Hormonal requirements for myogenesis of striated muscle in vitro: insulin and somatotropin.

The development of muscle in vitro from embryonic presumptive myoblasts is known to have complex nutritional requirements.' One of the necessary ingredients for myogenesis in vitro, and indeed for the growth of many cell types, is serum. During the course of studies aimed at understanding the mechanisms involved in the fusion of myoblasts, it became of interest to delete serum from the medium and to determine whether myogenesis could be restored by the addition of known factors. In view of the fact that muscle is dependent upon insulin for its normal function,2 this hormone was introduced into the culture medium in the absence of serum. It was observed that myotube formation was restored, although not to the extent obtained if serum were present. When somatotropin and insulin were both added to replace serum, an even greater degree of myotube formation took place. This report is therefore concerned with the effects of insulin and somatotropin on myogenesis. Materials and Methods-Materials: Crystalline bovine insulin and bovine somatotropin (1 IU/mg) were purchased from the Sigma Chemical Company. Highly purified collagenase, free of noncollagen proteolytic activity, was purchased from the Worthington Biochemical Company. Four-times-recrystallized bovine serum albumin was purchased from the Nutritional Biochemicals Corporation. Stock solutions were prepared by dissolving insulin at a concentration of 0.5 mg/ml in 0.001 N HCl, 0.1% NaCl, and somatotropin at the same concentration in 0.02 M potassium phosphate buffer, pH 8.0. Each hormone was added to the culture medium at a final concentration of 5 ;&g/ml. Serum albumin was dissolved in a modified Simm's balanced salt solution (BSS) at a concentration of 7% and the pH adjusted to 7.0; collagenase was dissolved in Ca++-Mg++free BSS at a concentration of 0.1%. All these protein solutions were sterilized by passage through type GS Millipore filters. The standard culture medium used consisted of eight parts Eagle's minimum essential medium (MEM), one part horse serum, one part 11-day chick embryo extract, 50 units/ml each of penicillin and streptomycin, and glutamine at a final concentration of 0.002 M. When horse serum and/or chick embryo extract were eliminated, they were replaced by one part of 7% serum albumin. In vitro development of muscle: Breast muscle from 11-day-old chick embryos was dissected free of connective tissue, teased into small pieces, and incubated in collagenase solution at 370C for 90 min. (This procedure was adopted in order to avoid exposure of the cells to serum which the trypsin method of releasing cells necessitates. The efficiency of this procedure is about 50% that obtained with trypsin.) Tissue removed from the collagenase solution was rinsed once with BSS and resuspended in 2 ml of MEM. The tissue was dispersed by repeated aspirations with a Pasteur pipette, and the cells were filtered through lens paper with the aid of a Swinney hypodermic adaptor. They were then centrifuged at 400 X g for 15 min and resuspended in a known volume of MEM; a cell count was made and the density adjusted to 4 X 107 cells per ml. To each milliliter of the medium to be tested, 0.025 ml (10 cells) of this suspension was added. One ml of each diluted suspension was added to individual Leighton tubes containing either two 10.5 X 22-mm cover slips or one 10.5 X 50-mm cover slip. The tubes were sealed with rubber stoppers and incubated at 370C. After 24 hr and every day thereafter, the medium was removed and replaced by an equal volume of fresh medium. Histological preparations: At 24-hr intervals the small cover slips were removed from the Leighton tubes, rinsed in cold BSS, and fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, for 90 min and stored in 70% ethanol in the refrigerator overnight. The tissues