The overall goal of this procedure is to image and quantify the number of 19 F labeled cells in vivo using 19 FMRI. This can be done in a clinical or preclinical setting. This is accomplished by first labeling cells with a suitable 19 F agent.
This is most often done ex vivo. The second step is to quantify the 19 F content per cell, which can be done using 19 F and MR with a known reference. The 19 F content per cell is assumed to remain constant during the experiment, otherwise correction factors must be calculated.
Next, the subject is imaged in vivo using one H and 19 FMRI to localize the transferred labeled cells. A known reference and appropriate imaging parameters are essential for quantification from the image data. The final step is to process the image data to calculate the number of cells.
Ex vivo histology or other techniques can be used to corroborate the data where possible. Ultimately, 19 FMRI for in vivo cell tracking can be used to study the transferred cells in terms of their localization and numbers over the relevant length of time, given certain assumptions as outlined in the text. The main advantage of this technique over existing methods like one HMRI using super paramagnetic cell labels, is that there's no confounding background signal and that the number of cells can be determined directly from the image data.
This method can help answer key questions in the field of cellular therapeutics, such as where are the cells and how many are present at various time points. To begin this protocol, first add a 19 F label to immature dendritic cells at a concentration of four milligrams per 1 million cells in two milliliters of medium. Then swirl gently to mix.
Next, incubate the dendritic cells with the label for three days before maturing and harvesting. Then collect the cells and remove excess label by washing at least three times. In PBS count, the resulting cells then resuspend in a small volume of PBS for injection or further testing.
Note that the protocol as described here is relevant for these specific dendritic cells and label. Hence, a protocol for another cell type will either need to be found in the literature or optimized by the researcher. Place a known number of labeled cells in an NMR tube.
Then add 10 microliters of 5%tri Fluor or acidic acid, or TFA. If the resonance frequency of TFA is unsuitable due to overlap with the label, use an alternative soluble compound. Place a sample in an NMR spectrometer and obtain the fluorine 19 spectrum.
Then calculate relative areas under the peaks of the reference and the sample or the main peak of the sample, and then calculate the average number of fluorines per cell in the sample. Note that this entire process should be repeated in average to reach statistical significance. This can also be done using spectroscopy directly at the MRI scanner.
Also note that this procedure reveals only the average fluorine 19 loading for a large number of cells, not variability between cells. Checking uniformity of cell uptake requires the presence of a fluorescent component with the 19 F agent. Here only scenario three shows homogenous labeling of the cells, although this is always assumed in the calculations, this can be verified using complementary techniques such as microscopy set up for imaging by placing a fully anesthetized mouse in a cradle and immobilizing it to prevent motion during the scan.
Also, connect temperature and breathing control for physiological monitoring during scanning. If required, position an external reference next to the animal, both the reference and the region of interest should be in the center of the coil. Begin acquisition with a conventional proton MRI scan as an anatomical reference for the 19 F imaging.
Then tune the system to the 19 F frequency of the cell marker and carry out a 19 FMRI scan. Ideally, the 19 FMRI scan will have the same field of view and slice selection as a conventional MRI, although likely with lower resolution. Next, determine the 19 F reference pulse gain using the formula seen here and acquire a 19 FNMR spectrum to determine the 19 F frequency.
Estimate the radio frequency coil dependent factor C in a separate in vitro experiment. Use of a radio frequency coil with an in homogenous B one profile such as a surface coil can hamper quantification of 19 F data. Since the signal depends not only on 19 F concentration, but also on the distance from the coil.
Therefore, acquire a B one map on the one H channel to retrospectively correct 19 F data. This requires that one H and 19 F coil profiles are identical except for the proportionality factor C and that sufficient one H background signal is provided in regions of 19 F.Here we see flip angle maps of tubes with TFA in water acquired at the one H and 19 F channel. To correct the 19 F data first acquire a one H flip angle map with the two flip angle method.
Use a gradient echo scan with a very long TR being over three times to one HT one with an estimated flip angle of just below 90 degrees close to the coil. Then acquire a second gradient echo scan with twice the flip angle of the first scan. Calculate the one H flip angle alpha at vle X, Y, Z via the signal and sens of the first and second gradient ECHO scans.
The 19 F flip angle will be identical except for the factor one C due to proportional B one profiles the attenuation of 19 F signal intensity from a spin echo sequence with excitation flip angle alpha refocusing flip angle two alpha during signal reception is seen on screen here. The attenuation due to imperfect excitation is seen here. Then the overall attenuation can be calculated.
Note that it is normalized to one in case of perfect 90 degrees, 180 degrees excitation refocusing flip angle, the B one corrected 19 F image signal intensities SI 19 F corrected are calculated via this formula to create one H and 19 F overlay images. Export the image data and open the files in an image processing program such as image J, apply image adjustments as necessary, being sure to keep the adjustments the same for all images. Then render the 19 F images in false color and then overlay onto the corresponding one H images.
In order to localize the signal, select the 19 F magnitude image with the relevant cells and the reference visible draw ROIs over the relevant cells, the reference and a region outside the subject for the noise value as shown in the sample. Note that here, the one H anatomic scan is shown for clarity. Simple proportionality and the value of 19 F per cell can then be used to estimate the number of cells in the ROI.
These images are from typical results of a protocol involving the transfer of 19 F labeled cells homing to a draining lymph node. Here we see a 19 FNMR spectrum of 1 million labeled cells using A TFA reference, and here we see a representative processed image for the in vivo imaging. A reference consisting of a sealed container of the same label used for the cells was placed between the feet of the mouse.
By applying the protocol described, we calculated a cell number of approximately 1.2 million based on the raw 19 F magnitude image. The 19 F image is superimposed in false color over the gray scale, anatomic proton image. When setting up a new experiment, it is important to validate fluorine.
MRI results with histology, optical imaging, or other methods. For example, it is important to note that fluorine label can remain in tissue when the cells die and lead to false positive signal.
