Genetic Manipulation of Mammalian Cells in Microphysiological Hydrogels

Area of Science:

• Biomaterials Engineering
• Tissue Engineering
• Synthetic Biology

Background:

• Engineering functional 3D tissue constructs is crucial for organ-like systems in research and drug testing.
• Complex multicellular structures require geometric, mechanical cues, and genetic programs for self-organization.

Purpose of the Study:

• To demonstrate GelMA hydrogels as effective matrices for 3D cell culture with genetic manipulation capabilities.
• To establish a versatile framework for engineered 3D cell systems with programmable genetic activity.

Main Methods:

• Utilized gelatin methacryloyl (GelMA) hydrogels for 3D cell culture of HEK293T cells.
• Implemented in situ gene delivery using plasmid DNA and mRNA.
• Applied in situ prime editing for permanent genetic modifications.
• Introduced gel-embedded channels for spatially confined, inducible gene expression via doxycycline and a Tet-On system.

Main Results:

• HEK293T cells exhibited viability and proliferation in GelMA hydrogels for over 16 days, forming cell clusters.
• Achieved efficient gene delivery and in situ prime editing within the 3D hydrogel environment.
• Demonstrated spatially controlled gene expression using localized doxycycline perfusion.

Conclusions:

• GelMA hydrogels effectively support 3D cell culture, genetic manipulation, and cell growth.
• The developed system integrates gene delivery, inducible expression, and spatial control for programmable genetic activity in engineered tissues.