This article describes a method for selective elimination of target cells in mixed spheroid cultures using photoimmunotherapy. The technique utilizes a monoclonal antibody conjugated with a photoabsorber dye, allowing for targeted cell death upon exposure to near-infrared light.
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.
To prepare the mixed 3D cell culture, add 1 milliliter of sterile water into the plate reservoir section of a 96-well hanging drop plate. Then, plate various ratios of the cell types of interest in the appropriate cell culture medium at, at least, 5000 cells per 50 microliters of medium per well for 5 to 7 days in a humidified incubator at 37 degrees Celsius and 5% carbon dioxide.
Change the culture medium every two days, and observe the morphology and size of the spheroids daily with an inverted Brightfield microscope at 10 to 40 times magnification. When the spheroids have reached 400 to 600 microns in diameter, replace the hanging drop plate medium with antibody-photoabsorber conjugate-containing medium, and return the plate to the incubator for six hours.
At the end of the incubation, wash the spheroids two times with fresh phenol red-free culture medium. Then, use a 200-microliter pipette tip with a cut-off end to transfer one spheroid at a time into individual glass bottom 50-millimeter dishes containing 100 microliters of fresh phenol red-free culture medium per dish. Use the inverted microscope to observe changes in the spheroid morphology.
To irradiate the spheroids, place a light-emitting diode no higher than 5 inches above the glass bottom dishes, and use an optical power meter to confirm the emission of a 670 to 710-nanometer wavelength at two joules per centimeter squared. After the near-infrared photoimmunotherapy, transfer the spheroids into a new hanging drop plate with 50 microliters of fresh culture medium per well for one day in the cell culture incubator.
Then, using another 200 microliter pipette tip with a cut-off end, gently transfer the spheroids into new glass bottom dishes containing 100 microliters of phenol red-free culture medium and assess the optical reporter expression under the fluorescent microscope. Finally, detect the dead cells by the addition of propidium iodide to the medium or by the loss of cytoplasmic GFP fluorescence.
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