Sizing It Up: Cellular MRI Using Micron-Sized Iron Oxide Particles Erik M. Shapiro, * Stanko Skrtic, and Alan P. Koretsky There is rapidly increasing interest in the use of MRI to track cell migration in intact animals. Currently, cell labeling is usually accomplished by endocytosis of nanometer-sized, dextran- coated iron oxide particles. The limitations of using nanometer- sized particles, however, are that millions of particles are re- quired to achieve sufficient contrast, the label can be diluted beyond observability by cell division, and the label is biodegrad- able. These problems make it difficult to label cells other than macrophages in vivo, and to conduct long-term engraftment studies. It was recently demonstrated that micron-sized iron oxide particles (MPIOs) can be taken up by a number of cell types. In this study we examined the MRI properties of single MPIOs with sizes of 0.96, 1.63, 2.79, 4.50, and 5.80 m. Further- more, the capacity of cells to endocytose these MPIOs was investigated, and the MRI properties of the labeled cells at 7.0 and 11.7 Tesla were measured as a function of image resolution and echo time (TE). Cells labeled with MPIOs generally con- tained iron levels of 100 pg, which is approximately threefold higher than those obtained with the best strategies to label cells using nanometer-sized particles. On occasion, some cells had levels as high as 400 pg. We demonstrate that these large particles and the cells labeled with them can be detected by spin echo (SE)-based imaging methods. These measurements indicate that MPIOs should be useful for improving cell tracking by MRI. Magn Reson Med 53:329 –338, 2005. Published 2005 Wiley-Liss, Inc. † Key words: MRI; iron oxide; cells; contrast agents; particles Cellular imaging with MRI has proven useful for many applications, including noninvasive monitoring of stem cell migration and homing (1–3), t-cell trafficking (4), and macrophage infiltration (5). Most often, cells are loaded with dextran-coated iron oxide nanoparticles (USPIO, MION) (6), but iron oxide-based dendrimers have also proven to be effective labeling agents (7). When there is sufficient iron oxide labeling, it is possible to detect single cells in vitro (8), and a few cells in vivo (9), creating the exciting possibility that the migration of single cells in an animal can be detected. There are a number of limitations in MRI cell tracking. The first is that millions of nanometer-sized particles are necessary to achieve detection. This requires highly effi- cient labeling schemes for robust detection (10). The need to get a large number of nanoparticles into cells for MRI has limited MRI-based cell tracking to requiring that most cells be labeled in vitro and then reintroduced into the animal. The only exception has been macrophages, which avidly take up injected dextran-coated iron oxide nanopar- ticles in vivo (11). Two other issues with MRI-based cell tracking that can limit its applicability are that cell division can dilute the label beyond detectability, and the dextran-coated parti- cles are biodegradable. After several weeks, the particles are broken down and the iron is recycled by the cell, which can cause loss of detection and toxicity (12,13). These disadvantages can hamper any study in which long- term engraftment and stability are being examined or very slow migration is anticipated. We recently reported that cells can be labeled with poly- mer-encapsulated 0.96-m-sized iron oxide particles (MPIOs) (14). These particles have higher relaxivity than USPIOs, based on equivalent iron content, by nearly 50% (14) and are readily available. We further demonstrated that a single MPIO can be reliably detected in single cells and 11.5-day-old mouse embryos (15). The ability to detect cells labeled with single particles may have a number of advantages over labeling cells with millions of nanometer- sized dextran-coated particles. Their polymer coating should be inert to the cell, allowing long-term studies of labeled cells and their progeny. Since cell division can only dilute particles down to single particles, at least some of the daughter cells can be followed after each cell divi- sion. Finally, the need to label cells with only one particle may open up possibilities for labeling cells in vivo. In our previous works, 0.96-m MPIOs were used (14,15). However, larger particles are now available and offer the possibility of greatly increasing iron content. In this study, we investigated the capacity of cells to endo- cytose particles of five different sizes (diameter = 0.96, 1.63, 2.79, 4.50, and 5.8 m), and performed MRI at 7.0 and 11.7 Tesla with different imaging resolutions and echo times (TEs). Additionally, we quantified the MRI proper- ties of single particles in agarose gels. Lastly, we explored the possibility of using T 2 -based contrast methods to visu- alize individual particles and labeled cells, as opposed to the more commonly used method of using susceptibility- based T 2 * measurements. MATERIALS AND METHODS Murine hepatocytes were isolated from female C57Bl/6 mice by the collagenase perfusion method followed by repeated centrifugations, according to Seglen (16). Isolated hepatocytes were suspended and cultured in DMEM/F-12 medium (GIBCO, Gaithersburg, MD) with 5% fetal bovine serum, 2 mM glutamine, 0.875 M bovine insulin, 100 nM dexamethasone, 5 ng/ml EGF, 100 U/ml penicillin, and 100 g/ml streptomycin. Cells were plated at a density of 1  10 6 cells/cm 2 on plastic culture flasks (TissueCulture; Laboratory of Functional and Molecular Imaging, National Institute of Neuro- logical Disorders and Stroke, National Institutes of Health, Bethesda, Mary- land. *Correspondence to: Erik M. Shapiro, National Institute for Neurological Dis- orders and Stroke, National Institutes of Health, Bethesda, MD 20892. E-mail: ShapiroE@ninds.nih.gov Received 24 June 2004; revised 30 August 2004; accepted 30 August 2004. DOI 10.1002/mrm.20342 Published online in Wiley InterScience (www.interscience.wiley.com). Magnetic Resonance in Medicine 53:329 –338 (2005) Published 2005 Wiley-Liss, Inc. † This article is a US Government work and, as such, is in the public domain in the United States of America. 329