CULTURE CONDITIONS FOR THE PRODUCTION OF PORCINE MYOTUBES AND MYOBALLS

The existence of a method for culturing porcine muscle cells would greatly facilitate the development of new breeding criteria for stress resistance and growth regulation in pig breeding. Also, many effects of nutritional or pharmacological components that influence animal performance could be studied first in muscle cell preparations. Therefore, we developed a specific procedure to culture porcine skeletal muscle cells from well-established methods for murine and human muscle cell culturing. Best results were obtained by isolating satellite cells from muscle tissue removed postmortem after normal slaughter procedure, using enzymatic dissociation. The satellite cells were allowed to proliferate for 3 to 5 d in a culture medium composed of 83% Ham's F-12 medium,.15% fetal calf serum (FCS), and 2% chick embryo extract (CEE). Well before reaching confluence, the cells were transferred to collagen-coated dishes filled with Dulbecco's modified Eagle's medium containing 5% horse serum (HS) for the differentiation to multinucleated myotubes. Also, .5% FCS can be used instead of HS. Besides the fusion to myotubes, the presence of voltage-sensitive Na+ channels is regarded as a specific feature of the muscle phenotype of the cells. To perform electrophysiological experiments of good quality, myotubes were converted into freely floating ''myoballs.'' Voltage-clamp experiments in the whole-cell mode showed transient inward currents that had kinetics and voltage dependences very similar to those of the Na+ currents in human myoballs. The porcine Na+ currents were almost completely blocked by 1 mumol/L of tetrodotoxin, indicative of the presence of the adult form of the Na+ channel. We conclude from this electrophysiological evidence that the cells cultured according to our method are really of muscular phenotype.