BIOREACTOR PERFUSION SYSTEM FOR THE LONG-TERM MAINTENANCE OF TISSUE-ENGINEERED SKELETAL-MUSCLE ORGANOIDS

Three-dimensional skeletal muscle organ-like structures (organoids) fo rmed in tissue culture by fusion of proliferating myoblasts into paral lel networks of long, unbranched myofibers provide an in vivo-like mod el for examining the effects of growth factors, tension, and space fli ght on muscle cell growth and metabolism. To determine the feasibility of maintaining either avian or mammalian muscle organoids in a commer cial perfusion bioreactive system, we measured metabolism, protein tur nover. and autocrine/paracrine growth factor release rates. Medium glu cose was metabolized at a constant rate in both low-serum- anti serum- free media for up to 30 d. Total organoid noncollagenous protein anti DNA content decreased approximately 22-28% (P < 0.05) over a 13-d peri od. Total protein synthesis rates could be determined accurately in th e bioreactors for up to 30 h and total protein degradation rates could be measured for up to 3 wk. Special fixation and storage conditions n ecessary for space flight studies were validated as part of the studie s. For example. the anabolic autocrine/paracrine skeletal muscle growt h factors prostaglandin F-2 alpha (PGF(2 alpha)) and insulin-like grow th factor-1 (IGF-1) could be measured accurately in collected media fi actions, even after storage at 37 degrees C for up to 10 d. In contra st, creatine kinase activity (a marker of cell damage) in collected me dia fractions was unreliable. These results provide initial benchmarks for long-term ex vivo studies of tissue-engineered skeletal muscle.