We show that a commercially available perfluorocarbon emulsion is a promising label for 19
F MRI-based cell tracking in a clinically relevant cell type. We found that the CS-1000 label had minimal effect on DC viability and function, as tested by expression of maturation markers, mRNA expression after electroporation and T cell activation. The minimum number of labeled cells per voxel detectable at 7T with our imaging parameters is ~2000, with 1.7 × 1013
fluorine atoms per cell. This labeling is consistent with previously published data using similar 19
F labels in murine cells.18,23
Uptake of label by mature DCs was much lower (data not shown) as maturation downregulates endocytic processes; hence, we labeled the cells in the immature stage. Labeling was observed within 24 hr after addition of label, although we cultured the cells for 6 days after label addition to minimize cell handling steps.
The detection limit using SPIO in vitro
is almost 10-fold lower than either the 19
F or the Gd labels ( and ). This is comparable with previous reports on the sensitivity of MRI for the detection of human DCs labeled with SPIO at 1,000 cells/mm3 in vitro
when loaded with 30 pg Fe/cell.24
SPIO agents can be extremely sensitive, and detection limits as low as single cells have been reported using SPIO agents in vitro.25–27
However, the addition of 1
H background from biological tissue complicates identification of labeled cells (), as endogenous regions of hypointensity are observed in various tissues, blood vessels or blood clots. In addition, the T1
effects are not independent of each other. Most importantly, the effect of contrast agents on relaxivity saturates at higher concentrations ().
agents are particularly sensitive to this saturation effect ().
Cell number quantification using
agents is prone to error due to the uncertainty in agent relaxivity in situ
and at higher cell densities due to the saturation of the
effect, as demonstrated in . Cheung et al.
carried out quantification of cells in phantoms using a ultrasmall SPIO agent and found that the cell numbers were underestimated at cell densities greater than approximately one million cells per voxel.28
This may be a limitation of the technique given that the typical cell numbers injected in a clinical DC vaccination trial is about 15 million12
and cell numbers can be even higher with other cell therapies. Other techniques to decipher the complex change in observed contrast with SPIO labels involving quantitative measures of relaxation rates are also susceptible to the same saturation effect.15
Recent advances with contrast agents include the use of “white markers” for cell localization using
In this technique, the background is made dark by dephasing gradients, and the intensity of the signal near the contrast agent dipoles is dependent on relaxivity, echo-time, slice thickness and gradient strength.30
Endogenous iron stores and blood clots at the site of injection also complicate relaxometric measurements at the site of cell transfer.32
It is also worth to mention that as we used monocytes-derived DCs, these cells do not divide.32
This fact makes the method used for cell quantification particularly suitable for tracking DCs since no signal reduction nor data misinterpretation are caused by the uncontrolled process of cell division. However, the technique of quantitative cell tracking using 19
F MRI has also been applied to actively dividing T cells, which were successfully tracked for up to 3 weeks.32
The underestimation of cell numbers that occurs due to cell division is often within tolerable limits, two–fourfold depending on the division rate and length of time. This error can be reduced if the cell division rate is known. Exo-cytosis of the label after cell death could also be a source of error in the quantification method, but only relevant when the number of dead cells is in the same order of magnitude as living cells. This issue is discussed in more detail elsewhere.13
Together, our findings demonstrate the feasibility of 19
F labeling of human DCs and cell quantification using MRI. The label shows comparable detection sensitivity to cells labeled in vitro
using Gd-complexes when imaged using conventional contrast mechanisms. The main limitation of 19
F imaging is the low concentration that necessitates the use of signal averaging and potentially longer scan times. Our current detection limit of 2,000 cells/voxel at 7T with 1.7 ± 0.1 × 1013 19
F atoms/cell translates to a limit of about 9,000 cells/voxel at 3T (considering a strong SNR∞Bo7/4
dependence with the magnetic field intensity Bo
for small coils), with other factors remaining constant. To put this into perspective, a typical DC vaccination study utilizes an intranodal or intradermal injection of about 10 million cells, with between 30,000 and 200,000 cells migrating to secondary lymph nodes, as detected using scintigraphy on 111
We note that the size of a voxel in MRI is determined by the practitioner, and high resolution imaging is not necessary for 19
F-based cell localization as these images are overlaid onto high resolution 1
H images for anatomy. Importantly, the SNR needed for 19
F images can generally be much lower (e.g.
, SNR~5 or less) than normally acceptable for 1
H images, as the 19
F is not used to provide tissue contrast or organ definition, but only cell population localization. Moreover, further improvements in 19
F MRI methodologies might yield significant imaging acceleration, for example using restricted k
-space acquisitions schemes.34
Currently, clinical 19
F MRS requires a concentration of 19
F in the millimolar range.35,36
Our current detection limits of 2,000 or 9,000 cells/voxel at 7 T and 3 T, respectively, translate to concentrations of 19
F at 1 and 5 mM. The clinical MR spectroscopy work suggests that these concentrations can be achieved and detected using human scanners.
Thus, to image a lymph node with 30,000–200,000 cells based on our system (acquired using the same coil) would take 30–0.7 min at 3T, while maintaining a minimum SNR of 3 for 19
F (sufficient for quantification18,19
) when using a single spin echo sequence. The use of a fast multiecho imaging sequence,18,19
typical in clinical applications, with a turbo factor (RARE factor) of 4 would result in imaging times of 8–0.2 min for 30,000–200,000 cells, which are within reasonable limits for clinical use. Overall, realistic advances in hardware and imaging protocols may allow in vivo 19
F-based cell tracking to become clinically relevant in the near future.