Genetic Manipulation of Mammalian Cells in Microphysiological Hydrogels

Engineering functional 3D tissue constructs is essential for developing advanced organ-like systems, with applications ranging from fundamental biological research to drug testing. The generation of complex multicellular structures requires the integration of external geometric and mechanical cues with the ability to activate genetic programs that regulate and stimulate cellular self-organization. Here, we demonstrate that gelatin methacryloyl (GelMA) hydrogels serve as effective matrices for 3D cell culture, supporting both in situ genetic manipulation and cell growth. HEK293T cells embedded in GelMA remained viable and proliferated over 16 days, forming clusters within the matrix. We achieved efficient gene delivery in the 3D hydrogel environment using both plasmid DNA and mRNA as gene vectors. Furthermore, we applied in situ prime editing to induce permanent genetic modifications in embedded cells. To achieve spatially confined gene expression, we introduced gel-embedded channels that allowed localized stimulation via doxycycline perfusion through a Tet-On system. Our findings establish GelMA hydrogel matrices as a versatile platform for generating spheroidal cell cultures while enabling precise genetic control and spatially resolved cellular manipulation through diffusible cues.