Two-photon induced polymerization (2PP) uses femtosecond-pulsed lasers to enable true 3D printing with high spatial control and resolutions in the sub-micrometer range. This allows fabrication of parts with ultra-small features such as photonic crystals, optical waveguides, microelectronic components and scaffolds for tissue engineering. 2PP-fabricated scaffolds have been used to microengineer 3D cell cultures, with the ultimate aim of studying stimulus - cellular response relationships, or testing of pharmaceutical compounds. In comparison to the approach of seeding cells onto pre-fabricated scaffolds, direct cell encapsulation into hydrogels supports higher initial cell loading, uniformity of cell distribution and a more intimate cell-matrix contact. Commercial initiators used in UV-encapsulation have proven inadequate for 2PP cell encapsulation strategies due to their low two-photon absorption, and the success of recently developed water-soluble two-photon initiators (2PIs), such as P2CK, has so far been limited because of their relatively high cytoand phototoxicity. This aim of this thesis was to design novel highly effective 2PIs that enable a biocompatible 2PP cell encapsulation process. To hinder 2PI transmembrane migration and prevent the intracellular formation of free radicals and reactive oxygen species (ROS) upon laser excitation, a macromolecular hyaluronan-based initiator HAPI was developed. Laser scanning microscopy imaging of 2PI autofluorescence demonstrated that in contrast to small molecule references, HAPI does not accumulate inside cells. While the cytoand phototoxicity of HAPI were lower in comparison to P2CK, some cell damage was still observed after 2PP encapsulation. A second strategy aimed at reducing the formation of ROS, which result from side reactions competing with the slow bimolecular electron transfer mechanism that is assumed to govern radical generation of conventional 2PIs. Thus, photocleavable compounds - which form radicals in a mechanism similar to biocompatible commercial UV-initiators - were studied as 2PIs. The cleavable trichloromethyl-triazine based system 2BC acted as efficient 2PI, but was impractical to use due to a tendency to cause overpolymerization as well as excessive visible light sensitivity. Investigation of cleavable diazosulfonates resulted in the development of AS7, which exhibits excellent biocompatibility and is highly effective as 2PI at fast writing speeds up to 1 m/s. ASC/TERT1 stem cells 2PP encapsulated in methacrylated gelatin hydrogels with AS7 showed no signs of photodamage, and were spreading and proliferating in the gel during a 5 day observation period after structuring.