Protein Metabolisiu of Tissue Cells in Vitro. 7. The Chemical Nature of Some Obligate Factors of Tissue Cell Nutrition

Fibroblasts from embryonic chicken heart tissue have been cultivated on a simple artificial medium containing dialyzed plasma, dialyzed serum, Tyrode solution and fructose-1.6-diphos-phate Ca-salt, glutamine, cystine and glycine. One drop per culture of dialyzed embryonal extract was used as clotting agent. By replacement experiments it could be shown that the four low-molecular components were obligate necessary to the growth and maintenance of the cultures. The experiments have been carried out both in Carrel flasks and in hanging drop cultures. Hypoxanthine added to the above medium, increased the growth ratio. Hanging drop cultures on this medium have been transferred 5 times during a period of 10 days without any visible change of morphological status. With aid of paper partition chromatography it has been found that reducing sugars (glucose) organic phosphates, especially aminoethanol phosphoric ester, glutamine, cystine, glycine and hypoxanthine are present in dialysate from calf embryo muscle extract, the latter previously known to contain a complete set of low-molecular accessory growth factors for the cultivation of fibroblasts from embryonal chicken heart tissue in vitro. The five components used in the synthetic medium: Glucose + a suitable phosphate source, glutamine, cystine, glycine and hypoxanthine, which also exist in extracts from embryonal tissue could be regarded as the main nutritional low-molecular components for the growth and maintenance of fibroblasts from embryonic chicken heart tissue in vitro and in vivo.