Automating the expansion process of human skeletal muscle myoblasts with suppression of myotube formation.

An intelligent culture system accompanied by automated operations (liquid transfer and cell passage) was newly developed to perform serial cultures of human skeletal muscle myoblasts. To realize a desired performance, a laminin-coated surface was applied to myoblast expansion in a culture flask. It was found that the laminin coating enhanced the overall growth ability attributable not to shortening of the doubling time but to prevention of differentiation toward myotube formation, compared with that on a conventional plain surface. In addition, the effects of seeding density and confluence degree on the growth were investigated quantitatively in terms of cell attachment and division as well as proliferative cell population in the culture on the laminin-coated surface. With increasing in seeding density, the number of proliferative cells decreased at the end of culture accompanied by an increase in the confluence degree, which caused poor attachment of the passaged cells on the surface in the subsequent culture. The quantitative analyses of these cell behaviors helped us determine the appropriate seeding density and attainable confluence degree during one passage, which were 1.0 x 10(3) cells/cm(2) and 0.5 as the initial and boundary conditions, respectively. An automated culture system that could manage two serial cultures by monitoring the confluence degree was constructed. The automated operation with the intelligent determination of the time for passage was successfully performed without serious loss of growth activity, compared with manual operation using conventional flasks. These results indicated that the monitoring of confluence degree is effective to perform the culture passage of myoblasts, being contributable to automating the cell expansion process.