We report on recent advances in the fabrication of three-dimensional (3D) scaffolds for tissue engineering and regenerative medicine constructs using a two-photon polymerization technique (2PP). 2PP is a novel CAD/CAM technology allowing the fabrication of any computer-designed 3D structure from a photosensitive polymeric material. The flexibility of this technology and the ability to precisely define 3D construct geometry allows issues associated with vascularization and patient-specific tissue fabrication to be directly addressed. The fabrication of reproducible scaffold structures by 2PP is important for systematic studies of cellular processes and better understanding of in vitro tissue formation. In this study, 2PP was applied for the generation of 3D scaffold-like structures, using the photosensitive organic-inorganic hybrid polymer ORMOCER (ORganically MOdified CERamics) and epoxy-based SU8 materials. By comparing the proliferation rates of cells grown on flat material surfaces and under control conditions, it was demonstrated that ORMOCER and SU8 are not cytotoxic. Additional tests show that the DNA strand breaking of GFSHR-17 granulosa cells was not affected by the presence of ORMOCER. Furthermore, gap junction conductance measurements revealed that ORMOCER did not alter the formation of cell-cell junctions, critical for functional tissue growth. The possibilities of seeding 3D structures with cells were analysed. These studies demonstrate the great potential of 2PP technique for the manufacturing of scaffolds with controlled topology and properties.