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Near-infrared fluorescence 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR)-labeled macrophages for cell imaging

DiR-Labeled macrophages
, PhD
National Center for Biotechnology Information, NLM, NIH
Corresponding author.

Created: ; Last Update: January 12, 2010.

Chemical name:Near-infrared fluorescence 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR)-labeled macrophages for cell imaging
Abbreviated name:DiR-labeled macrophages
Synonym:DiR-labeled Mø, DiR-Mø, DiR-macrophages
Agent Category:Cells
Target:Inflammation
Target Category:Others
Method of detection:Near-infrared fluorescence optical imaging
Source of signal / contrast:DiR
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
No structure is available.

Background

[PubMed]

Personalized diagnosis and treatment with allogenic or autologous cells are becoming a reality in the field of medicine (1, 2). Cytotoxic or engineered T cells are under clinical trial for the treatment of hematopoietic or other malignant diseases (3). Contrast agent–tagged macrophages are used as cellular probes to image the early inflammatory processes in macrophage-rich conditions such as inflammation, atherosclerosis, and acute cardiac graft rejection (1, 4). The roles of stem cells are under intensive investigation in therapeutic and regenerative medicine such as regenerating cardiomyocytes, neurons, bone, and cartilage (2, 5). Genetically modified cells are used to treat genetic disorders (6). With the promising results from these studies, a critical issue is how to monitor the temporal and spatial migration and homing of these cells, as well as the engraftment efficiency and functional capability of the transplanted cells in vivo (7-9). Histopathological techniques have only been used to obtain the information on the fate of implanted cells at the time of animal euthanization or via biopsy or surgery. To understand the temporal changes of cell location, viability, and functional status, cell imaging techniques have been introduced during the last few years. Cells of interest are labeled with reporter genes, fluorescent dyes, or other contrast agents that transform the tagged cells into cellular probes or imaging agents (10). There are three fundamentally different routes for labeling cells of interest (7, 8). One route is to label the cells through systemic contrast agent application, as seen in the systemic use of superparamagnetic iron oxides (SPIO), subsequent phagocytosis of the SPIO by macrophages, and accumulation in macrophage-rich lesions. The second route is to label the cells in situ by injecting contrast agents into the tissue area of interest to monitor target cell migration after phagocytosis. The more widely used route is to label the cells in vitro, which is achieved by in vitro incorporation of contrast agents or by transfecting one or more reporter genes into cells. Each labeling method has its own limitations.

In vivo optical cell imaging is a rapidly developing field in small animal imaging that depends on the use of reporter genes and fluorescent dyes (9). The Reporter gene–based approach is crucial for molecular imaging, but it strongly depends on the stable, persistent, and long-term expression of desired proteins. Long-term expression of the reporter genes may lead to host immune response and may carry on to the daughter cells in the proliferating population. The fluorescence-based approach is simple, cost-effective, and relatively sensitive, but issues of tissue-to-detector geometry, auto-fluorescence, and tissue absorption and scattering remain to be solved. Accurate quantification may only be possible when measurements are properly controlled and signals are normalized. Organic dyes are also less valuable for long-term cell tracking strategies. Nevertheless, the fluorescence-based approach is easily used for a variety of straightforward short-term labeling applications in cell imaging. In an attempt to noninvasively trace and monitor macrophages for better localization, visualization, and quantification of inflammation processes, Eisenblatter et al. developed a protocol for rapid and safe macrophage labeling with near-infrared fluorescent dye, and the investigators further tested the feasibility to image inflammation in a mouse granuloma inflammation model (1). Imaging results showed that the tagging of macrophages with the lipophilic tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR) allowed the noninvasive tracking of inflammatory cells for several days in vivo. DiR has an excitation spectrum of 750 nm and an emission spectrum of 782 nm.

Synthesis

[PubMed]

Macrophages were obtained from mouse bone marrow stem cell progenitors that were differentiated into macrophages in vitro (1, 4). Macrophages were then incubated with DiR-labeling solution for 5 min to achieve labeling. The labeling efficiency varied in the labeling intensity between different preparations of primary murine macrophages and between different concentrations of DiR. In general, the labeled fluorescence intensity increased gradually with increasing DiR concentrations up to 2 µl (19.7 µmol/L)/1 × 105 cells, with a slight drop at the higher dye concentrations. The standard dose of DiR was then set to 2.0 µl/1 × 105 cells for cell labeling. The amount of DiR per labeled macrophage was not reported. In the phantom experiments, as few as 10,000 cells distributed in 1 ml agarose gel could be detected easily with a near-infrared fluorescent scanner and an acquisition time of 3 s. Increasing numbers of labeled cells in the phantom resulted in a linear increase of signal intensity (data not shown). Co-culture of the DiR-labeled macrophages in a 1:1 ratio with T cells for 2 days resulted in <0.5% of the CD3-positive T cells exhibiting DiR fluorescence. The DiR-labeled macrophages were uniform, and the fluorescent signal was intense after 2 days. These results indicated that the DiR-labeled macrophages were stable and that there was no relevant transfer of the dye from macrophages to T cells.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Vitality and functionality assays of the DiR-labeled macrophages showed that DiR staining did not result in a higher rate of apoptosis, nor did it reduce the ability of macrophages to adhere to plastic surfaces. The phagocytosis of complement-opsonized Leishmania major parasites was not compromised in labeled macrophages compared with control cells. Equal nitric oxide production was obtained between labeled and control macrophages in response to interferon-gamma and lipopolysaccharide (LPS), indicating that the activation of DiR-labeled macrophages was unaffected (1, 4). However, the relationship between the loaded DiR amount per macrophage and macrophage functionality was not described in detail.

Animal Studies

Rodents

[PubMed]

Eisenblatter et al. tested the DiR-labeled macrophages as an optical probe in mice bearing cutaneous granuloma induced with LPS-containing polyacrylamide gel (PAG) (1). DiR-labeled macrophages were injected intravenously 24 h before PAG pellet implantation. The mice were examined daily for 7 days after injection. The signal/noise ratio (SNR) was calculated as signal intensity of pellet/standard deviation of noise. Optical imaging revealed the homing of labeled macrophages in different body compartments including the liver, lung, and bone marrow. The control PAG pellets without LPS displayed slightly higher fluorescence signals than did the reference tissue (skin neck area). Inclusion of LPS in the pellets resulted in significantly higher SNRs (529 ± 292 at 72 h after injection) than the control pellets (314 ± 168 at 72 h) (P < 0.05, n = 5). Tomographic imaging showed that the DiR-labeled macrophages distributed mainly in the periphery of the pellets, with a fluorescence signal in LPS-containing pellets ~2.7 times higher than in controls (459 ± 295 versus 253 ± 168; P < 0.05, n = 10). The injection of increasing numbers of labeled macrophages resulted in significantly increased signal intensities, presenting an almost linear correlation between tomographic signal intensity and the number of injected cells.

To verify extravasation and to quantify the number of DiR-stained macrophages inside the pellets, mice were euthanized and infiltrated cells in pellets were separated. Up to 27% of the 1 × 107 injected cells could be recovered from the pellets. In agreement with the tomographic imaging data, increasing numbers of injected DiR-labeled macrophages resulted in increasing numbers of DiR-positive cells recovered from the pellets. Up to 23% of the recovered cells were DiR-positive macrophages. The DiR-positive cells were uniformly positive for the macrophage differentiation marker F4/80 and the macrophage colony-stimulating factor receptor (data not shown). The DiR- and F4/80-negative cells were exclusively granulocytes. There was no evidence that in vivo macrophages lost their label to other cells (granulocytes) during the observation period. On the basis of the results, ~5 × 105 cells had to be distributed in the pellet to provide a strong signal for the inflammation visualization with optical imaging (1).

Other Non-Primate Mammals

[PubMed]

No references are currently available.

Non-Human Primates

[PubMed]

No references are currently available.

Human Studies

[PubMed]

No references are currently available.

References

1.
Eisenblatter M., Ehrchen J., Varga G., Sunderkotter C., Heindel W., Roth J., Bremer C., Wall A. In vivo optical imaging of cellular inflammatory response in granuloma formation using fluorescence-labeled macrophages. J Nucl Med. 2009;50(10):1676–82. [PubMed: 19759121]
2.
Mathiasen A.B., Haack-Sorensen M., Kastrup J. Mesenchymal stromal cells for cardiovascular repair: current status and future challenges. Future Cardiol. 2009;5(6):605–17. [PubMed: 19886787]
3.
Cesco-Gaspere M., Morris E., Stauss H.J. Immunomodulation in the treatment of haematological malignancies. Clin Exp Med. 2009;9(2):81–92. [PubMed: 19238515]
4.
Ehrchen J., Helming L., Varga G., Pasche B., Loser K., Gunzer M., Sunderkotter C., Sorg C., Roth J., Lengeling A. Vitamin D receptor signaling contributes to susceptibility to infection with Leishmania major. FASEB J. 2007;21(12):3208–18. [PubMed: 17551101]
5.
Kuhn N.Z., Tuan R.S. Regulation of stemness and stem cell niche of mesenchymal stem cells: implications in tumorigenesis and metastasis. J Cell Physiol. 2010;222(2):268–77. [PubMed: 19847802]
6.
Bachoud-Levi A.C. Neural grafts in Huntington's disease: viability after 10 years. Lancet Neurol. 2009;8(11):979–81. [PubMed: 19833293]
7.
Himmelreich U., Dresselaers T. Cell labeling and tracking for experimental models using magnetic resonance imaging. Methods. 2009;48(2):112–24. [PubMed: 19362150]
8.
Hoehn M., Wiedermann D., Justicia C., Ramos-Cabrer P., Kruttwig K., Farr T., Himmelreich U. Cell tracking using magnetic resonance imaging. J Physiol. 2007;584(Pt 1):25–30. [PMC free article: PMC2277052] [PubMed: 17690140]
9.
Arbab A.S., Janic B., Haller J., Pawelczyk E., Liu W., Frank J.A. In Vivo Cellular Imaging for Translational Medical Research. Curr Med Imaging Rev. 2009;5(1):19–38. [PMC free article: PMC2746660] [PubMed: 19768136]
10.
Loebinger M.R., Kyrtatos P.G., Turmaine M., Price A.N., Pankhurst Q., Lythgoe M.F., Janes S.M. Magnetic resonance imaging of mesenchymal stem cells homing to pulmonary metastases using biocompatible magnetic nanoparticles. Cancer Res. 2009;69(23):8862–7. [PMC free article: PMC2833408] [PubMed: 19920196]

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