Methods for reducing the serum requirement for growth in vitro of nontransformed diploid fibroblasts

The amount of serum protein required for clonal growth of normal diploid human and chicken fibroblasts has been reduced more than 90% by development of improved synthetic media and culture conditions. The following has contributed significantly to the reduction: (1) replacement of specific serum proteins with defined small molecular nutrients; (2) precise quantitative balance of all defined nutrients for optimal growth of single cells of each type under consideration; (3) modification of the culture substrate; (4) improvement of the cell harvesting technique. By combining these improvements, optimal growth of human fibroblasts (fetal lung, newborn foreskin, amniotic fluid) and chicken embryo fibroblasts occurs in synthetic media supplemented with 500 microgram/ml dialyzed fetal bovine serum protein [equivalent to 1% (v/v) whole serum]. The human fibroblasts form large colonies at a suboptimum rate at 25 microgram/ml dialyzed serum protein [equivalent to 0.05% (v/v) whole serum] and several divisions can be obtained in the complete absence of serum protein. Human and chicken fibroblasts differ both in their quantitative requirements for defined nutrients, and in the type and number of incompletely characterized serum components that they continue to require under the improved growth conditions. The improved systems provide a sensitive assay for the remaining undefined growth-promoting components of serum and other biological extracts.