Cell-assembled tissue engineering strategies hold great potential in regenerative medicine, as three-dimensional tissue-like modules can be produced, even from a patient's own cells. However, the development of such implantable devices requires prolonged culture time, which is associated with cell phenotypic drift. Considering that the cells are subjected to numerous stimuli, multifactorial approaches are continuously gaining pace towards controlling cell fate during expansion. Herein, we assessed the synergistic effect of simultaneous and serial growth factor supplementation (insulin growth factor-1, platelet-derived growth factor growth differentiation factor 5 and transforming growth factor 3) to macromolecular crowding (carrageenan) in human tenocyte function; collagen synthesis and deposition; and gene expression. TGF3 supplementation (without/with carrageenan) induced the highest (among all groups) DNA content. In all cases, tenocyte proliferation was significantly increased as a function of time in culture, whilst metabolic activity was not affected. Carrageenan supplementation induced significantly higher collagen deposition than groups without carrageenan (without/with any growth factor). Of all the growth factors used, TGF3 induced the highest collagen deposition when used together with carrageenan in both simultaneous and serial fashion. At day 13, gene expression analysis revealed that TGF3 in serial supplementation to carrageenan upregulated the most and downregulated the least collagen- and tendon- related genes and upregulated the least and downregulated the most osteo-, chondro-, fibrosis- and adipose- related trans-differentiation genes. Collectively, these data clearly advocate the beneficial effects of multifactorial approaches (in this case, growth factor and macromolecular crowding supplementation) in the development of functional cell-assembled tissue surrogates.