(3) limiting factor approach consisting of lowering the serum level in the medium till growth stops and then supplementing the medium with vitamins, amino acids, hormones, etc. till growth resumes.

These approaches have resulted in several elaborate media formulations in which serum in sought to be replaced by a mixture of amino acids, vitamins, several other organic compounds, etc.; hormones, growth factors and other proteins are supplemented when required. However, addition of 5-20% of serum even in these media is essential for optimum growth.