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).

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)
<ol>
	<li>Add 19F label8 to immature DCs at a concentration of 4 mg/106 cells (in 2 ml medium). Swirl gently to mix.</li>
	<li>Incubate the cells with the label for 3 days before maturing and harvesting the DCs.</li>
	<li>Collect the DCs and remove excess label by washing at least 3x in PBS. Count the cells.</li>
	<li>Resuspend in a small volume of PBS for injection or further testing.</li>
</ol>
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
<ol>
	<li>Place a known number of labeled cells (typically more than 0.5 x 106, or a number that results in sufficient signal) in a NMR tube.</li>
	<li>Add 10 &#956;l of 5% trifluoroacetic acid (TFA). If the resonance frequency of TFA is unsuitable due to overlap with the label, use an alternative soluble compound, preferably with a single 19F resonance.</li>
	<li>Place the sample in an NMR spectrometer and obtain the 19F spectrum. Ensure that the bandwidth is sufficient for both the TFA (reference) and the agent.</li>
</ol>
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&#160;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.
<ol start="4">
	<li>Calculate the 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 19F&#39;s/cell in the sample.</li>
</ol>
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
<ol>
	<li>Use an external reference containing a known amount of 19F signal for quantification12. Choose the signal intensity of the reference to be roughly comparable to that expected from the subject.</li>
</ol>
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.
<ol start="2">
	<li>Modify the placement, size and shape of the reference as necessary, dependent on the region of interest (ROI). Note: It is generally easiest to use tubes with a uniform cross-section for the reference.</li>
</ol>
5. Imaging and Mouse Preparation
<ol>
	<li>Dilute commercially available ketamine and xylazine 1:10 v/v with physiological saline to yield stock solutions of 10 mg/ml ketamine and 2 mg/ml xylazine.</li>
	<li>Turn on the heating system in the scanner (typically warm air) to ensure the bore will be warm before the mouse is imaged.</li>
	<li>Inject 100 mg/kg ketamine and 10 mg/kg xylazine intraperitoneal&#160;to initiate anesthesia. Note: These doses have been tested for CD1(ICR) and NU-Foxn1nu 8-week old male mice. Other strains may need slightly different doses, which should be optimized in a separate experiment. This dose will keep the mouse anesthetized for about 45 min. Note: The depth of anesthesia is normally sufficient if the animal shows no reaction to a foot pinch.</li>
	<li>Once anesthetized, place the animal in an animal cradle and immobilize to prevent motion during MRI. If required, position the external reference next to the animal. Both the reference and the ROI (expected location of the injected cells) should be in the center of the coil. The animal should be connected to temperature and breathing control for physiological monitoring during scanning.</li>
	<li>Cover the eyes of the mouse with sterile&#160;eye ointment to prevent drying&#160;out during long imaging sessions.</li>
	<li>If the imaging time exceeds 40 min, prepare additional catheters for injection of additional ketamine/xylazine before the animal may wake up. Position two separate subcutaneous&#160;catheters with the working solutions of ketamine and xylazine. Use this catheter to give the mouse an additional 2.5 mg of ketamine every 20 min after the first 40 min. If necessary, further prolong anesthesia by injection of additional 0.5 mg xylazine through the catheter, 100 min after the initial injection. In all cases, experimental time should not exceed 3 hr, except possibly under terminal anesthesia.</li>
	<li>Ensure that the mouse is warmed directly after the first injection and maintained at 37 &#176;C body temperature. With this protocol, &#60;80% blood oxygenation was detected under atmosphere air breathing. To prevent side effects from the lowered blood oxygenation, use 100% oxygen (0.3 L/min). Body temperature and breathing should be controlled throughout the whole experiment and until full recovery of the animal. Note that the spontaneous breathing rate under ketamine/xylazine is very high (200-250/min). Irregularities in the breathing pattern, rather than the breathing rate, can indicate that a higher dose is necessary to maintain a suitable state of anesthesia.&#160;&#160;The mouse will awaken spontaneously within 20-30 min&#160;after the last dose of anesthetic.</li>
</ol>
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 
<ol>
	<li>Position the animal inside the scanner so that the ROI is in the isocenter using a low-resolution, anatomical scan with different slice orientations (a localizer).</li>
	<li>Use the regular shimming protocol on 1H, e.g. a global shim on the whole volume inside the coil.</li>
	<li>Adjust the reference pulse gain (RG), i.e. the transmitter power measured in dB for a 1 msec Hermite 90&#176; RF pulse, using the adjustment scan provided by the vendor.</li>
</ol>
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.
<ol start="4">
	<li>The following protocol of 19F reference pulse gain adjustment works only if both 1H and 19F RF transmission is carried out with the same (single or double-tuned) RF coil. For such an imaging setup, the coil profile (B1 transmit field) on 1H should be proportional to the 19F coil profile, when a fixed RG is applied, i.e. B1,1H=C*B1,19F with a proportionality constant C.
	<ol>
		<li>To confirm that coil profiles are proportional for a specific setup, acquire a 1H and a 19F flip angle map (see following section on B1 correction) on a tube with highly concentrated 19F, e.g. TFA in water (1:1 v/v) using identical fields-of-view (FOVs), beforehand (Figure 3).</li>
		<li>Using the same RG, check that both maps are identical except for the global factor C.</li>
		<li>Once the 1H RG is determined, note this number down.</li>
	</ol>
	</li>
	<li>Carry out a conventional 1H MRI scan as an anatomical reference for the 19F imaging. Tune the system to the 19F frequency of the cell marker and carry out a 19F MR scan. Ideally the 19F MRI scan will have the same field-of-view and slice selection as the 1H MRI, although usually with lower resolution.&#160;The animal can be revived as soon as the imaging is complete. Image processing can then be carried out offline.&#160;</li>
	<li>Determine the 19F RG via the relation dB19F=dB1H-20*log10(C). Estimate the 19F agent frequency in a separate in vitro experiment.</li>
</ol>
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 &#956;m3, 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 &#956;m3, TA = 57 min, BW = 10 kHz).
<ol start="7">
	<li>B1 correction 
	Using an RF coil with inhomogeneous B1 profile, e.g. a surface coil, can hamper quantification of 19F data since the signal depends not only on 19F concentration but also on the distance from the coil. Acquire a B1 map on the 1H channel in order to retrospectively correct 19F data. This requires that 1H and 19F coil profiles are identical, except for the proportionality factor C, and that sufficient 1H background signal is provided in regions of 19F. An example protocol for correction of a 19F spin echo sequence is presented, using a single-tuned Tx/Rx surface coil tunable to both 1H and 19F and with proportional B1 profiles13.
	<ol>
		<li>Acquire a 1H flip angle map with the two flip angle method 14. Acquire a gradient echo scan with very long TR (&#62;3 times 1H T1) with an estimated flip angle of just below 90&#176;. Close to the coil. Acquire a second gradient echo scan with a RG=dB1H-6, i.e. twice the flip angle of the first scan.</li>
		<li>Calculate the 1H flip angle, &#945;, at voxel (x,y,z) via the signal intensities (SI) of the first and second gradient echo scans: &#945;(x,y,z)=arcos(SIGE,1(x,y,z)/2SIGE,2(x,y,z))14. The 19F flip angle is identical (except for the factor 1/C) due to proportional B1 profiles. According to Faraday&#39;s principle of reciprocity15, the attenuation of 19F signal intensity from a spin echo sequence (excitation flip angle &#945;, refocusing flip angle 2&#945;) during signal reception is attRx(x,y,z)~&#945;(x,y,z). The attenuation due to imperfect excitation is attTx(x,y,z)~sin3(&#945;(x,y,z))16. The overall attenuation is written as att=attRx*attTx= &#945;*sin3(&#945;)/90&#176;. Note that it is normalized to 1 in case of perfect 90&#176;/180&#176; excitation/refocusing flip angle. The B1 corrected 19F image signal intensities SI19F,corr are calculated via SI19F,corr(x,y,z)= SI19F(x,y,z)/att(x,y,z).</li>
		<li>In order to compare signal intensities from different experiments, place a reference tube with defined 19F concentration in the field of view, and normalize the SI19F,corr to the mean signal in the reference.</li>
	</ol>
	</li>
</ol>
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
<ol>
	<li>Making 1H and 19F overlay images
	<ol>
		<li>Export the image data for the final 1H and 19F magnitude images from the scanner.</li>
		<li>Open the files in an image processing program, such as ImageJ (freeware, NIH).</li>
		<li>Carry out image adjustments as necessary (cropping, brightness, contrast) keeping the adjustments the same for all images. The 19F images will typically need to be upscaled to a higher resolution to match the corresponding 1H images.</li>
		<li>Render the 19F images in false color (select a different look-up table, under the &#34;image&#34; menu in Image J) and then overlay on the corresponding 1H images in order to localize the signal.</li>
	</ol>
	</li>
	<li>19F Quantification
	<ol>
		<li>Select a 19F image (magnitude image) with the relevant cells and the reference visible.</li>
		<li>In a program such as Image J, draw ROIs over the relevant cells, the reference and a region outside the subject (noise).</li>
		<li>Use the command Analyze and then Measure (or simply Ctrl+M) to calculate the mean pixel intensity within these regions, and their area. Let S(cells) refer to the mean pixel intensity in the ROI over the cells. Multiply that by the volume (= area x slice thickness) to calculate the total signal.</li>
		<li>Calculate the SNR, for example using the formula SNR = 0.65 x S(cells) / &#963;, where &#963; is the standard deviation of pixel intensity in an ROI outside the sample which is free of any chemical shift artifact (typically in the background air)17. If the SNR is under 5, a correction for the signal due to the noise may be necessary and can be carried out as described elsewhere11 10.</li>
		<li>Otherwise, calculate the total amount of 19F responsible for the signal in the ROI over the reference by multiplying the area of the reference in the slice by slice thickness (to calculate the volume) by the concentration of 19F in the reference, which is known and assumed to be homogeneous.</li>
		<li>Multiply that value by the total signal in the ROI over the cells, divided by the total signal over the reference to calculate the amount of 19F in the cells.</li>
		<li>Calculate the number of cells by dividing that number by the value for 19F/cell, which was calculated in section 2.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interests to disclose.

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 ...

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.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>MFCD00131855</td>
			<td></td>
		 </tr>
		 <tr>
			<td>TFA</td>
			<td>Sigma-Aldrich</td>
			<td>76-05-1 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ketamine (Ketavet)</td>
			<td>Pfizer</td>
			<td>778-551</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Xylazine (Rompun)</td>
			<td>Bayer</td>
			<td>QN05 cm92</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ophtosan </td>
			<td>Produlab Pharma</td>
			<td>2702</td>
			<td>eye ointment</td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Material name </td>
		 </tr>
		 <tr>
			<td>MRI scanner</td>
			<td>Bruker Biospec</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>NMR spectrometer</td>
			<td>Bruker Biospec</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

in vivo 19f mri for cell tracking

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.

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 pro...

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In vivo 19F MRI for Cell Tracking

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 for Cell Tracking

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. ...

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JoVE Journal

Medicine

14.6K Views.  Radboud University Medical Center. 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.

Video: In vivo 19F MRI for Cell Tracking

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&#8212;Application in Premanifest Huntington's Disease

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy begins many years prior to the motor onset (during the &#34;premanifest&#34; period), but the spatiotemporal pattern of regional atrophy across the brain has not been fully characterized. Here we demonstrate an online cloud-computing platform, &#34;MRICloud&#34;, which provides atlas-based whole-brain segmentation of T1-weighted images at multiple granularity levels, and thereby, enables us to access the regional features of brain anatomy. We then describe a regression model that detects statistically significant inflection points, at which regional brain atrophy starts to be noticeable, i.e. the &#34;change-point&#34;, with respect to a disease progression index. We used the CAG-age product (CAP) score to index the disease progression in HD patients. Change-point analysis of the volumetric measurements from the segmentation pipeline, therefore, provides important information of the order and pattern of structural atrophy across the brain. The paper illustrates the use of these techniques on T1-weighted MRI data of premanifest HD subjects from a large multicenter PREDICT-HD study. This design potentially has wide applications in a range of neurodegenerative diseases to investigate the dynamic changes of brain anatomy.

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&amp;#8212;Application in Premanifest Huntington&#039;s Disease

This paper describes a statistical model for volumetric MRI data analysis, which identifies the &#34;change-point&#34; when brain atrophy begins in premanifest Huntington&#39;s disease. Whole-brain mapping of the change-points is achieved based on brain volumes obtained using an atlas-based segmentation pipeline of T1-weighted images.

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy ...

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...