The development and regulatory approval of continuous cell lines for manufacturing viral vaccines has brought numerous benefits to production processes. We and others have contributed to upstream advances by improving cell culture media with the development of animal-free and chemically-defined recombinant protein supplements. The supplements developed include recombinant insulin-like growth factor-I (LONG®R3IGF-I), epidermal growth factor (LONG®EGF), transforming growth factor-α (LONG®TGF-α), transferrin (CellPrime™ rTransferrin AF), and albumin (CellPrime ™rAlbumin AF-G or -S). Extensive literature on the action of these bioactive proteins on the cell types relevant to viral vaccine production, including Vero, MDCK, PerC6 ®, and HEK293 cells, supports their use to media design for these cell lines. In this report we present our initial results evaluating the effect of protein supplements on cell growth in several of these cell types. Recombinant supplements were added either alone or in various combinations and growth measured. Results indicated that individual supplements enhanced the growth of some cell types and various combinations of the supplements stimulated growth to a greater extent. The conclusion is that these animal-free recombinant bioactive protein supplements have the potential to improve growth performance of cell culture media in the absence of serum for cell types important in viral vaccine production.