The preclinical drug screening of pancreatic cancer treatments suffers from the absence of appropriate models capable to reproduce in vitro the heterogeneous tumor microenvironment and its stiff desmoplasia. Driven by this pressing need, we describe in this paper the conception and the characterization of a novel 3D tumor model consisting of a triple co-culture of pancreatic cancer cells (PANC-1), fibroblasts (MRC-5) and endothelial cells (HUVEC), which assembled to form a hetero-type multicellular tumor spheroid (MCTS). By histological analyses and Selective Plain Illumination Microscopy (SPIM) we have monitored the spatial distribution of each cell type and the evolution of the spheroid composition. Results revealed the presence of a core rich in fibroblasts and fibronectin in which endothelial cells were homogeneously distributed. The integration of the three cell types enabled to reproduce in vitro with fidelity the influence of the surrounding environment on the sensitivity of cancer cells to chemotherapy. To our knowledge, this is the first time that a scaffold-free pancreatic cancer spheroid model combining both tumor and multiple stromal components has been designed. It holds the possibility to become an advantageous tool for a pertinent assessment of the efficacy of various therapeutic strategies.