Optimizing culture conditions and ground controls of C2C12 myoblasts for FLUMIAS-ISS live-cell imaging microscope

Abstract

In the first part of this thesis differentiated murine C2C12 muscle cells were exposed for up to 2h to clino-rotation induced simulated microgravity. Western blot analyses were used to investigate the protein levels of the Chaperon-Assisted Autophagy pathway related proteins BAG3 and p62. Samples exposed to microgravity were compared to controls kept at standard gravity conditions. Regarding BAG3 expression a stable increase of approximately 18% above control levels was detected throughout the entire experimental duration. Levels of p62 were also found to be increased by 15%, however, solely after 2h of exposure to simulated microgravity. These results present evidence for an effect of stimulated microgravity on autophagy regulation and hint at a disbalance of the CASA mediated autophagy pathway for short term exposure to microgravity. For future planned space-flight experiments utilizing the novel FLUMIAS-ISS live-cell imaging microscope, intended to investigate Chaperon-Assisted Autophagy pathway components SYNPO2 and BAG3 in terminally differentiated muscle cells, it is a prerequisite to be able to reliably differentiate murine C2C12 myoblasts into myotubes. Part of this thesis tried to establish and optimize automated culture systems using comparable hardware as will be used with the FLUMIAS microscope. Thereby the influence of pump induced fluid shear forces, medium composition, surface treatments and medium volume on myotube fusion was assessed. Throughout all technical iterations of this cell culture system we finally were able to produce low numbers of differentiated myotubes. However, we concluded that it basically will be possible to significantly enhance the myotubes formation process after further adaptation of the technical microenvironment that was beyond this work.