Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.
Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.
The tracking of cells in vivo is a crucial aspect in several fields of biomedicine. For this, noninvasive imaging techniques that can selectively localize cells over a period of time are extremely valuable. Prior to the development of three-dimensional magnetic resonance imaging (MRI), the tracking of immune cell migration was limited to microscopic analyses or tissue biopsies. Cell tracking with the help of MRI has gained immense attention in the past few years, not only for immunologists studying immune cell behavior in vivo, but also for clinical and stem cell researchers. During the mid-90s, the first studies on iron oxide nanoparticles 1 initiated a cascade of developments for tracking cells with MRI. Iron oxide particles shorten the MR relaxation time (T2*) of the labeled cells and thus cause signal depletion in MR images. Iron oxide particles have been employed to label macrophages 2, oligodendrocyte progenitors 3 and many other cell types. Some of these particles have also been clinically approved by the FDA for labeling cellular vaccines in melanoma patients 4. Since in vivo or ex vivo labeling of cells with iron oxide particles relies on a shortening of the T2* signal and the latter could be also brought about by in vivo susceptibility-related T2* effects such as micro bleeds, iron deposits or air bubbles, it might be difficult to identify labeled cells in vivo from other background T2* signal extinctions 5.
In this article, we describe a technique for tracking dendritic cells (DC) in vivo by employing 19F/1H magnetic resonance imaging (MRI). This cell tracking technology was only introduced in 2005 6, several years after the first recognized applications for 19F in MRI had been reported 7. One important advantage of 19F over iron oxide particle cell labeling is the low biological occurrence of 19F in tissue; this makes it possible to track cells very selectively with basically background-free images. Furthermore, it is possible to overlay the 19F MR signal from the transplanted labeled cells with anatomical images obtained from conventional 1H MRI. 19F/1H MRI is therefore considerably relevant for studies investigating cell migration in vivo. Cells studied with this method are labeled with 19F-rich particles. Synthetically-derived perfluorocarbons (PFCs) consisting primarily of carbon and fluorine atoms are commonly used to prepare the particles. These compounds are insoluble in water and need to be emulsified prior to application in vitro or in vivo. The usual size of the PFC particles that have been employed by other groups for in vivo 19F-MRI tracking experiments ranges between 100 nm and 245 nm 6,8-10. We have however shown that the efficiency in labeling dendritic cells with perfluoro-15-crown-5-ether (PFCE) particles increases with increasing particle size (>560 nm).11
All animal procedures must be approved by the local institutional animal welfare committee prior to execution. During the MR measurements an adequate level of anesthesia and physiological monitoring (body temperature, respiratory rate) are indispensable requirements.
1. Generation of Mouse Bone Marrow-derived Dendritic Cells
2. Labeling of Dendritic Cells with 19F-rich Particles
3. In Vivo Application of 19F-labeled Dendritic Cells
4. In Vivo 19F/1H Magnetic Resonance Imaging
The detailed instructions for setting up the MR scans refer to a Bruker MR scanner using the control software Paravison (version 5.1). Names referring to vendor-specific functions and items have been highlighted in italics. For other MR scanners these steps may have to be adjusted according to the manufacturers’ guidelines.
5. Lymph Node Magnetic Resonance Spectroscopy (MRS)
Eighteen to twenty-one hours following intracutaneous application, 19F-labeled dendritic cells (DC) migrate into the draining popliteal lymph node. The movement of DC via the lymphatic ducts into the draining politeal lymph node can be appreciated by overlaying the 1H anatomical images with the 19F DC images (Figure 2A). We have previously reported on the migration of these cells in vivo, as well as the impact of 19F-particle size on DC immunobiology, including uptake efficiency 11. In order to quantify the extent of DC migration into the lymph nodes, we extract the draining lymph nodes and perform 19F MRS (Figure 2B). When we compare the 19F signal obtained from each lymph node with the 19F signal obtained from different numbers of DC labeled with the same PFCE particles (calibration curve, Figure 2C) we can deduce the number of 19F-labeled DC that reach the draining lymph node. In the representative experiment shown in Figures 2A and 2B, we can deduce that 3.6 x 105 antigen-loaded DC reached the right lymph node while 7.5 x 104 DC that were not loaded with antigen reached the right lymph node.
Figure 1. Position of the mouse on the mouse holder of the small animal MR scanner.
Figure 2. Quantification of 19F-labeled DC migration in vivo using 19F MRS. (A) DC were labeled with 1 mM 560 nm PFCE particles, loaded with (right) or without (left) antigen and administered intracutaneously in hind limb. Whole chicken ovalbumin (OVA) was employed as antigen; OVA was incubated with the DC together with the 19F particles. Dendritic cells are shown as 19F MR signal (red), whereas lymph nodes and lymphatic ducts are shown in the 1H anatomical MR image (grayscale). Images were acquired with a 19F/1H dual-tunable volume birdcage resonator. (B) Lymph nodes from mouse shown in A were extracted and placed in an NMR tube. The 19F signal was measured by 19F MRS (see Protocol Text) and the amplitude was calculated by performing a fast Fourier transformation (FFT) of the acquired free induction decay (FID). (C) Over a period of 24 hr, different amounts of DC were labeled with 1 mM 560 nm PFCE particles and 19F signal was measured by 19F MRS as in B.Click here to view larger figure.
This method of employing 19F/1H MRI to follow the movement of DC into the lymph node gives the opportunity to study the migration patterns of immune cells in vivo. Dendritic cells are excellent examples of rapidly migrating immune cells that are able to maneuver through three-dimensional structures without tightly adhering to specific substrates 17. Although the low spatial resolution (μm range) of the described technique is not comparable with the high resolution (nm range) that can be achieved with multiphoton microscopy, with this technique it is still possible to study the nature of cell migration in vivo and over a longer period of time. Furthermore, microscopy has a limited depth penetration and a limited field of view, making it currently unsuitable for imaging large areas within a living organism.
Due to the noninvasiveness of the technique, it is possible to monitor the migration of immune cells for several days without sacrificing the mouse after investigation. Another advantage of the technique is the possibility to overlay the 19F images that capture the migrating cells with anatomical 1H scans, thereby allowing an accurate localization of the cells within the living organism. This technique will enable us to study molecular mechanisms underlying DC migration. Unlike typical in vitro migration assays, this method enables the study of cell migration in a physiological 3D environment.
The potential application of 19F in MRS and MRI have long been recognized 7,18. The high gyromagnetic ratio, high spin and natural isotopic abundance makes 19F an ideal candidate for MR imaging 7. In addition, the similarity between 19F and 1H (regarding their NMR properties) is a prerequisite for a meaningful overlay between the anatomical 1H MRI with the 19F MRI of labeled cells. Indeed one advantage of 1H/19F MRI over other 1H/X nuclei MRI is that the same RF resonator can be used for both 19F and 1H nuclei. In most applications used for 19F/1H MRI, a volume resonator is employed that can be tuned to both 19F and 1H. Due to the almost identical B1 field for both channels, a transfer of power settings from the 1H channel (that is easier to measure) to the 19F channel is therefore possible.
One important factor to be considered when labeling cells with particles of any size (from ultra-small to large micro-sized particles) is the potential of biological manipulation and toxicity. We have recently shown that by increasing the size of the labeling particles, we could promote the maturation status of DC 11. A possible explanation for our finding is the preponderance for particles larger than 500 nm to be taken up by phagocytosis rather than endocytosis 19; the former uptake mechanism defining the nature of DC. Other physical characteristics (e.g. particle shape and surface topology) could also alter the biological function of the labeled cells and should also be carefully considered 20. For any given experimental setup that requires labeling of DC with particles, it is thus highly recommended that typical cell biology assays are performed in parallel to the experiment to determine/exclude any influence of the particles on the biological properties of these cells. Several assays can be performed, depending on the experimental study in question. To exclude an influence on DC maturation, measurements of cell surface marker (e.g. CD80, CD86) expression by FACS is recommended. To exclude an influence on antigen uptake and presentation, phagocytosis experiments and T cell priming experiments, respectively, are recommended. In the case of migration experiments, the expression of chemokine (CC) receptors (such as CCR7) and in vitro migration assays are recommended.
Although 19F/1H MRI holds promise for studying cell migration particularly for clinical purposes, there are currently a number of limitations. These include (i) a spatial resolution that precludes direct visualization of individual cells, (ii) insufficient 19F sensitivity (detection limit of several 105 cells within one ROI), (iii) increased scan durations as a result of increased averaging to compensate for the previous limitation, (iv) a limited range of 19F RF resonators on the market and (v) possible undesirable background 19F signal by other fluorine-containing elements such as isofluorane and polytetrafluoroethylene (PTFE) within MR hardware components (e.g. capacitors and connecting cables).
Apart from factors such as magnetic field strength and gradient strengths, one main determinant that dictates the level of spatial resolution is the sensitivity of the radio frequency (RF) resonator used 21. MRI resolution is closely related to the signal-to-noise ratio (SNR) 21 and upon reducing voxel size to amplify spatial resolution, a loss in SNR is to be expected. One way to increase spatial resolution without compromising signal sensitivity is to employ cryogenically-cooled coils that boost SNR by reducing thermal noise 22. With the aid of a 1H head coil that uses a cryogenic system, we could recently visualize cellular infiltrates in the experimental autoimmune encephalomyelitis after using an in plane resolution of (35×35) μm2 23. The application of this cryogenically-cooled technology for 19F/1H MRI would be an opportunity to overcome some of the MRI limitations associated with cell signal detection and resolution.
One important issue for in vivo tracking of cells by MRI is the quantification of these cells in a particular location within a living organism. For this it is possible to perform 19F MRS (see 5.1-5.9). By employing calibration curves that are obtained from 19F MRS measurements of different numbers of 19F-labeled DC, it is possible to deduce the number of cells reaching the lymph node. The 19F MRS measurements of the lymph nodes can be compared to the 19F MRS DC calibration curve. Furthermore, by comparing the MRS data from the DC cell calibration curves with the pure PFCE, we can deduce that it is possible to detect approximately 1013 19F spins per cell following labeling. According to MR principles, the lowest detection limit is 1018 spins per voxel 24. Thus, we can assume that we are able to detect a minimum number of 105 DC per voxel using a 9.4 T MRI.
In summary, the protocol that we describe here is beneficial for in vivo investigations studying cell migration during pathophysiological processes. The noninvasiveness of the technique enables longitudinal studies without the necessity of sacrificing large numbers of animals; the ability to overlay 19F images from the labeled cells on 1H anatomical images promotes optimal and highly selective tracking of the migrating cells; and 19F MRS provides an opportunity to estimate the number of cells localizing to specific anatomical areas in vivo.
The authors have nothing to disclose.
This study was funded by the Deutsche Forschungsgemeinschaft to S.W. (DFG WA 2804) and a university grant to S.W. from the Experimental and Clinical Research Center, a cooperation of the Max Delbrück Center for Molecular Medicine and Charité Medical Faculty in Berlin. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. We thank Mr. Robert Westphal for technical support during his internship in our laboratory.
REAGENTS | |||
C57BL/6 mice | Charles River, Berlin | ||
RPMI | Gibco | 21875-091 | |
FBS Superior | Biochrom AG | S 0615 | |
HEPES | Gibco-Invitrogen | 15630-056 | |
Penicillin-Streptomycin | Gibco | 15140-122 | |
L-glutamine | Gibco | 25030-024 | |
Dulbecco’s PBS | Sigma Aldrich | D8662 | |
PFA | Santa Cruz | sc-281692 | |
Perfluoro-15-crown-5-ether | ChemPur | 391-1996 | |
Pluronic F-68 | Sigma Aldrich | P5556 | |
Petri dishes (35 x 10 mm) | VWR, Germany | 391-1996 | |
27 ½ G syringes | VWR, Germany | 612-0151 | |
Nylon cell strainers (100 μm mesh) | VWR, Germany | 734-0004 | |
NMR tubes | VWR, Germany | 634-0461 | |
EQUIPMENT | |||
Dissection tools | FST | ||
CO2 incubator | Binder | ||
Small animal MR system | Bruker Biospin | 9.4T BioSpec 94/20 USR, ParaVision Acquisition and Processing Software | |
1H/19F dual-tunable volume RF coil | Rapid Biomed, Würzburg, Germany | 35 mm inner diameter, 50 mm length | |
19F spectroscopy coil | in-house | tune/match loop coil, 4 turns, inner diameter 5 mm, 10 mm long, two capacitors for tuning and matching | |
Isoflurane inhalation system | Föhr Medical Instruments GmbH | ||
Animal monitoring system Model 1025 | SA Instruments Inc., New York, USA |