Our data showed that (a) labeling hMSCs with Ferucarbotran at the level of roughly 7pg iron/cell did not attenuate viability of the cells over 12 days post labeling; (b) the amount of iron oxide taken up by the cells was reproducible over multiple labeling procedures; (c) the quantity of iron oxide inside single cells declined as the cells divided, but the total intracellular iron of all progeny was not diminished over this time; (d) substantial Ferucarbotran-induced effects on MR relaxation of hMSC solutions were evident up to 14 days after labeling; and (e) sonication-induced cell lysis of viable cells to disperse the internalized Ferucarbotran caused a marked decline in MRI signal intensity and increase in T2-relaxation rate. In our in vitro study, this effect could be used to differentiate intracellular iron oxides in viable cells from iron oxides, which had been released from lysed cells.
The large reduction in MRI signal intensity caused by disruption of cells and liberation of incorporated SPIO particles implies a big change in T2 relaxivity for extracellular versus intracellular particles. It is well known that relaxivity of susceptibility contrast materials is highly dependent on geometry and microscopic distribution of the magnetic centers. The observed differences on SE T2- versus GE T2*-weighted images in our study may be explained by many iron oxide particles being sequestered inside a few cells. Upon lysis of the cells the iron oxide particles disperse more uniformly throughout the sample causing a substantial reduction in distance between iron oxide particles. Using nominal density of 5.1 g/ml for iron oxide, iron fractional mass of approximately 72% and assuming a 4.2 nm sphere diameter for the iron oxide crystal in Ferucarbotran, we have an estimated iron mass of 1.43×10−19 g per crystal. Since the iron assay yielded approximately 7 pg of Fe loaded into each cell, we estimate roughly 4.9×107 crystals per cell. We do not know how many crystals comprise each SPIO particle, but if there are 8–10 crystals per particle it is still possible that distance between superparamagnetic centers, which would be proportional to the cubed root of the number of particles, may be reduced by roughly hundred-fold as a consequence of cellular disruption. Consequently an increased fraction of water molecules would be close to steep field gradients near the particles and would visit microscopic regions of the inhomogeneous magnetic field while diffusing during the TE interval.
On the other hand, the T2
* weighted images could exhibit much less effect upon disruption because the static field inhomogeneity would be less altered by cell disruption (22
). For example, if only a few very large magnetic particles were internalized into each cell, one would not expect to detect any change on T2
weighted SE images upon cell disruption. The observed signal characteristics are in accordance with previous studies: Simon et al. found increased R1
-values for free versus intracellularly compartmentalized USPIO in vitro (20
). In that study, monocytes were labeled with Ferumoxides USPIO particles and the cellular iron content was 10-fold less than the labeled hMSCs used in the current study. The difference in iron labeling can be attributed to a combination of factors including that monocytes are physically smaller cells than hMSCs, greater uptake efficiency for larger SPIO versus USPIO, and a larger per-particle iron content for SPIOs versus USPIOs. However the cell solutions used in the study of Simon et al contained roughly 10-fold more cells than the current study, so the total iron content of the cell solutions were similar. Yet in the former study uncompartmentalized USPIOs caused an increase of T1
- and T2
relaxation rates in comparison to intracellular USPIO, while in the current study disruption of cells containing SPIOs caused an overwhelming T2
–effect that severely attenuated signal, such that T2
could not be evaluated and their relaxation rates could not be measured. In addition, Tanimoto et al. showed in vivo that R2
* effects can be evoked by clustering of iron oxides, which would be comparable to the effect evoked by compartmentalization (33
). To our knowledge, this is the first study that applies this effect for the differentiation of viable and lysed cells.
We found that the described different T2-signal characteristics of iron oxides in viable cells and iron oxides released from dead cells were consistently observed (A) at different time points over 14 days and (B) at two different field strengths, 1.5 T and 3T.
Within the 14-day observation period, we found consistently different signal characteristics of intracellular iron oxides and free iron oxides. It can be deduced that neither iron oxide metabolization nor cell proliferation altered this effect. The cellular iron content and T2
-effect stayed nearly constant for the whole proliferating cell population and slowly decreased for individual cells. This can be explained by a distribution and dilution of the internalized iron oxides in proliferating progenies and minimal or absent metabolization of the contrast agent for up to 14 days after labeling. The positive staining for the dextran coat of the iron oxides for up to 4 weeks after labeling further supports the conclusion that no major metabolism of the contrast agent particles occurred during our experiment (). Other authors described that they found a persistent T2
-effect of iron oxide labeled mesenchymal stem cells in vivo 7 days after localized injection in the myocardium (34
) or in the kidney after injection into the renal artery (9
). Even after injection of highly proliferative neural stem cells detection was possible for up to 32 days (35
). However, Walczak et al reported that the proliferation-induced dilution of the contrast agent in cell lines with a high rate of proliferation - neural stem cells in that case - limits the long-term detectability by MR imaging (36
). In slowly proliferating cell types like the mesenchymal stem cells used in this study, this effect should not affect the detectability.
Studies with longer follow up intervals showed that iron oxides are slowly metabolized within the lysosomes and incorporated into the body’s iron metabolism (37
). Arbab and coworkers reported, that the intracellular iron oxide metabolization is dependent on the contrast agent type, the applied labeling technique and the pH of the cells cytoplasm (38
). All these discussed mechanisms, cell proliferation, dilution in progenies, iron metabolization, and potential cellular iron elimination, are expected to result in a slow decline in cellular iron content, and consequently, a decline in the cells T2
-signal effect over time. We hypothesize, that any increase in T2
-effect at the transplantation site after local transplantation of labeled cells would be an indicator of iron oxides release and cell death.
Another important factor for potential in vivo applications will be the proton density and proton diffusion capacity within the target organ. Since the observed T2-effects are apparently dependent on the degree of interaction between protons and iron oxides, our results are more likely reproducible in vivo in a proton-rich pathology (e.g. edema) than in a proton-deprived environment.
In vivo applications of our technique are also dependent on additional biologic factors. Our technique to differentiate viable and dead cells is based on differences in T2
-effects and dependent on a lysis and iron release of dying cells. There are two major mechanisms how cell death can occur – necrosis and apoptosis (39
). Necrosis results in cell lysis and subsequent inflammatory response (40
). It is to be expected that processes resulting in necrosis and cell lysis will produce a similar signal behavior as we have shown. On the other hand, apoptosis is a regulated cell death where controlled degradation occurs leading to fragmentation of the cell. This type of cell death will result in the same iron content being compartmentalized among many cell fragments. This would be similar to the behavior seen in our scans with increasing cell counts – the R2
relaxation rate would be the parameter most sensitive to change in this case. In many instances, both mechanisms may be involved after cell transplantation.
In addition, necrotic or apoptotic cells as well as released iron oxide particles may undergo a secondary phagocytosis by macrophages. The relation between the number of lysed cells and the number of macrophages would determine the resulting rate of compartmentalization and thereby a possible contrast agent effect. Therefore, the consistency of the surrounding tissue (fluid necrosis vs solid scar tissue) in which the release and the re-compartmentalization of iron oxide particles takes place and the number of macrophages will determine the possibility to detect a graft failure. Thus in vivo applications will be more complex and will have to evaluate various different pathologies in order to clarify, if the results from our in vitro model are applicable for certain pathologies and target organs in vivo.