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DOI: 10.1148/radiol.2432060120
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(Radiology 2007;243:467-474.)
© RSNA, 2007

Molecular Imaging

Comparison of SPIO and USPIO for in Vitro Labeling of Human Monocytes: MR Detection and Cell Function1

Raoul D. Oude Engberink, MSc, Susanne M. A. van der Pol, Ed A. Döpp, Helga E. de Vries, PhD, and Erwin L. A. Blezer, PhD

1 From the Image Sciences Institute, University Medical Center Utrecht, Bolognalaan 50, 3584 CJ Utrecht, the Netherlands (R.D.O.E., E.L.A.B.); and Department of Molecular Cell Biology and Immunology, VU Medical Center, Amsterdam, the Netherlands (S.M.A.v.d.P., E.D., H.E.d.V.). Received January 20, 2006; revision requested March 22; revision received April 20; accepted May 17; final version accepted September 18. Supported by the Dutch MS Research Foundation (MS 01-470) and the European Commission (HPRI-CT-2001-50028). Address correspondence to R.D.O.E. (e-mail: raoul{at}invivonmr.uu.nl).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Purpose: To label human monocytes with superparamagnetic iron oxide (SPIO) and compare labeling efficiency with that of ultrasmall SPIO (USPIO) and evaluate the effect of iron incorporation on cell viability, migratory capacity, and proinflammatory cytokine production.

Materials and Methods: The study was approved by the institutional ethics committee; informed consent was obtained from donors. Freshly isolated human monocytes were labeled with iron particles of two sizes, USPIOs of 30 nm and SPIOs of 150 nm, for 1.5 hours in culture medium containing 0.1, 0.5, 1.0, and 3.7 mg of iron per milliliter. Labeling efficiency was determined with relaxation time magnetic resonance (MR) imaging (4.7 T) and Prussian blue staining for presence of intracellular iron. Cell viability was monitored; migratory capacity of monocytes after labeling was evaluated by using an in vitro assay with monolayers of brain endothelial cells. Levels of proinflammatory cytokines, interleukin (IL) 1 and IL-6, were measured with enzyme-linked immunosorbent assay 24 hours after labeling. Data were analyzed with Student t test or two-way analysis of variance followed by a multiple-comparison procedure.

Results: R2 relaxation rates increased for cell samples incubated with SPIOs, whereas rates were not affected for samples incubated with highest concentration of USPIOs. Labeling monocytes with SPIOs (1.0 mg Fe/mL) resulted in an R2 of 13.1 sec–1 ± 0.8 (standard error of the mean) (7 sec–1 ± 0.2 for vehicle-treated cells, P < .05) and had no effect on cell viability. On the basis of T2 relaxation times, the in vitro MR detection limit of 58 labeled monocytes per 0.05 µL was calculated. Migration of labeled monocytes was not different from that of vehicle-treated cells. Intracellular iron had no effect on production of IL-1 and IL-6 24 hours after labeling.

Conclusion: In vitro labeling of human monocytes is effective by using SPIOs, not USPIOs. Incubation with SPIOs (1.0 mg Fe/mL) results in efficient labeling detectable on MR images and does not affect cellular viability and activation markers such as cell migration and cytokine production.

© RSNA, 2007


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Neuroinflammation is an important event in neurologic diseases such as stroke, Alzheimer disease, and multiple sclerosis. The infiltration of monocytes into the central nervous system plays a dominant role (1–3). Noninvasive visualization of monocyte trafficking provides insight into the pathologic aspects of neuroinflammatory conditions and may possibly provide a valuable tool to monitor cell-directed interventional strategies aimed to inhibit cell entry into the central nervous system.

The development of new contrast agents such as particles of superparamagnetic iron oxide (SPIO) extended the use of magnetic resonance (MR) imaging to cellular imaging (4). Researchers in prior studies have evaluated the use of intravenously administered ultrasmall SPIO (USPIO) as MR contrast agents for imaging macrophage activity in animal models of neuroinflammation (5–7). However, transport to and uptake of USPIOs in the central nervous system and its cellular compartments remain unclear. As a result, hypointense areas as seen on T2*-weighted images of the affected areas may not only represent infiltrates of macrophages but also reflect leakage of USPIOs across the blood-brain barrier or nonspecific uptake by subsets of immune cells.

Investigators in some studies have reported the labeling of stimulated (in macrophage medium or endotoxin activated) (8,9) and unstimulated (10) human monocytes with SPIOs and have shown the uptake of iron particles and MR relaxivities of labeled cells. Findings in these studies suggested that in vitro labeling of target cells with SPIOs is an interesting concept for in vivo tracking of monocytes (macrophages). It is important that the labeling procedure allows in vivo detection of a small number of cells and that the incorporation of contrast agents does not affect cell function. Optimal MR detection of prelabeled monocytes requires a biological balance between iron incorporation and cell function. We hypothesized that labeling efficiency and the effect on cell function are dependent on particle size, concentration, and incubation time. The purpose of our study was to label human monocytes with SPIOs and compare the labeling efficiency for SPIOs with that for USPIOs and to evaluate the effect of iron incorporation on cell viability, migratory capacity, and proinflammatory cytokine production.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Isolation of Primary Monocytes
Human monocytes were isolated from buffycoats of four healthy donors (Sanquin Blood Bank, Amsterdam, the Netherlands). Informed consent was obtained from each donor, and the study was approved by the Ethics Committee of VU Medical Center, Amsterdam, the Netherlands. Whole-blood samples of 15 mL were layered on 12.5-mL lymphocyte separation medium (Lymphoprep; Fresenius Kabi Norge, Halden, Norway) and centrifuged for 40 minutes at 400g and 18°C. The mononuclear interphase cells were isolated and washed with phosphate-buffered saline (pH 7.4). Finally, monocytes were purified with positive selection by using immunomagnetic beads coated with a monoclonal antibody directed against the monocytic CD14 molecule (MACS-CD14; Milteny Biotec, Bergisch Gladbach, Germany).

MR Contrast Agent: USPIO versus SPIO
Two iron oxides with superparamagnetic properties of different sizes were compared: USPIO (Sinerem; Guerbet, Paris, France) and SPIO (Endorem; Guerbet). These contrast agents were provided by the manufacturer. The authors had control of all data and information submitted for publication. The SPIO particles are composed of an iron oxide crystalline dextran-coated core, with a mean hydrodynamic diameter of 150 nm, and are supplied in suspension at a concentration of 11.2 mg of iron per milliliter. The USPIO particles are similar dextran-coated iron oxides, with a mean hydrodynamic diameter of 30 nm (11–13), and are supplied as lyophilized powder.

Monocyte Labeling
Monocytes (n = 4) were incubated in the absence (vehicle treated) or presence of USPIO and SPIO at 37°C. Iron concentrations in culture medium (supplemented RPMI-1640) were adjusted to 0.1, 0.5, 1.0, and 3.7 mg Fe/mL. The concentration of cells (monocytes) in the culture medium was 2 x 106 cells per milliliter. The effects of incubation time (n = 3) and the presence of transfection agents (TAs) (n = 3) were investigated for cell samples incubated with SPIOs at a standard concentration of 1.0 mg Fe/mL. After 0.5, 1.5, 3 and 6 hours, monocytes were processed for in vitro MR imaging and viability measurements. In separate experiments, both iron oxides (1.0 mg Fe/mL) were preincubated at room temperature with three TAs: a multicomponent lipid-based reagent (FuGene 6; Roche Diagnostics, Basel, Switzerland) at a 0.1% vol/vol ratio for 60 minutes; poly-L-lysine (Sigma, St Louis, Mo) at a 1 µg/mL wt/vol ratio for 60 minutes; and a ready-to-use reagent (SuperFect; Qiagen, Valencia, Calif) at a 0.025% vol/vol ratio for 10 minutes. After preincubation, the mixture of USPIOs, SPIOs, and TA was added directly to the cell samples. After the incubation period of 1.5 hours, cell samples were washed three times in cold phosphate-buffered saline and centrifuged for 8 minutes at 1190 rpm. All procedures with regard to cells were performed by two individuals (S.M.A.v.d.P. and R.D.O.E.).

In Vitro MR Imaging
Labeling efficiency was determined with T1 and T2 relaxation time MR imaging performed with a 4.7-T horizontal-bore spectrometer (Varian Instruments, Palo Alto, Calif). Relaxation times were measured from agarose gel (0.4%) suspensions containing free labeled (n = 2) and all samples of labeled monocytes described in the previous paragraph (0.5 x 106 cells per 250 µL) in 96-well plates. The following MR imaging data sets were collected (field of view, 4 x 4 cm; matrix, 128 x 128; receiver bandwidth, 42.5 kHz; number of transitions, two): T1 maps were a result of a monoexponential fit of seven saturation-recovery single-section T1-weighted spin-echo MR images with increasing repetition times (repetition time msec/echo time msec, 55–3000/18; one section of 20-mm thickness). T2 maps were a result of a monoexponential fit of 10 spin-echo MR images with increasing echo times (3200/17.5, 35, 52.5, 70, 87.5, 105, 122.5, 140, 157.5, 175; nine sections of 0.5-mm thickness). For analysis of relaxation time, regions of interest (circular, 7.5 mm in diameter) were placed in the center of a well, and special care was taken to exclude areas with air-susceptibility artifacts. Quantitative T1 values were obtained by using the single section. Quantitative T2 values were obtained by averaging the middle five regions of interest in the phantom volume. These procedures generated the most stable values for relaxation time, with minimum noise levels. Analysis was performed by experienced MR technologists (R.D.O.E. and E.L.A.B., with 3 and 10 years of experience, respectively).

In Vitro Detection Limit
Monocytes were incubated in the presence of SPIOs at a concentration of 1.0 mg Fe/mL for 1.5 hours. T2 relaxation times were measured of four concentrations of labeled monocytes in agar phantoms. An exponential equation (y = ex) describes the decrease in T2 (variable y) as a function of increasing cell numbers (variable x). We chose the T2 value of vehicle-treated cells (minus two times the standard deviation) as the minimum relaxation time that can be detected in this specific phantom. Solving the equation y = T2 (vehicle minus two times the standard deviation) results in the minimum number of cells that must be present in this volume to give rise to a substantial MR signal intensity.

Histochemical Detection of USPIOs and SPIOs
Immediately after labeling, cell samples were washed, and cytospots were prepared on glass slides with centrifugation at 68 rpm for 5 minutes and air dried. The presence of USPIOs and SPIOs was detected by using Prussian blue staining of iron, and cell spots were counterstained with Nuclear Fast Red. Briefly, cell spots were fixed in acetone and incubated with a 1:1 vol/vol mixture of 2% potassium ferrous cyanide (kaliumhexacyanoferrat [II]) and 2N HCl for 30 minutes. Glass slides were rinsed in distilled water and counterstained with Nuclear Fast Red for 5 minutes. The presence of iron oxides was qualitatively assessed with a microscope by estimating iron-positive cells from a total of approximately 150 cells. This procedure was performed by an experienced research technician (S.M.A.v.d.P., with 8 years of experience).

Cell Activation Assays
Cell viability was determined by using trypan blue exclusion assays for all samples described in the paragraph about monocyte labeling. Dead cells were counted with the microscope (S.M.A.v.d.P.) in a total area of 25 mm2 (approximately 150 cells) by using a calibrated counting chamber.

Monocyte migration was monitored by using a time-lapse video-microscopy migration assay (14–16). Briefly, 7.5 x 105 monocytes suspended in serum-free culture medium were added to brain endothelial monolayers (established from rat brain endothelial cell line GP8/3) grown in 96-well plates and were allowed to migrate for 4 hours. Monocyte migration (n = 4 for all samples) was monitored for SPIO-labeled (1.0 mg Fe/mL) and vehicle-treated monocytes. Freshly isolated monocytes served as a negative control and monocytes stimulated with lipopolysaccharide (100 ng/mL) for 24 hours served as a positive control. The level of migration was quantified as the percentage of migrated cells of the total number of monocytes present within a field of 200 µm2. In this experiment, two individuals (R.D.O.E. and H.E.d.V., with 15 years of experience) each analyzed some samples.

Major histocompatibility complex class II receptor expression on labeled monocytes was tested by using flow cytometric analysis (n = 3) of SPIO-labeled cells (1.0 mg Fe/mL) compared with vehicle-treated cells after 24 hours. Cells were incubated with mouse-antihuman HLA-DR monoclonal antibody (1 µg/mL) for 1 hour at 4°C. Binding of these monoclonal antibodies was detected by using phycoerythrin-coupled rabbit-antimouse F(ab)2 (1 µg/mL). Omission of the primary antibody served as negative control. Fluorescence intensity was determined by using a flow cytometer (FACScan; Becton & Dickinson, San Jose, Calif). Experimental procedures and analysis were performed by one individual (R.D.O.E.).

Production of proinflammatory cytokines interleukin (IL) 1 and IL-6 was measured 24 hours after labeling with SPIO with a concentration of 1.0 mg Fe/mL (n = 5) by using standard enzyme-linked immunosorbent assay protocols. Briefly, 96-well enzyme-linked immunosorbent assay plates were coated with 1 µg/mL mouse-antihuman IL-1ß or IL-6 diluted in phosphate-buffered saline (pH 7.4, 100 µL per well). After overnight incubation at 4°C, plates were washed in phosphate-buffered saline with 0.1% Tween and blocked with phosphate-buffered saline containing 0.5% bovine serum albumin at 37°C for 1 hour. After washing, diluted serum samples (1:1, 1:10, 1:100 vol/vol ratios) were added to the plates in duplicate and were incubated for 1 hour at 37°C in the presence of detecting antibodies, 0.5 mg/mL mouse-antihuman IL-1ß-biotin and IL-6-biotin. After washing, samples were incubated with peroxidase-labeled streptavidin (Vector Laboratories, Burlingame, Calif) to enhance the detecting antibodies. After final washing, binding of secondary antibodies was detected by adding the substrate tetramethylbenzidine. After 10–20 minutes, the reaction was stopped by using 1 mol/L H2SO4, and optical density in the wells was measured in an enzyme-linked immunosorbent assay plate reader at 450 nm. Freshly isolated monocytes served as a negative control, and monocytes stimulated with lipopolysaccharide (100 ng/mL) for 24 hours served as a positive control. Experimental procedures and analysis were performed by one individual (R.D.O.E.).

Statistical Analysis
The effects of increasing iron concentrations on relaxation rates and cell viability were evaluated with two-way analysis of variance, followed by a multiple-comparison procedure (Student-Newman-Keuls test) by using statistical software (Sigmastat, version 3.11, 2004; Systat Software, Erkrath, Germany). The effects of incubation time, presence of TAs, and cell activity assays were analyzed by using Student t tests. A difference with P < .05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
In Vitro MR Imaging: MR Properties of Free Label
The R1 relaxation rate is similar for both iron particles (Fig 1a) and shows a linear detection up to 20 µg Fe/mL (r1 = 0.035 mL/[sec · µg]). No substantial differences were observed for R2 relaxation rates (Fig 1b), but in this case, linear detection was up to 10 µg Fe/mL (r2 [USPIO] = 1.58 mL/[sec · µg] and r2 [SPIO] = 1.55 mL/[sec · µg]). Because r2 relaxivity was 50 times more sensitive for the presence of small amounts of iron, T2 relaxation time MR imaging was used in all subsequent experiments.


Figure 1A
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  		Figure 1a: Graphs show (a) R1 and (b) R2 relaxation rates determined at 4.7-T MR imaging for increasing concentrations of USPIOs ({square}) and SPIOs ({diamondsuit}). Linear regression analyses were performed to calculate relaxivity values r1 and r2 presented as mL/(sec · µg).

 

Figure 1B
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  		Figure 1b: Graphs show (a) R1 and (b) R2 relaxation rates determined at 4.7-T MR imaging for increasing concentrations of USPIOs ({square}) and SPIOs ({diamondsuit}). Linear regression analyses were performed to calculate relaxivity values r1 and r2 presented as mL/(sec · µg).

 
In Vitro MR Imaging: Monocyte Labeling
A typical T2 map of monocytes in a 96-well plate (Fig 2a) shows the difference in signal intensity loss between vehicle-treated cells and cells incubated with SPIOs versus USPIOs for similar iron concentrations. Corresponding R2 relaxation rates show the effects of different iron concentrations on labeling efficiency (Fig 2b). Cells incubated with SPIOs resulted in an increasing R2 relaxation rate for increasing iron concentrations; R2 of monocytes labeled with SPIO with a concentration of 1.0 mg Fe/mL was 13.1 sec–1 ± 0.8 (standard error of the mean), and R2 of vehicle-treated monocytes was 7 sec–1 ± 0.2. In contrast, cells incubated with USPIOs at a maximal iron concentration (3.7 mg Fe/mL) did not show a significant increase in R2 as compared with vehicle-treated cells (P = .18). Relaxation rates of agar phantoms containing vehicle-treated monocytes were not different from empty agar phantoms (P = .57, data not shown).


Figure 2A
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  		Figure 2a: (a) In vitro T2 relaxation time MR image of agar-gel-suspended monocytes labeled with USPIO and SPIO at varying iron concentrations in a 96-well plate. The vehicle-treated sample contained unlabeled monocytes. (b) Graph shows R2 relaxation rates of cell samples. (c) Graph shows T2 relaxation times of increasing concentrations of SPIO-labeled monocytes. Data in b and c are presented as mean ± standard error of the mean. * = P < .05 versus vehicle-treated cells, # = P < .05 versus USPIO-labeled cells, 128e0.67x = 128 · ex.

 

Figure 2B
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  		Figure 2b: (a) In vitro T2 relaxation time MR image of agar-gel-suspended monocytes labeled with USPIO and SPIO at varying iron concentrations in a 96-well plate. The vehicle-treated sample contained unlabeled monocytes. (b) Graph shows R2 relaxation rates of cell samples. (c) Graph shows T2 relaxation times of increasing concentrations of SPIO-labeled monocytes. Data in b and c are presented as mean ± standard error of the mean. * = P < .05 versus vehicle-treated cells, # = P < .05 versus USPIO-labeled cells, 128e0.67x = 128 · ex.

 

Figure 2C
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  		Figure 2c: (a) In vitro T2 relaxation time MR image of agar-gel-suspended monocytes labeled with USPIO and SPIO at varying iron concentrations in a 96-well plate. The vehicle-treated sample contained unlabeled monocytes. (b) Graph shows R2 relaxation rates of cell samples. (c) Graph shows T2 relaxation times of increasing concentrations of SPIO-labeled monocytes. Data in b and c are presented as mean ± standard error of the mean. * = P < .05 versus vehicle-treated cells, # = P < .05 versus USPIO-labeled cells, 128e0.67x = 128 · ex.

 
In Vitro MR Imaging: Incubation Time and TAs
In regard to the effect of increasing incubation times up to 6 hours on MR detection and cell viability for monocytes incubated in the presence of SPIOs at a concentration of 1.0 mg Fe/mL (Table), incubation times longer than 1.5 hours did not further increase R2 (P = .009 for 1.5 hours vs 0.5 hour and P = .16 for 3 hours vs 1.5 hours). Increased cell death was observed after 6 hours of incubation. Preincubation of both iron oxides (1.0 mg Fe/mL) with previously described TAs did not enhance the R2 relaxation rate of labeled monocytes.


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  		Effect of Incubation Time and TAs on R2

 
In Vitro MR Imaging: Detection Limit
On the basis of the plot for T2 relaxation times of the increasing number of labeled monocytes (Fig 2c), voxel size (0.3 x 0.3 x 0.5 mm3 or approximately 0.05 µL), and phantom volume (250 µL), we calculated an MR detection limit of 58 labeled monocytes per 0.05 µL. T2 relaxation time of vehicle-treated monocytes minus two times the standard deviation (y = 105 msec) was used in the trend line equation to solve x as the minimum number of cells that could be detected theoretically in a T2 relaxation time image of this specific phantom.

Histochemical Detection of USPIOs and SPIOs
Vehicle-treated monocytes showed no positive staining for iron (Fig 3a). Approximately 1% of the monocytes incubated in the presence of USPIOs (1.0 mg Fe/mL) contained intracellular iron (Fig 3b). In contrast, 70%–80% of monocytes incubated with SPIOs (1.0 mg Fe/mL) showed intracellular iron present in the cytosol of the monocytes (Fig 3c, 3d). Qualitative analysis of cell samples incubated in the presence of SPIOs with a concentration higher than 2.0 mg Fe/mL revealed an extracellular clustering of SPIO particles.


Figure 3
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  		Figure 3: Detected iron oxides in monocytes with Prussian blue staining. Iron oxide is blue, and cell nuclei are red. A, Vehicle-treated monocytes. B, Monocytes incubated with USPIOs at a concentration of 1.0 mg Fe/mL. C, Monocytes incubated with SPIOs at a concentration of 1.0 mg Fe/mL. D, Higher magnification of SPIO-labeled monocytes. (Prussian blue stain; original magnification, x40 [A–C], x100 [D].)

 
Effect of Iron Incorporation on Monocyte Function
No decrease in monocyte viability was observed after USPIO and SPIO incubation up to a concentration of 1.0 mg Fe/mL (Fig 4). SPIO incubation at a concentration of 3.7 mg Fe/mL significantly reduced cell viability to 76% ± 3.3 (P = .015, SPIO-labeled cells vs vehicle-treated cells). Cell viability after USPIO incubation (3.7 mg Fe/mL) decreased to 83% ± 2.3 (P = .08, USPIO-labeled cells vs vehicle-treated cells).


Figure 4
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  		Figure 4: Graph shows cell viability measured by using trypan blue exclusion for monocytes labeled in the presence of USPIOs ({circ}) or SPIOs (bullet) at varying iron concentrations for 1.5 hours. Data are presented as mean ± standard error of the mean. * = P < .05 versus vehicle-treated cells.

 
No significant difference was found (Fig 5) in the number of migrated cells for SPIO-labeled monocytes (23.5% ± 2) compared with vehicle-treated (21.8% ± 0.8, P = .33) and freshly isolated (23.2% ± 0.1; P = .41) cells. Lipopolysaccharide-stimulated monocytes showed an increased migratory capacity (30.3% ± 2.5, P = .003 vs freshly isolated cells).


Figure 5
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  		Figure 5: Graph shows percentage of total number of added monocytes that migrated across monolayers of brain endothelium after SPIO labeling at a concentration of 1.0 mg Fe/mL. Freshly isolated monocytes (Ctrl) and vehicle-treated cells served as negative controls. Monocytes stimulated with lipopolysaccharide (LPS) for 24 hours served as a positive control. Data are presented as mean ± standard error of the mean. * = P < .05 versus freshly isolated monocytes.

 
IL-1 production (Fig 6) of labeled cells (545 pg/mL ± 100) was not significantly different from freshly isolated cells (580 pg/mL ± 80; P = .77). Similarly, IL-6 levels were not significantly different for labeled cells (18 200 pg/mL ± 6000, P = .58) compared with freshly isolated monocytes (14 550 pg/mL ± 3800). In both cases, lipopolysaccharide incubation increased cytokine levels by twofold (IL-1, P = .005; IL-6, P = .04 vs freshly isolated cells). Major histocompatibility complex class II expression on SPIO-labeled monocytes was measured and was not affected by the labeling procedure or the presence of intracellular iron (P = .83 for vehicle-treated vs freshly isolated cells and P = .91 for SPIO-labeled vs freshly isolated cells).


Figure 6
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  		Figure 6: Graph shows IL-1 and IL-6 production of labeled monocytes 24 hours after incubation with SPIOs at a concentration of 1.0 mg Fe/mL. Freshly isolated monocytes (Ctrl) served as negative control. Monocytes stimulated with lipopolysaccharide (LPS) for 24 hours served as a positive control. Data are presented as mean ± standard error of the mean. * = P < .05 versus freshly isolated monocytes.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ADVANCES IN KNOWLEDGE
 References
 
Our study findings suggest that monocytes are labeled more efficiently by using SPIOs instead of USPIOs. The larger diameter of SPIO particles (150 nm) compared to that of USPIO particles (30 nm) may result in cellular uptake by monocytes. Earlier reports confirm that phagocytotic uptake of iron oxide increased with an increase in particle size (17,18). Similar results were reported in a study concerning macrophage endocytosis of SPIO nanoparticles by mouse peritoneal macrophages (19), and it is suggested that uptake of SPIOs involves scavenger receptor A–mediated endocytosis. Although our study results indicate that SPIO is far more suitable for in vitro labeling of primary monocytes, the long blood pool half-life of USPIO makes it the most frequently used iron oxide for in vivo labeling. The concept of in vivo labeling by using USPIOs has been studied in animals and patients with several diseases, which include stroke (20,21) and multiple sclerosis (5,7,22). In humans, the blood pool half-life of USPIOs is more than 24 hours (23), whereas the half-life of SPIOs appears to be shorter than 6 minutes (24), and such a half-life limits their possibility to label endogenous monocytes in circulation. In a recent study in animals, researchers showed that, with an increase in the circulation time of SPIOs by means of elimination of blood-borne macrophages, similar MR abnormalities may be observed as with USPIO labeling (25).

We have shown that only a small percentage of monocytes (<1%) is labeled by using USPIOs in vitro. In most in vivo labeling studies, the iron concentration in the plasma is relatively low (34 µg/mL) (10). This leads us to question the efficiency of in vivo labeling by using USPIOs. Although some studies have reported the presence of iron oxides within macrophages, hypointense areas as seen on MR images might reflect the presence of extracellular iron or other labeled phagocytic cells (5). Thus, to ensure reliable detection of target cells in vivo, in vitro SPIO labeling of cells may be a better tool.

In our study, labeling of monocytes in the presence of SPIOs with a concentration of 1.0 mg Fe/mL for 1.5 hours led to an increase in the R2 relaxation rate up to 13.1 sec–1 (vehicle-treated cells, 7 sec–1) measured at 4.7 T. We have to note that the relaxation rate of agar phantoms containing vehicle-treated cells is not different from empty agar phantoms. This finding helps to rule out the possibility of contribution to the MR signal of the beads used for monocyte isolation. For this labeling condition, cell viability was not affected. This established labeling concentration is five to 10 times higher than iron concentrations used for labeling other cell types in conjunction with TAs (18,26). Increasing the iron concentration in the medium is necessary to label primary human monocytes. We have also shown that TAs in combination with USPIOs and SPIOs with a concentration of 1.0 mg Fe/mL do not further increase labeling efficiency. It might be possible that longer incubation (24–48 hours) times and lower iron concentrations in combination with TAs result in comparable iron incorporation. With respect to monocyte function and application in cell-tracking studies, however, a relatively short incubation time is required.

We used an exponential fit of T2 relaxation times versus increasing numbers of SPIO-labeled monocytes in agar phantoms to calculate a detection limit of 58 labeled monocytes in 0.05 µL. However, we must emphasize the fact that this is a mathematic estimation from measurements in agar phantoms at 4.7 T. It is likely that, at a clinical field strength of 1.5 T, this detection limit will be lower because of reduced susceptibility effects of iron oxide. Qualitative visual detection by an actual observer will be limited. However, with the recent emergence of clinical high-field-strength imagers of 3 T and higher, we believe that their detection levels may eventually be comparable with our results. When MR sequences that are more sensitive for susceptibility artifacts of iron oxides, such as gradient-echo–based sequences (27), are applied, we anticipate that the detection level could be further improved to near single-cell detection. This opens up the possibility to detect a few labeled macrophages present in an inflammatory lesion. For example, in an animal model of multiple sclerosis, numerous active lesions are associated with a massive influx of monocyte-derived macrophages (7,28,29). When we ignored issues such as physiologic noise and MR artifacts, our in vitro data suggested that SPIO labeling of monocytes is an accurate tool to monitor cell dynamics within an area of inflammation.

With a view to future in vivo tracking experiments, we used unstimulated freshly isolated human monocytes to conduct the labeling studies. To assess the effect of iron incorporation on monocyte function, in vitro migration over brain endothelial cells and IL-1 and IL-6 production were studied. Activated monocytes (macrophages) show increased levels of cytokines IL-1 and IL-6 (30). For the established SPIO concentration (1.0 mg Fe/mL), no effect of the labeling procedure and intracellular iron on the migratory capacity and cytokine production of monocytes was found. This finding may indicate that, when in vivo tracking studies are conducted, labeled monocytes still migrate toward an area of inflammation and that the intracellular presence of iron does not activate the monocytes. In a previous study, labeled human monocytes were injected into the brains of immunodeficient mice, and intracerebral migration was studied with MR imaging (8). Areas of hypointensity were detected even after day 14 after injection; this finding suggests the possibility for monitoring monocyte (macrophage) dynamics in vivo. However, it was unclear whether iron particles remained inside the target cells.

Cellular MR imaging has a unique window of opportunity to monitor cellular mechanisms that underly several diseases. For accurate imaging of target cells, efficient incorporation of contrast agents is required but not at the expense of cellular function. One of the limitations of our study was that in vitro data on cell samples in agar phantoms imaged at 4.7 T did not provide an ideal replacement for an in vivo situation; thus, further studies should therefore focus on measurements at clinical field strengths. Another limitation of our study was that the effect of iron incorporation on cell viability was assessed directly after incubation. There might be a possibility that iron uptake is toxic after a longer period of time. Future studies should focus on cell viability at later times. Furthermore, to elucidate the precise mechanism of preferred SPIO uptake, specific receptor binding and inhibition assays should be performed. We are currently adopting the labeling protocol for rat monocytes and exploring in vivo monocyte tracking in animal studies. Our in vitro study with human monocytes will contribute to the design of cell labeling protocols and emphasizes the need for a balance between iron incorporation and cell function.

In summary, freshly isolated human monocytes were labeled more efficiently by using SPIOs at a concentration of 1.0 mg Fe/mL. With this established iron concentration in the incubation medium, intracellular iron did not affect migratory capacity and proinflammatory cytokine production. Tuning into the fine balance between iron incorporation and cell function, we think that in vitro labeling of monocytes contributes to a more cell-specific and accurate way of in vivo cell tracking both in humans and animal models of neurologic disease.


   ADVANCES IN KNOWLEDGE
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
References
 


   FOOTNOTES
 

Abbreviations: IL = interleukin • SPIO = superparamagnetic iron oxide • TA = transfection agent • USPIO = ultrasmall SPIO

Author contributions: Guarantors of integrity of entire study, H.E.d.V., E.L.A.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.D.O.E., H.E.d.V., E.L.A.B.; experimental studies, all authors; statistical analysis, all authors; and manuscript editing, R.D.O.E., H.E.d.V., E.L.A.B.See Materials and Methods for pertinent disclosures.


   References
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 

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