We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.
In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.
In vivo cell tracking is essential for the optimization and monitoring of cellular therapeutics1. Due to its noninvasive nature, imaging offers excellent opportunities to monitor cells in vivo. Magnetic Resonance Imaging (MRI) is independent of ionizing radiation and allows a superior imaging resolution and intrinsic soft tissue contrast. MRI-based cell tracking has already been used clinically to follow dendritic cells in melanoma patients2. Conventional clinical MRI is carried out on the 1H nucleus, present in mobile water in tissues. It is also possible to carry out MRI on other active nuclei, such as 13C, 19F and 23Na. However, only 19F MRI has been applied successfully to in vivo cell tracking as it offers the highest sensitivity after 1H. The absence of MRI-detectable endogenous 19F in tissues permits high signal selectivity for the detection of exogenous 19F contrast agents and allows quantification of fluorine concentration directly from the image data. For a detailed discussion on 19F MRI, see3-5. A key issue with 19F MRI is the need to develop and optimize suitable 19F cell labels, although several labels have been developed, with a trend towards multimodal agents6.
The protocol we describe here is based on studies by our groups7-9, including the first articles that described in vivo quantitative 19F MRI-based cell tracking10,11. The general procedure of cell tracking using 19F MRI is summarized in Figure 1. We describe a general protocol for labeling and imaging of dendritic cells (DCs) using a custom-made perfluorocarbon contrast agent8. The imaging protocol is generally applicable to different cell types, labels and animal models. The cell type and animal model described here should only be taken as an example, and thus we do not provide details on the cell isolation and labeling, but rather focus on the imaging protocol. Modifications will be necessary for each label, cell type, animal model, and imaging setup, and these can be found in the literature or may need to be optimized by the researchers. Some common modifications are included in the discussion.
Note: All experiments and procedures involving animals must be carried out in accordance with relevant ethical guidelines and conform with standard animal care and humane requirements.
1. Cell Labeling (Standard Protocol with Coincubation)
Note: This protocol is relevant for these specific DCs and label. The procedure was optimized in another publication8 and included here are only the steps to prepare a sample to image, since the focus of this protocol is on the imaging. Hence, the protocol for isolation, culture and labeling of a specific cell type will either need to be found in the literature or optimized by the researcher. A summary of all other 19F labels that have been used in the literature is presented elsewhere6.
2. Determination of 19F/cell Using 19F NMR
Note: The TR should be long enough for full relaxation of the reference and the sample spins (around 5 times T1 relaxation time of the longest T1 component in the tube, typically TR ~5-8 sec). Some spectrometers require D2O for a frequency lock signal. A standard 19F spectrum is sufficient. Extensive averaging or higher cell numbers will be required if cell uptake of the label is poor or if cell numbers are low. The spectrum should be centered between the reference and relevant label peaks, unless corrections are made to account for partial excitation.
Note: The entire process must be repeated and averaged extensively enough to reach statistical significance. This can also be done using spectroscopy directly at the MRI scanner. However, note that this procedure reveals only the average 19F loading for a large number of cells, not the variability between cells. Checking uniformity of cell uptake requires the presence of a fluorescent component at the 19F agent, so that cell uptake can be analyzed using microscopy or flow cytometry.
3. Considerations for Experimental Design – Cell Injection
Due to the relatively poor sensitivity of 19F MRI for cell tracking, an animal model where the cells will localize in large numbers is necessary for successful in vivo imaging. Typical sensitivity values range from 1,000-100,000 cells/voxel/1 hr scan time6, with a cell loading in the range of 1011-1013 fluorine atoms.
The number and frequency of imaging sessions must be carefully planned, as this influences the choice of anesthetic. Note that isoflurane may not be suitable for 19F MRI if the 19F resonance frequency of isoflurane is close to that of the label compound used. isoflurane may still be suitable if the imaging sessions are short enough to prevent significant build-up. Several alternative anesthetics have been used in the literature10,11; an example is included in the imaging protocol. Anesthetics may need to be optimized for different mouse strains and disease models.
4. Design of an External 19F Reference
Note: Generally, it is simplest if the reference compound is the same as the cell label, to match the relaxation parameters. If spectroscopic sequences or sequences without spatial localization are used, then a compound with a different chemical shift may be necessary.
5. Imaging and Mouse Preparation
Note: For cell tracking, it is necessary to image the same animal more than once. In that case, the imaging sessions should be scheduled with consideration to the time necessary for clearance of anesthesia from the system and animal use guidelines of the institute.
6. Imaging
1H adjustments and imaging
Note: This adjustment will vary with the type of RF coil and is essential for 19F MRI since the signal on the 19F frequency is usually too low for direct adjustments. As a consequence, an estimate of the RG must be made from measurements on the 1H signal. For RF transmission with a surface coil, the 1H RG should be adjusted to a plane parallel to the coil through the part of interest, for a volume coil with homogenous B1 profile, the exact settings of the adjustment are less important.
Note: In vivo, the exact frequency can vary slightly from experiment to experiment due to different shimming conditions. To prevent chemical shift artifacts the exact frequency should therefore be determined in each experiment by 19F MRS, if possible. A typical scan for very low 19F signal would be global (pulse-acquire) spectroscopy (TR=200 msec, NEX=3,000, TA=10 min, BW=50 kHz). NEX, thus TA, can be dramatically reduced for high 19F signal. The 19F agent frequency is determined from the spectrum after Fourier transformation and phase correction. NEX refers to the number of excitations, TA is the acquisition time and BW is the bandwidth.
Note: Typical imaging parameters9 on a 11.7T/16 cm dedicated animal scanner are: An anatomic 1H imaging scan using a turbo spin echo (TSE) sequence with TR/effective echo time (TEeff) = 2,200 msec/42.8 msec, 8 echoes per excitation, NEX = 2, 10 consecutive, 1 mm thick slices, FOV = 1.92 x 1.92 cm2, 128 x 128 matrix, i.e. a resolution of 150 x 150 x 1,000 μm3, TA = 1 min, BW = 50 kHz and a linear phase encoding scheme. 19F images were acquired with the same sequence and matching geometry, but at lower in-plane resolution and lower BW (NEX = 256, 48 x 48 matrix, i.e. a resolution of 400 x 400 x 1,000 μm3, TA = 57 min, BW = 10 kHz).
Note: Other 1H B1/flip angle mapping methods can be used and different 19F pulse sequences require modifications of attTx,19F. Any kind of B1 correction scheme will introduce additional error in the cell quantification procedure. If accurate cell quantification is the main premise, an RF coil with homogenous B1 profile can be used to circumvent this part of the protocol.
7. Image Processing
Here we show the typical results for a protocol involving the transfer of 19F-labeled cells homing to a draining lymph node. Figure 2 shows a 19F NMR spectrum of 106 labeled cells using a TFA reference. The imaging setup was carried out as described in the protocol (Figure 3). For the in vivo imaging, we used a reference consisting of a sealed cylinder of the same label used for the cells placed between the feet of the mouse. A representative processed image is shown in Figure 4. By applying the protocol described in section 7.2, we calculated a cell number of approx. 1.2 x 106 based on the raw 19F magnitude image.
Figure 1. Key steps for 19F MRI-based cell tracking. These include the appropriate selection of label and labeling protocol is crucial for success of the experiment (Step 1). Cell labeling (Step 2), may require enhancement through the use of coatings or transfection agents. After suitable preparation, including removal of any excess label, (Step 3) the cells can be imaged. Post-processing can be used to generate quantitative data from the images, if acquired suitably (Step 4). Finally, various ex vivo analysis can be carried out (Step 5) to corroborate the in vivo data. Figure reproduced with permission6. Click here to view larger figure.
Figure 2. Representative 19F NMR spectrum. The spectrum shows the 19F signal obtained due to the known amount of reference, in this case TFA, and the known number of cells, labeled "sample". This can be used to calculate the amount of fluorine per cell, which is necessary for in vivo quantification from MR image data.
Figure 3. Flip angle maps of tubes with TFA in water were acquired at the 1H and 19F channel. The proportionality factor was calculated to be 1.03±0.08 for this setup. FA: flip angle.
Figure 4. In vivo 1H/19F image of dendritic cells homing to a draining lymph node. The figure shows a representative image of a mouse, with 19F signal visible, in false color, in the reference (R) and the labeled cells. Only the legs of the mouse were imaged here. In this example, the injected labeled cells migrated to the draining lymph node. The imaging parameters are summarized in the main text. The figure is for illustration purposes only.
The protocol outlined here describes the general procedure for in vivo 19F MRI cell tracking. Despite the described co-incubation method, there are several different protocols for labeling cells with a 19F agent. However, the co-incubation is most often used and can be further optimized e.g. by addition of transfection agents6. The actual cell labeling protocol will depend on the cell type. Only key labeling steps are described in the protocol here. Generally, the label is prepared and added to the cells for a selected time ranging from a few hours to a few days. Finally, the cells must be washed carefully to remove any excess of label, especially if quantification from the image data will be carried out3. If the 19F agent includes a fluorescence tag, the presence of label outside (or not bound to) the cells can be detected using microscopy. After labeling, it is necessary to study the cell viability and functionality, in relation to nonlabeled cells (e.g. by trypan blue exclusion assay). Cell-specific functionality tests, such as the ability to activate T cells in the case of DCs, must also be carried out. Finally, as some non-19F MRI labels have an effect on cell migration18, such tests should also be included for 19F labels, especially with high cell loading.
The protocol for the animal model is also dependent on the user´s needs. However, it is important to remember the strict sensitivity limits of 19F MRI when planning experiments. Published sensitivity values range from 1,000-00,000 cells/voxel/1 hr time6, with a cell loading in the range of 1011-1013 19F. Sensitivity and expected label concentration in the region of interest also impact the imaging parameters. For example, the slice thickness used for the 19F image can be adjusted to match that of the 1H images or to cover a much thicker area (such as the entire lymph node or region of interest in a single slice). Typically, the 19F imaging is carried out at lower resolution to maximize signal detection, especially since high resolution is not usually necessary to distinguish structures such as lymph nodes. If the signal is sufficient, a larger number of thinner slices can be acquired and the total signal over the volume of interest calculated from these. However, the optimal parameters will need to be derived for each application individually.
We describe an injection anesthesia protocol with ketamine/xylazine avoiding fluorinated inhalation gases like isoflurane. Other protocols have been used in the literature10,11. However, in all cases, these protocols will need to be adjusted for the mouse strain, age, gender, health and the length and frequency of the imaging sessions.
Several 19F MRI imaging sequences have been used for cell tracking, for a review see6. Our imaging protocol can be readily modified to include other imaging sequences. However, the quantification method described here does not take into account any partial volume effects, discrepancies in selection of ROIs, changes in relaxation parameters of the label in vivo, or changes in cellular 19F loading. These issues are described elsewhere3. In brief, the error has been found to be within tolerable limits for longitudinal cell tracking10. Compared to these earlier works we additionally propose an image correction scheme for the use of surface RF coils by mapping the B1 field. Depending on the specific RF coil setup it is important to be aware of limitations of B1 mapping techniques, which have been well-studied in the context of 1H MRI16. For example, for the double angle method presented here the B1 map is most accurate for flip angle pairs just below 90°-180° 14 and significant error can be introduced in other regions. Still, after proper validation in vitro, such retrospective correction can further improve cell quantification. In general, we recommend corroboration of the data with more established techniques, at least initially. This is typically done in combination with other in vivo imaging techniques, e.g. optical imaging, to help exclude large errors. In most cases, histological evaluation is necessary to assess 19F label location precisely and to allow correlation with the imaging data. This may require the addition of a fluorescent dye to the 19F agent.
Finally, although 19F MRI has not yet been applied to clinical cell tracking, it would be possible to modify this protocol for human use. The main modifications necessary would be to carry out as much of the optimization and setup beforehand as possible (to minimize the length of time the subject is in the scanner), and to adjust imaging parameters to keep within clinical specific absorption rate (SAR) restrictions.
The authors have nothing to disclose.
We would like to acknowledge Nadine Henn and Arend Heerschap for their valuable assistance. This work was supported by the Netherlands Institute of Regenerative Medicine (NIRM, FES0908), the EU FP7 program ENCITE (HEALTH-F5-2008-201842) and TargetBraIn (HEALTH-F2-2012-279017), a grant from the Volkswagen Foundation (I/83 443), the Netherlands Organization for Scientific Research (VENI 700.10.409 and Vidi 917.76.363), ERC (Advanced Grant 269019), and Radboud University Nijmegen Medical Centre (AGIKO-2008-2-4).
REAGENTS | |||
PBS | Sigma-Aldrich | MFCD00131855 | |
TFA | Sigma-Aldrich | 76-05-1 | |
Ketamine (Ketavet) | Pfizer | 778-551 | |
Xylazine (Rompun) | Bayer | QN05 cm92 | |
Ophtosan | Produlab Pharma | 2702 | eye ointment |
Material name | |||
MRI scanner | Bruker Biospec | ||
NMR spectrometer | Bruker Biospec |