This work was supported by NIH 5R21HL127389-02, NIH 4T32HL007111-39, NIH R01HL134664, and DOE DE-SC0008947 grants, International Atomic Energy Agency, Vienna, Mayo Clinic Division of Nuclear Medicine, Department of Radiology, and Mayo Clinic Center for Regenerative Medicine, Rochester, MN. All figures were created using BioRender.com.

Dendritic cells and melanoma cells were obtained commercially18. Hepatocytes were isolated from the liver of pigs following laparoscopic partial hepatectomy22,24. Stem cells were isolated from bone marrow aspirates18,19,26. The adipose tissue-derived stem cells were obtained from the Human Cellular Therapy Laboratory, Mayo Clinic Rochester<sup cla...

<ol>
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Authors have no financial competing interest but are the inventors of this technology (Patent # US20210330823A1).

Following are critical steps in the protocol that need optimization for effective cell radiolabeling. In protocol steps 1.2 and 1.3, depending on the volume of [89Zr]Zr(HPO4)2 or [89Zr]ZrCl4 employed, an appropriate volume (microliters) of base must be used; 1.0 M K2CO3 solution must be used for the neutralization of [89Zr]Zr(HPO4)2 and 1.0 M Na2CO3 solution for the neutralization of [8...

Stem cell and chimeric antigen receptor (CAR) T-cell therapies are gaining popularity and are under active investigation for the treatment of various diseases, such as myocardial failure1,2, retinal degeneration2, macular degeneration2, diabetes2, myocardial infarction3,4,5, and cancers6,7,8,9,10. Among the two plausible approaches of stem cell therapies, stem cells can either be directly engrafted on the disease site to cause a therapeutic response, or cause changes in the microenvironment of the disease site without adhering to the disease site to initiate an indirect therapeutic response. An indirect therapeutic response could cause changes in the microenvironment of the disease site by releasing factors that would repair or treat the disease5. These approaches of stem cell therapies could be evaluated by noninvasive imaging of radiolabeled stem cells. Noninvasive imaging could correlate the uptake of the radiolabeled cells on the disease site with a therapeutic response to decipher the direct versus indirect therapeutic response.
Additionally, immune cell-based therapies are being developed to treat various cancers using CAR T-cell6,7,8,9,10and dendritic cell immunotherapy11,12. Mechanistically, in CAR T-cell immunotherapy6,7,8,9,10, T-cells are engineered to express an epitope that binds to a specific antigen on tumors that needs to be treated. These engineered CAR T-cells, upon administration, bind to the specific antigen present on the tumor cells through an epitope-antigen interaction. After binding, the bound CAR T-cells undergo activation and then proliferate and release cytokines, which signals the immune system of the host to attack the tumor expressing the specific antigen. In contrast, in the case of dendritic cell therapies11,12, dendritic cells are engineered to present a specific cancer antigen on their surface. These engineered dendritic cells, when administered, home to the lymph nodes and bind to the T-cells in the lymph nodes. The T-cells, upon binding to the specific cancer antigens on the administered dendritic cells, undergo activation/proliferation and initiate an immune response of the host against the tumor expressing that specific antigen. Hence, the assessment of trafficking of administered CAR T-cells to a tumor site9,10 and homing of dendritic cells to the lymph nodes11,12 is possible by imaging radiolabeled CAR T-cells and dendritic cells to determine the efficacy of immunotherapy. Furthermore, noninvasive cell trafficking can help to better understand the therapeutic potential, clarify the direct versus indirect therapeutic response, and predict and monitor the therapeutic response of both stem cell and immune cell-based therapies.
Different imaging modalities for cell trafficking have been explored3,4,9,10,12, including optical imaging, magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), and positron emission tomography (PET). Each of these techniques has its own advantages and disadvantages. Among these, PET is the most promising modality due to its quantitative nature and high sensitivity, which are essential for the reliable quantification of cells in imaging-based cell trafficking3,4,9,10.
The positron-emitting radioisotope 89Zr, with a half-life of 78.4 h, is suitable for cell labeling. It allows PET imaging of cell trafficking for over 1 week and is readily produced by widely available, low-energy medical cyclotrons13,14,15,16,17. Additionally, an appropriately functionalized, p-isothiocyanatobenzyl-desferrioxamine (DFO-Bn-NCS) chelator is commercially available for the synthesis of a 89Zr-labeled, ready-to-use, cell labeling synthon, [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine, also known as [89Zr]Zr-DBN18,19,20,21,22,23,24,25.&#160;The principle of [89Zr]Zr-DBN-mediated cell labeling is based on a reaction between primary amines of cell membrane proteins and the isothiocyanate (NCS) moiety of [89Zr]Zr-DBN to produce a stable covalent thiourea bond.
[89Zr]Zr-DBN-based cell labeling and imaging have been published to track a variety of different cells, including stem cells18,23,25, dendritic cells18, cardiopoietic stem cells19, decidual stromal cells20,&#160;bone marrow-derived macrophages20, peripheral blood mononuclear cells20, Jurkat/CAR T-cells21, hepatocytes22,24, and white blood cells25. The following protocol provides step-by-step methods of preparation and cell radiolabeling with [89Zr]Zr-DBN and describes changes that may be required in the radiolabeling protocol for a specific cell type. For greater clarity, the method of cell radiolabeling presented here is divided into four sections. The first section deals with the preparation of [89Zr]Zr-DBN by chelating 89Zr&#160;with DFO-Bn-NCS. The second section describes the preparation of a biocompatible formulation of [89Zr]Zr-DBN that can be readily used for cell radiolabeling. The third section covers the steps needed for the preconditioning of cells for radiolabeling. The preconditioning of cells involves washing the cells with protein-free phosphate buffered saline (PBS) and HEPES buffered Hanks balanced salt solution (H-HBSS) to remove external proteins, which might interfere or compete with the reaction of [89Zr]Zr-DBN with primary amines present on the cell surface proteins during radiolabeling. The final section provides steps involved in the actual radiolabeling of the cells and quality control analysis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetonitrile</td>
			<td>Thermo Fisher Scientific, Inc., Waltham, MA, USA</td>
			<td>A996-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha Minimum Essential Medium</td>
			<td>Thermo Fisher Scientific, Inc., Waltham, MA, USA</td>
			<td>12571063</td>
			<td></td>
		</tr>
		<tr>
			<td>Anion exchange column</td>
			<td>Macherey-Nagel, Inc., D&#252;ren, Germany</td>
			<td>731876</td>
			<td>Chromafix 30-PS-HCO3&#160;SPE 45 mg cartridge</td>
		</tr>
		<tr>
			<td>Conical centrifuge tubes (15 mL)</td>
			<td>Corning Inc., Glendale, AZ, USA</td>
			<td>352096</td>
			<td>Falcon 15 mL high-clarity polypropylene (PP) conical centrifuge tubes</td>
		</tr>
		<tr>
			<td>Dendritic cells</td>
			<td>The American Type Culture Collection, Manassas, VA, USA</td>
			<td>CRL-11904</td>
			<td></td>
		</tr>
		<tr>
			<td>DFO-Bn-NCS</td>
			<td>Macrocyclics, Inc., Plano, TX, USA</td>
			<td>B-705</td>
			<td>p-SCN-Bn-Deferoxamine</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich, Inc., St. Louis, MO</td>
			<td>276855</td>
			<td></td>
		</tr>
		<tr>
			<td>Dose calibrator</td>
			<td>Mirion Technologies (Capintec), Inc., Florham Park, NJ, USA</td>
			<td>5130-3234</td>
			<td>CRC -55tR Dose Calibrator&#160;</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium&#160;</td>
			<td>The American Type Culture Collection, Manassas, VA, USA</td>
			<td>30-2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>The American Type Culture Collection, Manassas, VA, USA</td>
			<td>30-2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt solution (HBSS)</td>
			<td>Thermo Fisher Scientific, Inc., Waltham, MA, USA</td>
			<td>14025092</td>
			<td>For preparation of H-HBSS</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid (trace metal basis grade)</td>
			<td>Thermo Fisher Scientific, Inc., Waltham, MA, USA</td>
			<td>A508P212</td>
			<td></td>
		</tr>
		<tr>
			<td>Melanoma cells</td>
			<td>The American Type Culture Collection, Manassas, VA, USA</td>
			<td>CRL-6475</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich, Inc., St. Louis, MO</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>30108442</td>
			<td>Protein LoBind microcentrifuge tube</td>
		</tr>
		<tr>
			<td>Murine GM-CSF</td>
			<td>R&#38;D Systems, Inc., Minneapolis, MN USA</td>
			<td>415-ML-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fisher Scientific, Inc., Waltham, MA, USA</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline without Ca2+ and Mg2+</td>
			<td>Thermo Fisher Scientific, Inc., Waltham, MA, USA</td>
			<td>10010023</td>
			<td>For washing cells</td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Covidien LLC, Mansfield, MA, USA</td>
			<td>1020</td>
			<td>0.9% Sterile Saline Solution</td>
		</tr>
		<tr>
			<td>Shaker&#160;</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>T1317</td>
			<td>Thermomixer</td>
		</tr>
		<tr>
			<td>Silica gel-rad-TLC paper sheet&#160;</td>
			<td>Agilent Technologies Inc., Santa Clara, CA, USA</td>
			<td>SGI0001</td>
			<td>iTLC-SG</td>
		</tr>
	</tbody>
</table>

positron emission tomography imaging of cell trafficking: a method of cell radiolabeling

Stem cell and chimeric antigen receptor (CAR) T-cell therapies are emerging as promising therapeutics for organ regeneration and as immunotherapy for various cancers. Despite significant progress having been made in these areas, there is still more to be learned to better understand the pharmacokinetics and pharmacodynamics of the administered therapeutic cells in the living system. For noninvasive, in vivo tracking of cells with positron emission tomography (PET), a novel [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN)-mediated cell radiolabeling method has been developed utilizing 89Zr (t1/2 78.4 h). The present protocol describes a [89Zr]Zr-DBN-mediated, ready-to-use, radiolabeling synthon for direct radiolabeling of variety of cells, including mesenchymal stem cells, lineage-guided cardiopoietic stem cells, liver regenerating hepatocytes, white blood cells, melanoma cells, and dendritic cells. The developed methodology enables noninvasive PET imaging of cell trafficking for up to 7 days post-administration without affecting the nature or the function of the radiolabeled cells. Additionally, this protocol describes a stepwise method for the radiosynthesis of [89Zr]Zr-DBN, biocompatible formulation of [89Zr]Zr-DBN, preparation of cells for radiolabeling, and finally the radiolabeling of cells with [89Zr]Zr-DBN, including all the intricate details needed for the successful radiolabeling of cells.

The representative results presented in this manuscript were compiled from the previous [89Zr]Zr-DBN synthesis and cell radiolabeling studies18,19,22,23,24,25. In brief, 89Zr can be successfully complexed with DFO-Bn-NCS in ~30-60 min at 25-37 &#176;C using 7.5-15 &#181;g of DFO-Bn-NCS (Table 2</...

Watch this Scientific Journal Video about Positron Emission Tomography Imaging of Cell Trafficking: A Method of Cell Radiolabeling at JoVE.com

Positron Emission Tomography Imaging of Cell Trafficking: A Method of Cell Radiolabeling

Presented here is a protocol to radiolabel cells with a positron emission tomography (PET) radioisotope, 89Zr (t1/2 78.4 h), using a ready-to-use radiolabeling synthon, [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN). Radiolabeling cells with [89Zr]Zr-DBN allows noninvasive tracking and imaging of administered radiolabeled cells in the body with PET for up to 7 days post-administration.

Stem cell and chimeric antigen receptor (CAR) T-cell therapies are emerging as promising therapeutics for organ regeneration and as immunotherapy for ...

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.

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Research

JoVE Journal

Cancer Research

1.3K Views.  Mayo Clinic. Questo protocollo descrive un metodo graduale per la radiomarcatura di cellule e zirconio-89-DBN e il suo tracciamento non invasivo mediante PET. Questa tecnica fornisce anche un metodo affidabile e stabile per la radiomarcatura delle cellule mediante una coniugazione covalente di zirconio-89-DBN ai mezzi primari delle proteine della superficie cellulare. I medici e gli scienziati che lavorano con le terapie cellulari per le malattie neurologiche e vari tipi di cancro troveranno questo protoc...