The interaction between different cardiac cells has shown to be important for critical biological properties including cell survival, proliferation, differentiation and function. The improvement of culture conditions with different cell types and to study their effects on cardiomyocyte viability and functionality is essential. For practical applications including general toxicity testing, drug development and tissue engineering it is important to study whether co-cultures have additional advantages over cardiomyocyte monoculture. Two multicellular in vitro cardiovascular constructs devoid of added biomaterial were developed in this study. In the first construct, neonatal rat cardiomyocytes (CM) were seeded on vascular-like network formed by human umbilical vein endothelial cells (HUVEC) and human adipose stromal cells (hASC). In the second construct, CMs were seeded on vascular-like network formed by HUVECs and human foreskin fibroblasts. The ability of these two vascular-like networks to support the viability and functionality of CMs was analyzed. Different culture media compositions were evaluated to support the development of optimal cardiovascular construct. Our results demonstrate that both vascular-like networks markedly improved CM viability and functionality. In the constructs, co-localization of CMs and vascular-like networks was seen. Multicellular constructs also allowed synchronized contractility of CMs. Serum-free medium supplemented with vascular endothelial growth factor and basic fibroblast growth factor was found to provide the most optimal conditions for cardiovascular construct as an entity. In conclusion, when combining a vascular-like network with CMs, the viability and functionality of CMs was markedly improved. The results suggest that the cardiovascular constructs developed provide a promising new tool for the assessment of toxicological and safety pharmacological effects of compounds in vitro.