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Published online before print September 11, 2007, 10.1148/radiol.2451061345
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(Radiology 2007;245:449-457.)
© RSNA, 2007

Experimental Studies

R2 and R2* Mapping for Sensing Cell-bound Superparamagnetic Nanoparticles: In Vitro and Murine in Vivo Testing1

Rebecca Kuhlpeter, MD, Hannes Dahnke, PhD, Lars Matuszewski, MD, Thorsten Persigehl, MD, Angelika von Wallbrunn, PhD, Thomas Allkemper, MD, Walter L. Heindel, MD, Tobias Schaeffter, PhD, and Christoph Bremer, MD

1 From the Department of Clinical Radiology, University Hospital of Muenster, Albert-Schweitzer-Str 33, D-48129 Muenster, Germany (R.K., L.M., T.P., A.v.W., T.A., W.L.H., C.B.); Philips Research Laboratories, Hamburg, Germany (H.D., T.S.); and Interdisciplinary Center for Clinical Research (IZKF Muenster, FG3), University of Muenster, Muenster, Germany (C.B.). Received August 3, 2006; revision requested October 10; revision received December 18; accepted January 6, 2007; final version accepted March 14. C.B. supported in part by the Deutsche Forschungsgemeinschaft (BR 1653/2-1) and the Bundesministerium für Bildung und Forschung (13N8896). Address correspondence to C.B. (e-mail: bremerc{at}uni-muenster.de).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
ADVANCES IN KNOWLEDGE
IMPLICATIONS FOR PATIENT CARE...
 References
 
Purpose: To prospectively determine the cellular iron uptake by using R2 and R2* mapping with multiecho readout gradient-echo and spin-echo sequences.

Materials and Methods: All experiments were approved by the institutional animal care committee. Lung carcinoma cells were lipofected with superparamagnetic iron oxides (SPIOs). Agarose gel phantoms containing (a) 1 x 105 CCL-185 cells per milliliter of agarose gel with increasing SPIO load (0.01–5.00 mg of iron per milliliter in the medium), (b) different amounts (5.0 x 103 to 2.5 x 105 cells per milliliter of agarose gel) of identically loaded cells, and (c) free (non–cell-bound) SPIOs at the iron concentrations described for (b) were analyzed with 3.0-T R2 and R2* relaxometry. Iron uptake was analyzed with light microscopy, quantified with atomic emission spectroscopy (AES), and compared with MR data. For in vivo relaxometry, agarose gel pellets containing SPIO-labeled cells, free SPIO, unlabeled control cells, and pure agarose gel were injected into three nude mice each. Linear and nonlinear regression analyses were performed.

Results: Light microscopy and AES revealed efficient SPIO particle uptake (mean uptake: 0.22 pg of iron per cell ± 0.1 [standard deviation] for unlabeled cells, 31.17 pg of iron per cell ± 4.63 for cells incubated with 0.5 mg/mL iron). R2 and R2* values were linearly correlated with cellular iron load, number of iron-loaded cells, and content of freely dissolved iron (r2 range, 0.92–0.99; P < .001). For cell-bound SPIO, R2* effects were significantly greater than R2 effects (P < .01); for free SPIO, R2 and R2* effects were similar. In vivo relaxometry enabled accurate prediction of the number of labeled cells. R2' (R2* – R2) mapping enabled differentiation between cell-bound and free iron in vitro and in vivo.

Conclusion: Quantitative R2 and R2* mapping enables noninvasive estimations of cellular iron load and number of iron-labeled cells. Cell-bound SPIOs can be differentiated from free SPIOs with R2' imaging.

© RSNA, 2007


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
 DISCUSSION
ADVANCES IN KNOWLEDGE
IMPLICATIONS FOR PATIENT CARE...
 References
 
The advent of cell-based tumor therapies and stem cell–based tissue repair has prompted the need for a high-spatial-resolution, sensitive imaging tool for in vivo cell tracking (1,2). To date, various strategies for magnetic resonance (MR)-based cell tracking have been described. Specifically, the use of iron oxides seems to be promising for in vivo cell tracking because the high-spatial-resolution capabilities of MR imaging can be exploited and strong susceptibility effects enable the detection of even small amounts of cells labeled with iron oxides (3–8). Various cell-tagging approaches have recently been designed and include simple cell incubation with contrast media, receptor targeting of iron oxides, and sophisticated methods of iron oxide surface modification with membrane translocation signals such as the human immunodeficiency virus–derived tat peptide or magnetodendrimers (3,9). Investigators in more recent works have described the use of clinically approved iron oxides for cell tagging with a simple transfection method, bringing this technique one step closer to clinical practice (10–12). The clinical application of iron oxide–based cell monitoring was recently described by de Vries et al (13).

For clinical as well as experimental applications, a reliable method of quantifying iron oxide load and thus a noninvasive means of measuring the amount of migrating cell populations would be desirable. The differentiation between cell-bound and free iron oxides would be especially desirable for in vivo applications. Thus, the purpose of our study was to prospectively determine the cellular iron uptake by using R2 and R2* mapping with multiecho readout gradient-echo and spin-echo sequences.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
ADVANCES IN KNOWLEDGE
IMPLICATIONS FOR PATIENT CARE...
 References
 
Two authors (H.D., T.S.) who are employees of Philips Research Laboratories (Hamburg, Germany) were involved in designing the MR sequences and developing the R2 and R2* mapping tool used in our study. All authors who are not employees of Philips Research Laboratories had control of the data and information submitted for publication that might present a conflict of interest for the authors who are employees.

Superparamagnetic Iron Oxides
A clinically approved carboxydextran-coated superparamagnetic iron oxide (SPIO) (SHU 555 A, Resovist; Schering, Berlin, Germany) was used for all experiments. These SPIO particles have an average hydrodynamic diameter of 65 nm, as measured with photon correlation spectroscopy. The size of the iron oxide core is 3–5 nm, as measured with electron microscopy. SPIO-based contrast agents mainly act on R2 (1/T2) and R2* (1/T2*) relaxation rates. The change in these relaxation rates after application of the SPIOs is determined according to the relaxivities r2 and r2*. R2 and R2*—these two terms cited together being succinctly referred to as R2(*)—are calculated as follows: R2(*) = Formula + [r2(*) · C], where Formula is the intrinsic R2(*), C is the SPIO concentration, and r2(*) is the term used to refer to the r2 and r2* relaxivities of the contrast agent cited together, each measured under standardized conditions. The r2 of SHU 555 measured at 0.47 T and 39°C in water has been reported to be 164 L · mmol–1 · sec–1 (14,15).

Cell Culture and Cell Labeling
Human lung carcinoma cells (CCL-185), which are commercially available from the American Type Culture Collection (Manassas, Va), were cultured under generally established cell culture conditions to confluence in 5% CO2 at 37°C (cell culture flasks, 175 cm2). The culture medium consisted of Ham F12 medium supplemented with 10% fetal bovine serum (Gibco, Paisley, England), 1% penicillin-streptomycin (Invitrogen, Paisley, England), and 0.5% glutamine (Invitrogen). The cells were labeled with iron by using a previously established lipofection protocol (10,11). Briefly, to facilitate cellular SPIO uptake, the contrast agent was incubated with a polycationic transfection medium (cytRoti-Fect; Carl Roth, Karlsruhe, Germany) to result in the formation of micelles with the SPIOs. The lipid-encapsulated SPIOs were then efficiently taken up into the cytosol by means of endocytosis such that they were interacting with the negatively charged cell membrane (10,11). For micellization of the SPIOs, 0.5 mmol of the SPIO particles, 21.5 µL of the transfection medium, and 9 µL of mercaptoethanol were incubated with 1.5 mL of serum-free medium for 30 minutes.

For cell labeling, first the confluent cells were trisected into equal parts—each containing 3 x 106 cells—and the serum-containing medium was replaced by the serum-free medium, which included the lipid-encapsulated SPIOs. The cells were then incubated in 5% CO2 at 37°C for 24 hours. After incubation, the cells were washed with phosphate-buffered saline until the supernatant was cleared from the free SPIOs. The cells were then trypsinized and repeatedly centrifuged at 1400 rpm for 5 minutes until the supernatant was cleared from the non–cell-bound SPIOs (R.K., L.M.).

Cell viability was assessed by using the trypan blue exclusion test (R.K., T.P.). With this assay, cell viability is determined by using trypan blue dye, which is excluded by live cells but accumulates in dead cells (16). The ratio of dead to live cells—that is, the cell viability (V, expressed as a percentage)—was calculated for each control and tagged cell batch as follows: V = (N – Ntb)/N, where N is the total number of cells and Ntb is the number of trypan blue–positive cells.

Quantification of Iron Uptake in Cells
Inductively coupled plasma atomic emission spectrometry (AES) was used to determine the intracellular uptake of iron in CCL-185 cells (R.K., L.M.). By measuring the light emitted from excited iron atoms, the iron concentrations in a sample can be determined by means of calibration with known standards. The emitted wavelength is characteristic of the material being investigated: The emitted wavelength for iron is 248.3 nm. For inductively coupled plasma AES analysis, one million cells were prepared in 500 µL of phosphate-buffered saline. We dissolved 100 µL of the cell suspension in 100 µL of nitric acid–hydrogen peroxide and diluted the solution to 700 µL by adding distilled water. Iron counts were automatically calculated and expressed in micrograms of iron per liter of solution by using an atomic absorption spectrophotometer (Perkin-Elmer, Ueberlingen, Germany).

Light Microscopy
Cellular SPIO uptake was visually analyzed by using light microscopy. CCL-185 cells were labeled with iron as described above and then allowed to reattach to the culture plates. Both iron-loaded and nonlabeled control cells were examined on phase-contrast microscopic images by using an inverted microscope (Nikon TE2000 [objective magnification, x10–x40]; Nikon, Düsseldorf, Germany). The reattached cells were then fixed on the culture plates with 99% acetone–99% methanol (1:1) for staining with Prussian blue and counterstaining with nuclear fast red. Digital images were acquired (R.K., A.v.W.) by using the Nikon DXM1200 camera (Nikon) and ACT1/DXM1200F software (Nikon).

Cell Phantoms and Cell Preparations for in Vivo Imaging
The labeled and washed cells were first resuspended in 1 mL of medium and then homogeneously distributed in a 2% agarose gel to a total volume of 6 mL. Each phantom consisted of five to seven glass tubes (16 mm in diameter, 100 mm long) filled with 2% agarose gel that contained different concentrations of free (non–cell-bound) SPIO or SPIO-labeled cells (R.K., A.v.W.). Three phantom setups were prepared for MR imaging: (a) To determine the effect of different SPIO loads, 1 x 105 CCL-185 cells per milliliter of agarose gel were incubated with increasing SPIO loads (0.01, 1.00, 2.00, and 5.00 mg of iron per milliliter of medium; three samples each) in the medium. (b) Different numbers (5.0 x 103 to 2.5 x 105 cells per milliliter of agarose gel, four samples each) of identically iron-loaded (incubated with 1.0, 3.0, or 5.0 mg/mL iron) cells were prepared; the exact number of SPIO-loaded cells was determined by using a Neubauer counting chamber. (c) Finally, the phantoms were prepared such that they contained the same quantities of SPIO as those described for (b) (four samples each), but the SPIOs were not bound to cells.

Animal Testing
To assess the performance of relaxometry in vivo, agarose gel pellets (0.2%) were injected subcutaneously into the flanks of three female CD1 NU/NU nude mice (Charles River Laboratories, Sulzfeld, Germany) that were about 8 weeks old and weighed 20–25 g (R.K., T.P.). The total volume of agarose gel subcutaneously injected (500 µL) contained (a) 2 x 105 CCL-185 cells per milliliter of agarose gel labeled with 1.0 mg/mL iron, (b) free (non–cell-bound) iron (at concentrations identical to those of the iron bound to the 2 x 105 cells), (c) 2 x 105 nonlabeled CCL-185 cells per milliliter of agarose gel, or (d) 500 µL of pure agarose gel, both of which served as control pellets (three each). All experiments were approved by the institutional animal care committee of the University Hospital of Muenster.

MR Imaging of Cell Phantoms
Imaging was performed with a 3.0-T clinical whole-body MR unit (Intera; Philips Medical Systems, Best, the Netherlands) by using a transmit-receive head coil for imaging the cell phantoms (H.D., T.S.). T2* maps were obtained by using a multi–gradient-echo sequence. For each gradient echo, full k-space was sampled and a series of images was reconstructed at different echo times. To avoid phase effects from eddy currents, the even and odd echoes were treated separately. For the phantom experiments, 36 gradient echoes were used to reconstruct a time series of images for T2* determination (420-msec repetition time [TR], 1.6-msec interval between two uneven echoes, 30° flip angle, 256 x 256 spatial resolution, 3-mm section thickness, 210-mm field of view, total examination time of 3 minutes 37 seconds). T2 maps were acquired by using a Carr-Purcell-Meiboom-Gill multiecho multisection sequence (32 echoes, 820-msec TR, 5.8-msec interval between two echoes, 128 x 128 spatial resolution, 6-mm section thickness, 210-mm field of view, 90° flip angle, total examination time of 7 minutes 2 seconds).

To suppress susceptibility influences in the gradient-echo sequence, measurements were performed with a high spatial resolution (256 x 256) and a low section thickness (3 mm). Because the spin-echo sequence is not strongly influenced by susceptibility artifacts, a lower spatial resolution (128 x 128) and a higher section thickness (6 mm) could be used to optimize the signal-to-noise ratio. These parameters did not influence the relaxation time results owing to our use of the homogeneous in vitro and in vivo agarose gel models, which made identical section thicknesses and spatial resolutions unnecessary.

The test tubes containing tagged cells or free SPIOs suspended in agarose gel were placed perpendicular to the main magnetic induction field (B0) in a 220-mm-diameter circular water bath. For T2* quantification, the section direction was set to be coronal to avoid macroscopic influences in section direction, from alterations of the B0, that corrupt R2*. For the qualitative determinations, the section direction was also set to be transverse for comparison of the standard deviations of the coronal and transverse relaxation times, with the assumption that the standard deviation should be identical in both orientations if the cells were distributed homogeneously (ie, no cell sedimentation occurred). From these measurements, the R2 and R2* were determined by using a monoexponential Levenberg-Marquardt algorithm for every voxel. The mean relaxation rate per glass tube was determined by calculating the mean R2* and R2 values within the diameter of the glass tube, which contained about 100 voxels.

Regions of interest (mean size, 90 pixels ± 15 [standard deviation]) were centered over the test tubes to determine the T2 and T2*. The homogeneous distribution of the cells in the agarose gel was qualitatively and quantitatively tested by means of visual inspection of the T2 and T2* maps to ensure that there were no hot spots of accumulated iron-labeled cells (R.K., H.D.).

MR Imaging of in Vivo Experiments
For the in vivo experiments, three female nude mice were anesthetized by means of intraperitoneal injection of ketamine (125 mg per kilogram of body weight) and xylazine (12.5 mg/kg), and 500-µL agarose gel pellets, prepared as described earlier, were subcutaneously injected bilaterally into the flanks. The animals were then transferred to the MR unit and into a custom-made small-animal solenoid coil (70 mm in diameter and 50 mm in length, resulting in a 70-mm field of view) (Philips Research Laboratories), and the head-to-tail symmetry line was placed perpendicular to B0 in the magnet bore. To visualize anatomic details, transverse high-spatial-resolution T2-weighted MR images of the injection site were acquired by using a fast spin-echo sequence with the following parameters: 2700/100 (TR msec/echo time msec), a fast spin-echo factor of 15, a 2-mm section thickness, a 0.2-mm intersection gap, a 384 x 384 matrix reconstructed to 512 x 512, and a 70 x 70-mm field of view. For in vivo T2* relaxometry, 15 gradient echoes were acquired by using a spectral inversion-recovery fat-suppressed multisection sequence (477-msec TR, 3-msec interval between two echoes, 256 x 256 spatial resolution, 1.1-mm section thickness, 55-mm field of view, flip angle 30°). For in vivo T2 relaxometry, the same location was measured with 28 spin echoes (1055-msec TR, 8.2-msec interval between two echoes, 112 x 112 spatial resolution, 1.7-mm section thickness, 55-mm field of view, 90° flip angle).

Regions of interest (mean size, 70 pixels ± 10) were centered over the injected pellets to determine the mean R2* and R2 values and to calculate the R2' (R2* – R2) (R.K., H.D.). To avoid through-plane susceptibility artifacts that hamper R2* determination, a susceptibility correction method was applied, as described in detail elsewhere (17). This method is used to correct for through-plane inhomogeneities of the main magnetic field, which occur at air-tissue interfaces. These inhomogeneities lead to a deviation from the exponential R2*, which depends on the strength of alteration in B0. Alteration in B0 is determined by calculating a frequency map, which can be obtained from the multi–gradient-echo data. Therefore, the influence of the through-plane B0 alteration inhomogeneities is corrected without the need for an additional measurement. An interactive display language (IDL, version 6.1; Research Systems, Boulder, Colo) implementation of the described method was applied for postprocessing on the basis of PAR/REC4 images.

Statistical Analyses
All data are presented as means ± standard deviations. Linear and nonlinear regression analyses were performed to analyze the MR data with the AES measurements. Viability assay results were compared by using the unpaired Student t test. P ≤ .05 was considered to indicate statistical significance. All calculations were performed by using commercially available statistics software (Prism 4, version 4, 2003; GraphPad Software, San Diego, Calif) (R.K., C.B., T.A., W.L.H.).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
ADVANCES IN KNOWLEDGE
IMPLICATIONS FOR PATIENT CARE...
 References
 
Cell Viability Assay, Light Microscopy, and AES
The trypan blue exclusion assay revealed the mean cell viability to be 93.5% of control cells ± 5.0. Even high SPIO concentrations in the incubation medium did not have a substantial effect on cell viability: With an SPIO concentration of 5.0 mg/mL iron in the medium, the mean cell viability was 94.5% of control cells ± 2.5 (P > .05). Prussian blue–stained specimens showed a dose-dependent nonlinear intracellular uptake of SPIOs into the cytosol (Fig 1). Quantitative measurements of SPIO uptake revealed a saturation of cellular iron load with a maximal cellular iron concentration by using the 0.5 mg/mL iron incubation medium. (The mean uptake was 26.77 pg of iron per cell ± 6.1.) (Fig 1).


Figure 1A
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  		Figure 1a: Labeling of CCL-185 cells with SPIOs. (a) Unlabeled control cells and (b) cells labeled with a clinically approved SPIO (1 mg/mL iron in incubation medium, labeled by using previously established transfection protocol) were stained with Prussian blue dye and counterstained with nuclear fast red (original magnification, x40). Labeled cells (b) show blue precipitates representing cytosolic SPIO inclusion. (c) Graph illustrating quantification of cellular SPIO load with AES shows a maximum iron uptake of 0.5 mg/mL in the incubation medium (sigmoidal dose response).

 

Figure 1B
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  		Figure 1b: Labeling of CCL-185 cells with SPIOs. (a) Unlabeled control cells and (b) cells labeled with a clinically approved SPIO (1 mg/mL iron in incubation medium, labeled by using previously established transfection protocol) were stained with Prussian blue dye and counterstained with nuclear fast red (original magnification, x40). Labeled cells (b) show blue precipitates representing cytosolic SPIO inclusion. (c) Graph illustrating quantification of cellular SPIO load with AES shows a maximum iron uptake of 0.5 mg/mL in the incubation medium (sigmoidal dose response).

 

Figure 1C
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  		Figure 1c: Labeling of CCL-185 cells with SPIOs. (a) Unlabeled control cells and (b) cells labeled with a clinically approved SPIO (1 mg/mL iron in incubation medium, labeled by using previously established transfection protocol) were stained with Prussian blue dye and counterstained with nuclear fast red (original magnification, x40). Labeled cells (b) show blue precipitates representing cytosolic SPIO inclusion. (c) Graph illustrating quantification of cellular SPIO load with AES shows a maximum iron uptake of 0.5 mg/mL in the incubation medium (sigmoidal dose response).

 
MR Imaging of Cell Phantoms
In all phantom experiments, the standard deviation of the mean relaxation rate was similar for coronal and transverse regions of interest, suggesting a homogeneous distribution of the cells within the phantom, which was also confirmed at visual inspection of the MR images.

The effects of SPIO cell labeling on R2 and R2* were nicely visualized on the phantom images (Fig 2a, 2b). Both the R2 and the R2* had a linear relation to the number of tagged cells (Fig 2c, 2d). However, the slope for the R2* effects was almost 20-fold higher (3.6 x 10–4 mL/cells · sec) than the slope for the R2 effects (1.9 x 10–5 mL/cells · sec) (Fig 2).


Figure 2A
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  		Figure 2a: In vitro relaxometry of cell phantoms. Coronal (a) T2*-weighted (420-msec TR, 1.6-msec interval between two uneven echoes, 30° flip angle) and (b) T2-weighted (820-msec TR, 5.8-msec interval between two echoes, 90° flip angle) relaxation time maps (of phantom measurements) containing different amounts of SPIO-tagged cells (0–2.5 x 105 cells per milliliter of agarose gel). (c, d) Graphs of quantitative data evaluation show a linear relationship between cell count and both R2* (c) and R2 (d). Note that changes in R2 are fundamentally smaller than changes in R2*.

 

Figure 2B
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  		Figure 2b: In vitro relaxometry of cell phantoms. Coronal (a) T2*-weighted (420-msec TR, 1.6-msec interval between two uneven echoes, 30° flip angle) and (b) T2-weighted (820-msec TR, 5.8-msec interval between two echoes, 90° flip angle) relaxation time maps (of phantom measurements) containing different amounts of SPIO-tagged cells (0–2.5 x 105 cells per milliliter of agarose gel). (c, d) Graphs of quantitative data evaluation show a linear relationship between cell count and both R2* (c) and R2 (d). Note that changes in R2 are fundamentally smaller than changes in R2*.

 

Figure 2C
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  		Figure 2c: In vitro relaxometry of cell phantoms. Coronal (a) T2*-weighted (420-msec TR, 1.6-msec interval between two uneven echoes, 30° flip angle) and (b) T2-weighted (820-msec TR, 5.8-msec interval between two echoes, 90° flip angle) relaxation time maps (of phantom measurements) containing different amounts of SPIO-tagged cells (0–2.5 x 105 cells per milliliter of agarose gel). (c, d) Graphs of quantitative data evaluation show a linear relationship between cell count and both R2* (c) and R2 (d). Note that changes in R2 are fundamentally smaller than changes in R2*.

 

Figure 2D
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  		Figure 2d: In vitro relaxometry of cell phantoms. Coronal (a) T2*-weighted (420-msec TR, 1.6-msec interval between two uneven echoes, 30° flip angle) and (b) T2-weighted (820-msec TR, 5.8-msec interval between two echoes, 90° flip angle) relaxation time maps (of phantom measurements) containing different amounts of SPIO-tagged cells (0–2.5 x 105 cells per milliliter of agarose gel). (c, d) Graphs of quantitative data evaluation show a linear relationship between cell count and both R2* (c) and R2 (d). Note that changes in R2 are fundamentally smaller than changes in R2*.

 
To determine whether the linear relationship between R2* and SPIO concentration in a given voxel was valid, the final iron concentrations in the phantom (regardless of high or low cellular iron load) were plotted against the relaxation rates measured with MR imaging (Fig 3). The linear slope indicated that within the range of the applied incubation concentration (medium containing 1.0–5.0 mg/mL iron), no deviation from a linear behavior occurred. Thus, linear regression analysis enabled us to determine the r2 and r2* values for free and cell-bound SPIOs (Table). Moreover, at analysis of the individual data cluster, it became apparent that the highly iron-loaded cells showed the same R2 and R2* effects as the higher number of cells with lower iron load did as long as the final iron concentration in a given voxel was equal.


Figure 3
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  		Figure 3: Graphs illustrate dependency of R2* and R2 on SPIO concentration in phantoms containing free (right) and cell-bound (left) SPIOs. R2 (dashed lines) and R2* (solid lines) have a linear relationship with SPIO concentration in the agarose gel phantom. Cell-bound SPIOs exhibit a substantially stronger increase in R2* ({blacksquare}) compared with free SPIOs (bullet). Interestingly, effects on R2 are less pronounced in cell-bound SPIOs ({square}) than in free SPIOs ({circ}).

 

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  		Relaxivity Measurements for Free versus Cell-bound SPIO

 
A similar linear relationship between SPIO concentration and R2(*) was observed for free iron (Fig 3). By comparing the cell-bound SPIOs with the free SPIOs (Fig 3), we observed that with identical concentrations of iron in the phantom, cell-bound SPIOs showed higher R2* values compared with free SPIOs. On the other hand, R2 measurements were higher for the free iron than for the cell-bound iron.

MR Imaging of in Vivo Experiments
In vivo measurements enabled the acquisition of high-spatial-resolution R2 and R2* maps of agarose pellets containing SPIO-tagged cells, unlabeled cells, free SPIOs, or pure agarose gel (Fig 4). The mean R2 for the pellets containing cell-bound SPIOs (7.49 sec–1 ± 3.46) was substantially lower than that for the pellets containing free SPIOs (24.13 sec–1 ± 7.58), whereas the mean R2* for cell-bound SPIOs (62.83 sec–1 ± 1.71) was fundamentally increased compared with that for free SPIOs (31.54 sec–1 ± 2.37). By calculating the R2' (Fig 5), we observed a clear difference between the free and cell-bound SPIOs. The pellets containing cell-bound SPIOs showed a mean R2' of 55.34 sec–1 ± 4.29, whereas the pellets containing free SPIOs showed a mean R2' that did not deviate substantially from zero within the error bars (7.68 sec–1 ± 4.84). The mean R2 and R2* values for the unlabeled control cells (27.06 sec–1 ± 0.17 and 42.47 sec–1 ± 4.72, respectively) did not differ fundamentally from the mean values for pure agarose gel (24.04 sec–1 ± 1.22 and 40.97 sec–1 ± 4.12, respectively). We observed no important differences in R2' values between unlabeled control cells (15.42 sec–1 ± 4.64) and pure agarose gel (16.93 sec–1 ± 2.9).


Figure 4
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  		Figure 4: Transverse R2 and R2* maps of implanted agarose gel pellets. Gel pellets containing free (upper pellet) or cell-bound (lower pellet) SPIOs at identical SPIO concentrations (2 x 105 cells per milliliter of medium labeled with 1 mg/mL SPIO) were injected into a female nude mouse. Note the high R2* (mean, 77.5 sec–1 ± 14) but rather low R2 (mean, 13.8 sec–1 ± 1.8) for the iron-labeled cells. The R2 (mean, 35.5 sec–1 ± 2.1) and R2* (mean, 39.1 sec–1 ± 5.9) for the free SPIOs are almost identical.

 

Figure 5
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  		Figure 5: Transverse parametric R2' (R2* – R2) map of implanted agarose gel pellets shows high R2' values for cell-bound SPIOs (mean, 68.9 sec–1 ± 18), whereas the R2' values for free SPIOs (mean, 3.2 sec–1 ± 6) show no substantial deviation from zero within the error bars. Thus, R2' mapping may facilitate the differentiation of cell-bound versus free SPIOs.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
ADVANCES IN KNOWLEDGE
IMPLICATIONS FOR PATIENT CARE...
 References
 
Tagging cells with SPIO has been shown to be a sensitive method of noninvasively tracking various cell populations in the brain (18–24), bone marrow (3,12,25–27), kidneys (28,29), and myocardial tissue (30–32). However, accurate determination of the cell population size and characterization of the iron distribution (cell bound or free) are not widely achieved since mainly T2- and T2*-weighted MR images, which enable only qualitative data analysis with arbitrary signal intensity measurements, are acquired. In addition, commonly used MR sequences have limited echo train length and poor dynamic range and are sensitive to susceptibility artifacts, which hamper valid quantification.

In our study, we used R2 and R2* relaxometry. Light microscopy revealed good label retention with no toxic effects, in accordance with other findings that were obtained by using commercially available contrast agents for cell tagging (18,33). Electron microscopy examinations revealed a lysosomal deposition of SPIOs, as observed in our previous studies (11).

In vitro we observed a linear relationship between R2 and R2* values and the number of SPIO-tagged cells. This finding is consistent with the observations of Kircher et al (1), who reported on T-cell tracking with a high-field-strength (8.5-T) system. T2* measurements at this high field strength are prone to susceptibility effects. Therefore, we exploited the T2 relaxivity to visualize 3 x 104 cells per 50 µL of medium, which translates to 6 x 105 cells per milliliter of medium. However, by using T2* relaxometry, we were able to visualize relaxation effects at cell amounts well below 1 x 105 cells per milliliter with a 3.0-T clinical MR unit. Because R2* effects strongly outweigh R2 effects, this technique has superior sensitivity for the detection of low amounts of labeled cells. These observations are supported by the data presented by Bowen et al (34), who found R2* measurements to have approximately 70-fold higher sensitivity compared with R2 measurements in the detection of cell-bound SPIOs. On the basis of our in vitro MR measurements, the R2* measured in vivo translates to a cell concentration of approximately 2.15 x 105 cells per milliliter, which is a good approximation for the actual cell number that was applied (2.0 x 105 cells).

True quantification of cell populations in vivo is complex, however, because baseline (ie, before cell injection) R2 and R2* values can differ substantially according to the tissue type. Thus, true in vivo quantification requires a preinjection measurement of baseline R2 and R2* values. Moreover, dilution of the labeling agent may occur over time—for example, owing to cell proliferation or, alternatively, apoptosis of tagged cells and consequently phagocytosis (11,35).

We observed a linear relationship between cell number (of identically labeled cells) or iron concentration in the phantom and R2(*) value. However, compared with the free SPIOs, the cell-bound SPIOs revealed substantially higher R2* and r2* values and substantially lower R2 and r2 values. This was also confirmed in exemplary in vivo experiments. Concomitant measurement of R2 and R2* can thus provide information about the amount of iron compartmentalized inside the cells versus the free fraction of SPIOs owing to the difference in behavior between R2 and R2*. For freely dissolved SPIOs, R2 and R2* showed similar relaxation rates. In fact, the free SPIOs showed negligible R2', whereas the cell-bound (ie, compartmentalized) SPIOs showed substantial R2' values.

When SPIO particles are compartmentalized—in cells, for example—a nonuniform field distribution that changes the influence of diffusion on the acquired signal is generated (36). This may be addressed theoretically by applying the static dephasing regime theory (37). The change in relaxivity, depending on the compartmentalization status, again reveals the difficulties in cell quantification because R2 and R2* values depend not only on the iron concentration but also on the SPIO distribution. Therefore, our approach to measuring R2 and R2* gives rise to the additional parameter R2', which has to be taken into account if an accurate cell quantification is sought. R2' values were exemplarily calculated for the in vivo experiments, but they can also be extracted from in vitro data sets, which show obvious differences in R2* and R2 between cell-bound and free SPIOs (equivalent to R2').

Our study also included measurement of high-spatial-resolution R2(*) maps in an in vivo setting. To obtain accurate R2' values, a correction of susceptibility-induced overestimations of R2* was applied, as previously described (17). Obtaining the correct R2* value is especially important for calculating the difference of R2 – R2*, as shown in this study. Overestimated R2* values lead to false predictions of free and cell-bound SPIOs. In addition, sensitive cell detection, as is achieved with R2* measurements, is optimized by using this correction algorithm.

It is important to note that imaging in mice—even looking at cell pellets—is fundamentally different from imaging test tubes, especially if one wishes to quantify R2 and R2* properties, because motion and susceptibility artifacts are substantial in the in vivo setting owing to breathing, vessel pulsation, and gut movement. It is also evident that in vivo MR sequences have to be different from in vitro MR sequences so that higher spatial resolution can be achieved by means of higher readout gradients.

A limitation of our study was the fact that the labeled cells were injected locally, unlike in other in vivo cell tracking studies, in which labeled cells typically are injected systemically (ie, intravenously). However, to prove our hypothesis, we needed to use very well-controlled experimental conditions. Specifically, it was mandatory that the number of cells or the amount of iron be exactly determined in the in vivo setting; this is virtually impossible with an approach involving intravenous cell injections.

On the basis of the obtained results, it will be possible to also show the R2' effects in tumor models, which better reflect biologic in vivo conditions. This will be the topic of future studies. Furthermore, the sample size for the in vivo measurements was rather small. However, clear-cut differences among the groups could be shown.

In summary, the results of our study show that R2 and R2* effects in cell-tagging studies depend on both the final iron concentration and the iron distribution. Thus, the concomitant measurement of R2 and R2*, leading to parametric R2' imaging, should be advantageous for quantifying cell populations in vivo. Further studies to determine whether this method is applicable to noninvasive cell quantification in in vivo MR cell-tracking studies are warranted.

Practical application: Visualizing cell migration has important implications for in vivo experimental studies and future clinical studies. Stem cell–based tissue repair has recently emerged as a new treatment paradigm—in example, for infarcted brain or myocardial tissue (21,31,38–40). Various cell types (eg, endothelial progenitor cells) homing to specific body sites can be exploited as drug delivery or treatment vehicles (41–43). Tagged immune cells can be used diagnostically to localize inflammatory processes in the body. Recently, SPIO-loaded dendritic cells were injected and traced by using MR imaging in patients with malignant melanoma (13). The technique described herein is a promising tool for iron quantification and for differentiation between cell-bound and free SPIOs; therefore, it may help to improve monitoring in clinical cell-tracking studies.


   ADVANCES IN KNOWLEDGE
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
ADVANCES IN KNOWLEDGE
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   IMPLICATIONS FOR PATIENT CARE
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
ADVANCES IN KNOWLEDGE
IMPLICATIONS FOR PATIENT CARE...
References
 


   FOOTNOTES
 

Abbreviations: AES = atomic emission spectroscopy • SPIO = superparamagnetic iron oxide • TR = repetition time

Guarantors of integrity of entire study, W.L.H., C.B.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors; literature research, R.K., H.D., L.M., T.P., A.v.W., T.A., T.S., C.B.; experimental studies, R.K., H.D., L.M., T.P., A.v.W., C.B.; statistical analysis, R.K., L.M., T.P., A.v.W., C.B.; and manuscript editing, all authors


   References
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
ADVANCES IN KNOWLEDGE
IMPLICATIONS FOR PATIENT CARE...
 References
 

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