The overall goal of this experimental procedure is to demonstrate the elimination of specific cells of interest from a mixed 3D cell culture using near infrared photoimmunotherapy. This method can help answer key questions in the tissue-engineering field about regenerating medicine. The main advantage of this technique that it can be used against a variety of target molecules and that the treatment can be restricted to specific lesions of interest.
Thus can minimize the side effect. For IR700 conjugation to monoclonal antibodies, incubate the antibodies of interest with IR700 in Disodium Phosphate in a microcentrefuge tube at room temperature for one hour covered in aluminum foil. At the end of the incubation, wash a PD10 column two times with 15 milliliters of PBS.
Then, load the antibody solution onto the column and purify the mixture by PBS elution according to the manufacturer's instructions. To confirm the number of fluoro-4 molecules conjugated to each monoclonal antibody molecule, use a spectrophotometer to determine the monoclonal antibody protein concentration. Then, store the conjugate at 4-degree Celsius.
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 5, 000 cells per 50 microliters of medium per well for five to seven days in a humidified incubator at 37-degree 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 bright-field 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 2 joules per centimeter squared.
After the NIR 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 flourescent microscope. Finally, detect the dead cells by the addition of propidium iodide to the medium.
Or by the loss of cytoplasmic GFP flourescence. In this representative experiment, mixed spheroids composed of various ratios of A431-luc-GFP and 3T3-RFP cells were prepared as just demonstrated. Repeated NIR infrared photoimmunotherapy was then performed with the incubation of panitumumab-IR700 as illustrated.
And the target cell elimination was monitored through the assessment of the flourescent and luciferase activities within the hanging drop cultures. Once mastered, this technique can be completed in one hour if it is performed properly. While attempting this procedure, it's important to remember that light energy decreases according to inverse-square law.
