the decrease in differentiation may be due, at least in part, to fluid stresses acting on the myotubes. In addition, constraints on aggregate size imposed by the action of fluid forces in the bioreactor affect differentiation. These results may have implications for muscle growth and repair during spaceflight.

A novel in vitro three-dimensional skeletal muscle model

“…No mitotic cells were observed in either culture condition. Reduced proliferation has been previously noted in RCCScultured myoblast aggregates (Molnar et al 1997;Torgan et al 2000); however, these reports used support structures (beads, membranes) for cell growth. Interestingly, the appearance of visibly large cells was noted at the periphery of RCCS 6+ d aggregates (see Figs.…”

“…These have included collagen lattice (Cheema et al 2003), collagencoated microcarrier beads (Molnar et al 1997;Torgan et al 2000), and fibrin gel (Huang et al 2005). The use of preexisting extracellular matrix (ECM) can stimulate endogenous myotube ECM production (Vandenburgh et al 1974).…”

“…Most current 3D skeletal muscle constructs are engineered using an exogenous matrix consisting of substances compatible with muscle function (Molnar et al 1997;Torgan et al 2000;Cheema et al 2003). These have included collagen lattice (Cheema et al 2003), collagencoated microcarrier beads (Molnar et al 1997;Torgan et al 2000), and fibrin gel (Huang et al 2005).…”

Effect of IR Laser on Myoblasts: Prospects of Application for Counteracting Microgravity-Induced Muscle Atrophy

“…Following these findings, we hypothesized that IR laser radiation could be a useful tool to counteract the microgravity-induced impairment of energetic metabolism , cytoarchitectural alterations (Pache et al 2010) and decrease in the levels of contractile proteins (Torgan et al 2000) observed in myoblasts exposed to microgravity.…”

“…The C2C12 cells are widely accepted as a model to study the behaviour of satellite cells (Burattini et al 2004), which play a crucial role in skeletal muscle regeneration and repair (Wang and Rudnicki 2012) and are capable to repopulate atrophied muscle (Hawke and Garry 2001). Moreover, C2C12 cells have been already used in previous studies regarding the effects of microgravity on mechanical signalling mechanisms in muscle plasticity (Torgan et al 2000) and myoblast behaviour (Slentz et al 2001;Pache et al 2010).…”