Near-Infrared Photoimmunotherapy for Targeted Cell Elimination in Mixed 3D Cultures

Take a culture of hanging drop mixed spheroids, which are heterogeneous cell aggregates consisting of target cells expressing growth factor receptors and a fluorescent protein, while non-target cells express a different-colored fluorescent protein.

Replace media with an antibody-photoabsorber conjugate comprising a target cell-specific monoclonal antibody and a photoabsorber dye.

Incubate to facilitate antibody binding to target cell receptors.

Wash the spheroids with media to remove unbound conjugates, then transfer them to a glass-bottom dish with media for light irradiation.

Expose the spheroids to near-infrared light for photoimmunotherapy, triggering a photochemical reaction of the photoabsorber dye on target cells, causing rapid cellular membrane damage and cell death.

This process eliminates target cells without damaging non-target cells.

Transfer the spheroids to a fresh hanging drop plate. Add media and incubate for spheroid growth.

Under a fluorescence microscope, the absence of target cell fluorescence indicates effective cell elimination, while the presence of non-target cell fluorescence confirms the selective nature of photoimmunotherapy.