To detect cells by contrast-enhanced US they can be labeled with microbubbles targeting e.g. specific receptors expressed on the surface of the target cells [6
The principle of PET is the detection of radioactive tracers. The cells can be directly labeled before transplantation e.g. by incubation with indium-111-oxine (111
] or fluorine-18-fluordeoxyglucose (FDG) [19
]. Another interesting approach of cell labeling for PET or SPECT imaging is the enhanced uptake of systematically applied tracers into the donor cells after transplantation. Before transplantation, the cells are transfected with a reporter gene like the sodium iodide symporter which promotes the in vivo cellular uptake of technetium 99m (99m
Tc) or iodine 124 (124
The generation of images by MRI is based on the variable arrangement of protons induced by magnetization and their re-arrangement (relaxation) after change of the magnetic field induced by the MRI scanner. Two relaxation time constants T1 and T2 are utilized to generate tissue contrast [21
]. Principally, there are two major ways to label cells for MRI: using paramagnetic substances or superparamagnetic substances. Paramagnetic substances such as gadolinium (Gd) reduce the T1 relaxation time producing a hyperintensive (positive = white) contrast [21
]. However, the current protocols basing on paramagnetic labeling reagents seem to be inferior to superparamagnetic agents in terms of cytotoxicity and ability of generating images of strong contrast [21
]. Superparamagnetic agents are mainly basing on iron (Fe). They reduce the T2 relaxation time leading to a strong hypointensive (negative = black) contrast [21
]. Iron labeling is usually performed using superparamagnetic small particles of iron oxide (SPIO) or ultrasmall particles of iron oxide (USPIO) [26
]. They differ mainly in their particle size (20–120 nm) and their coating ((carboxy)dextran). SPIO like Endorem® (Guerbet, Paris, France) or Resovist® (Schering AG, Berlin, Germany) are already approved for clinical purposes (liver imaging). Labeling of various cell entities like immune cells and hematopoietic and non-hematopoietic stem cells with superparamagnetic agents such as particles of iron oxide has been established previously [29
]. So far, many Fe labeling protocols are based on passive Fe incorporation by endocytosis whilst in vitro incubation of the cells with the labeling agent [32
]. However, the total iron load (TIL) of each cell declines by each cell division. As the TIL correlates with the imaging quality [33
], transfection agents (TA) are applied being helpful with respect of transporting Fe into the target cell. Currently used TA are lipofectamine [34
], poly-L-lysine [35
], protamine sulphate [36
] and polethylenimine (PEI) [37
]. The positively charged TA, coating the negatively charged SPIO, leads to an enhanced SPIO-cell binding via electrostatic interactions and finally to an enhanced uptake into the cell [29
]. Interestingly, although significantly enhancing the TIL, complexes of TA and SPIO may remain for a certain time on the cellular surface before being taken up [37
]. If this phenomenon may have an impact e.g. on extracellularly expressed receptors and the interaction with their ligands remain to be elucidated. Moreover, with respect to the principles of good manufacturing practice (GMP), a prolonged in vitro incubation time with all its risks of cell culture may not be desirable. Therefore, advanced techniques have been developed in order to deliver the labeling agent as quick as possible into the cell. Exposing the cells to an electrical pulse (130 V) provokes a temporal increase of the permeability of the cell membrane, leading to an enhanced uptake of the labeling agent into the cell. This method called magnetoelectroporation is effective in terms of labeling of many cells in a very short time [38
]. Magnetosonoporation can also increase the permeability of the cell membrane in order to enhance the uptake of the labeling agent. Exposure to low frequency US (1–3 MHz) can create temporary pores into the cell membrane of up to 100 nm by the acoustic cavitation effect [40
]. Liu et al. [42
] effectively labeled human mesenchymal stem cells (MSCs) with SPIO by microbubble enhanced US exposure, referring to previous reports that US exposure in the presence of microbubbles can increase the transfection efficiency [43
]. An alternative approach combining stem cell labeling for MRI with specific positive selection techniques is the use of magnetic beads linked to specific antibodies such as anti-CD34 [45
] or specific aptamers [46
Referring to the sensitivity, using clinical MRI scanners 5,000 SPIO+TA labeled stem cells could be detected in vitro [33
], and the sensitivity of Fe labeling of cells in vivo allowed even single cell detection by MRI in small animal models [47
Even if ‘simple’ Fe labeling appears attractive and potent, one major limitation is that the detected signal refers only to the applied Fe particles and gives no information about the viability and the biological status (differentiation, metabolic or mitotic activity) of the cell labeled prior to the transplantation. Moreover, the signal may be generated from Fe particles which were detached from the labeled donor cells and/or ingested into host cells. In order to overcome these problems of direct cell labeling, reporter gene methods were also developed for cell imaging by MRI.
Introducing a metalloprotein from the ferritin family as a reporter gene, the transduced cells sequestered endogenous (superparamagnetic) Fe from the organism [49
]. No exogenous metal complexed contrast agent was required, thereby simplifying intracellular delivery. Following focal inoculation of the vector into the mouse brain, the reporter activity was monitored using in vivo MRI.
Gilad et al. [50
] designed a non-metallic, biodegradable, lysine rich protein reporter, expressing an artificial protein with frequency selective contrast. This endogenous contrast produced only by transfected viable cells is based on transfer of radiofrequency labeling from the reporter's amide protons to water protons.
It is assumed that more reporter gene based technologies for ‘smart imaging’ of distinct properties of transplanted stem cells will be developed in the near future.