Current conventional cancer drug screening models based on two-dimensional (2D) cell culture have several flaws and there is a large need of more in vivo mimicking preclinical drug screening platforms. The microenvironment is crucial for the cells to adapt relevant in vivo characteristics and here we introduce a new cell culture system based on three-dimensional (3D) printed scaffolds using cellulose nanofibrils (CNF) pre-treated with 2,2,6,6-tetramethylpyperidine-1-oxyl (TEMPO) as the structural material component. Breast cancer cell lines, MCF7 and MDA-MB-231, were cultured in 3D TEMPO-CNF scaffolds and were shown by scanning electron microscopy (SEM) and histochemistry to grow in multiple layers as a heterogenous cell population with different morphologies, contrasting 2D cultured mono-layered cells with a morphologically homogenous cell population. Gene expression analysis demonstrated that 3D TEMPO-CNF scaffolds induced elevation of the stemness marker CD44 and the migration markers VIM and SNAI1 in MCF7 cells relative to 2D control. T47D cells confirmed the increased level of the stemness marker CD44 and migration marker VIM which was further supported by increased capacity of holoclone formation for 3D cultured cells. Therefore, TEMPO-CNF was shown to represent a promising material for 3D cell culture model systems for cancer cell applications such as drug screening.