Scale-up of a myoblast culture process

The effects of different types of cell carriers, strategies for cell transfer on carriers, and of several fusion inhibitors on the growth kinetics of primary human myoblasts culture were studied in order to develop a bioprocess suitable for the treatment of Duchenne muscular dystrophy based on the transplantation of unfused cells. Our results indicate that myoblast production is larger on Cytodex 1 and 3 than on polypropylene or polyester fabrics and on a commercial porous macrocarrier. Myoblast growth conditions with Cytodex 1 were further investigated to establish the bioprocess operating conditions. It was found that microcarrier density of 3 g DW l(-1), inoculum density of 2x10(5) cells ml(-1), and continuous agitation speed of 30-rpm result in final myoblast production comparable to static cultures. However, for all the culture conditions used, myoblasts growth kinetics exhibited a lag phase that lasted a minimum of 1 week prior to growth, the end of the lag phase correlating with the appearance of microcarrier aggregates. Based on this observation, we propose that aggregation promotes cell growth by offering a network of very large inter-particular pores that protect cells from mechanical stress. We took advantage of the presence of these aggregates for the scale-up of the culture process. Indeed, using myoblast-loaded microcarrier-aggregates instead of myoblast suspension to inoculate a fresh suspension of microcarriers significantly reduced the duration of the lag phase and allowed the scale-up of the bioprocess at the 500-ml scale. In order to ensure the production of unfused myoblasts, the efficiency of five different fusion inhibitors was investigated. Only calpeptin (9.1 microg ml(-1)) significantly inhibited the fusion of the myoblasts, while TGFbeta (50 ng ml(-1)) and LPA (10 microg ml(-1)) increased myoblasts growth but did not affect fusion, sphingosine (30 microg ml(-1)) induced a 50% death and NMMA (25 microg ml(-1)) had no effect on either growth or fusion. Finally, transplantation trials on severe combined immunodeficient mice showed that microcarrier-cultured human myoblasts grown using the optimized bioprocess resulted in grafts as successful as myoblasts grown in static cultures. The bioprocess, therefore, prove to be suitable for the large-scale production of myoblasts required for muscular dystrophy treatment.