This protocol describes a stepwise method for radiolabeling of cells and zirconium-89-DBN and its non-invasive tracking by PET. This technique also provides a reliable and a stable method for radiolabeling cells by a covalent conjugation of zirconium-89-DBN to the primary means of the cell surface proteins. Physicians and scientists working with cell therapies for neurological diseases and various cancers will find this protocol instrumental in understanding the pharmacokinetics of cell-based therapies via non-invasive PET imaging.
The detailed protocol provides guidance to new users to learn and adopt this radiolabeling technique. Additional nodes included at the critical steps will help troubleshooting problems for a new user. To begin, prepare a column with approximately 100 milligrams of hydroxamate resin, and activate it by washing the column with eight milliliters of pure anhydrous acetonitrile followed by a flush with five to six milliliters of air.
Pass two milliliters of 0.5 normal hydrochloric acid to the column. After passing an additional flush of five to six milliliters of air, load the solution containing both zirconium-89 and natural yttrium to the hydroxamate resin slowly. Wash the unbound natural yttrium from the hydroxamate resin with 20 milliliters of two normal hydrochloric acid, followed by 10 milliliters of deionized water.
For eluding zirconium in the form of zirconium-89 hydrogen phosphate, add 0.5 milliliters of 1.2 molar dipotassium phosphate potassium dihydrogen phosphate buffer to the column and allow it to sit on the column for 30 minutes. Add an additional 1.5 milliliters of 1.2 molar buffer to elute the zirconium from the column. Next, take around 120 microliters of zirconium-89 hydrogen phosphate formulated in the buffer and neutralize the solution with HEPES-KOH and potassium carbonate to achieve a pH of 7.528.
Add four microliters of freshly prepared five millimolar DFO-Bn-NCS to around 285 microliters of neutralized zirconium-89 hydrogen phosphate. And mix the solution by pipetting. After isolating zirconium from yttrium, add 0.5 milliliters of one molar oxalic acid to the column and allow it to sit on the column for one minute to elute zirconium in the form of zirconium-89 oxalate.
Add an additional 2.5 milliliters of one molar oxalic acid to the column. To convert zirconium-89 oxalate into zirconium-89 chloride, wash the anion exchange column with six milliliters of acetonitrile, followed by a flush with five to six milliliters of air. Then, wash the column with 10 milliliters of saline followed by another flush of air.
Slowly, load the solution containing zirconium-89 oxalate onto the activated anion exchange column before rewashing it with air. Then, wash the column with 50 milliliters of deionized water to remove the unbound oxalate ion. For eluding zirconium in the form of zirconium-89 chloride, add 0.1 milliliters of one normal hydrochloric acid to the column and allow it to sit on the column for one minute.
Elute the zirconium from the column with an additional 0.4 milliliters of one normal hydrochloric acid. Dry the eluded zirconium-89 chloride in a V-shaped vial by placing it in a heating block at 65 degrees Celsius under a steady flow of nitrogen gas for 10 to 30 minutes, and then reconstitute the dried zirconium-89 chloride in water. Prepare 500 microliters of cell suspension with approximately 6 million cells in 500 microliters of HEPES-buffered HBSS in a 1.5-milliliter micro centrifuge tube and add approximately 100 microliters of the formulated zirconium-89-DBN.
Mix the zirconium-89-DBN and the cell suspension by gently pipetting the solution up and down with a micro pipette. Incubate the cells and zirconium-89-DBN mixture on a shaker at around 550 RPM at 25 to 37 degrees Celsius for 30 to 45 minutes for cell labeling. Perform radioactive TLC on the cell radiolabeling reaction using freshly prepared radioactive TLC solvent.
Calculate the percentage of radioactivity at Rf equals 0.01 to 0.02 using the equation shown on the screen. In a separate 1.5 milliliter micro centrifuge tube, mix around 100 microliters of the formulated zirconium-89-DBN with around 500 microliters of HEPES-buffered HBSS to use it as a no-cell control for background correction. Calculate the percentage of radioactivity at Rf equals 0.01 to 0.02 using the equation shown on the screen, then calculate the effective cell radiolabeling by subtracting the two calculated radioactivity percentages.
After confirming the completion of radiolabeling, add 600 microliters of chilled cell-appropriate complete medium to perform the quenching of the radiolabeling reaction. Centrifuge the cells at 96G for 10 minutes at four degrees Celsius. And discard the supernatant.
Gently resuspend the pelleted cells in 500 microliters of the chilled medium by pipetting the medium up and down with a micro pipette. Centrifuge the cells at 96G for 10 minutes at four degrees Celsius and discard the supernatant. Next, calculate the final radiolabeling efficiency after all the washes using the equation shown on the screen.
To ensure the quality of the radiolabeled cells, visually inspect the final suspension of radiolabeled cells, and if no clumps are present, perform a trypan blue exclusion viability test using 0.4%trypan blue solution prepared in PBS within one hour of radiolabeling and the washing steps. The chelation efficiency of DFO-Bn-NCS for zirconium-89 using zirconium-89 hydrogen phosphate and zirconium-89 chloride are different as measured by radioactive TLC. The stability of various cells radiolabeled with zirconium-89 over seven days is shown here.
The retention of zirconium-89 radioactivity by radiolabeled cells was found to be stable in all the cells studied, with a negligible efflux observed over seven days post-labeling. The cell radiolabeling efficiencies in different cell types with zirconium-89-DBN are shown in this table. The cell radiolabeling efficiency varies from 20 to 50%as an uncorrected yield observed in the radiolabeled cell pellet after washing.
The most critical step of this protocol are the addition of DFO-Bn-NCS to the chelation mixture and the quality assessment of radiolabeled cells. The application of this technique will help to understand the pharmacokinetics of cell-based therapies. It would also raise questions about any clinical need to alter the clearance profile of cell therapies.
