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

Podsumowanie

Presented here is a protocol to radiolabel cells with a positron emission tomography (PET) radioisotope, ⁸⁹Zr (t_1/2 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.

Streszczenie

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 (t_1/2 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.

Wprowadzenie

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 failure^1,2, retinal degeneration², macular degeneration², diabetes², myocardial infarction^3,4,5, and cancers^6,7,8,9,

Protokół

Dendritic cells and melanoma cells were obtained commercially¹⁸. Hepatocytes were isolated from the liver of pigs following laparoscopic partial hepatectomy^22,24. Stem cells were isolated from bone marrow aspirates^18,19,26. The adipose tissue-derived stem cells were obtained from the Human Cellular Therapy Laboratory, Mayo Clinic Rochester²³. Human white blood cells were isolated from the collected blood received from the Division of Transfusion Medicine, Mayo Clinic Roc....

Wyniki

The representative results presented in this manuscript were compiled from the previous [⁸⁹Zr]Zr-DBN synthesis and cell radiolabeling studies^{18,19,22,23,24,25}. In brief, ⁸⁹Zr can be successfully complexed with DFO-Bn-NCS in ~30-60 min at 25-37 °C using 7.5-15 µg of DFO-Bn-NCS (Table 2

Dyskusje

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.......

Materiały

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