Primary culture of mouse myoblasts.

The literature has revealed variations in the protocols for myoblast cultures, and little information is available on myoblast and fibroblast proliferation. Therefore, the purposes of this study were to establish a prudent protocol for myoblast cultures by comparing a variety of culturing procedures used in previous research and to quantitate myoblast proliferation and fusion under different culture conditions. In addition, the growth status of myoblasts and fibroblasts was investigated. Results indicate that the requirements for an ideal myoblast culture should include a combined enzyme of 0.25% trypsin and 0.2% collagenase type IV (1:1), a preplating time of approximately 15-20 minutes, and a seeding density at 1 x 10(5) cells/ml. Furthermore, the mouse sample should be those of newborns. A better proliferative capacity of myoblasts was noted in an incubator of 10% CO2, coupled with Dulbecco's MEM plus 15% fetal calf serum. The doubling times of myoblasts were shorter than those of fibroblasts, and myoblast number reached its highest at 4 and 5 days. The findings of this study are valuable in understanding the growth status of myoblasts and fibroblasts in primary cultures. Moreover, the establishment of requirements for a good growth of myoblast cultures will facilitate myoblast transfer therapy.