Substrates composition and surface features of materials rule adhesion control of cells to surfaces. As a result, most of the aspects of cell functions, such as spreading, migration, proliferation, and differentiation, can be significantly influenced in biomedical applications. Cell cultures make possible to understand cell biology, tissue morphology, mechanisms of diseases, drug action, and tissue engineering development, among others. Recent techniques related to culturing 3D cell aggregates in the presence of very low wettable surfaces represent an innovative field for in vitro experimentation aimed at more reliable conditions to investigate both tumor and non-tumor cell lines. Matching in particular cell biology to innovative materials, this work reviews the recent literature available on promoting cell aggregates formation strongly influenced by the high surface hydrophobicity. In particular, for spheroid formation, the highest water repellent coatings seem to be required for the significant effectiveness of the process. In this way, 3D cell culture has become a reliable method for reproducing in vitro cellular growth in more realistic physiological conditions.