Intracellular Magnetic Labeling of Lymphocytes for In Vivo Trafficking Studies

It is necessary to note that although there is a considerable number (several thousands) of publications regarding the use and role (presupposed) of bone marrow mononuclear cells in the therapy of different diseases (Futterman and Lemberg, 2004;Uzan, 2004) and reparation of posttraumatic lesions, we have been unable find information on their morphology. References such as "identification of morphology, size and shape is difficult" were mentioned in some papers (Dalrup-Link et al., 2003). Therefore the description of the morphology of this cell type is the aim of our study. These cells will be put to morphometric measurement, and their distribution whithin different regions of experimental myocardial infarction in the next phase of the experiment. MATERIAL AND METHODS Animal modelRabbits (Hy-Plus, weight 2.5 to 3.5 kg) were used as an experimental animal model. Experimental protocol was approved by the Ethics Committee of University of Veterinary Medicine, Brno. Bone marrow collectionMononuclear cells were harvested from rabbit bone marrow on day -14, before the ligation of the coronary artery. Bone marrow was obtained by multiple aspiration from iliac bone into heparin flushed syringes under general anesthesia. Samples were collected into tubes containing heparin and 0.9%NaCl (1:1) under sterile conditions. A sufficient amount of bone marrow was collected in order to obtain the required numbers of enriched cells (approximately 5-10 ml). Selection of mononuclear cellsMononuclear cells were enriched from bone marrow by density gradient centrifugation. Bone marrow was diluted with Hanks/4% albumin solution (1:1) and overlaid on a sterile density medium ABSTRACT: Mononuclear cells from rabbit bone marrow were cultured for 14 days in cell-free medium for hematopoietic cells together with iron oxid nanoparticles, and then they were processed by technique for free cells for TEM (transmission electron microscopy). Staining with turnbull blue was used for the detection of iron using a light microscope. It was shown that iron nanoparticles were incorporated into the cytoplasm of mononuclear cells during 14 days cultivation. Here they were localized within different sized vacuoles with distinct membranes. Appearance of iron-labeled blood mononuclear cells in electron microscopy

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“…Schoepf et al (1998) and Lewin et al (2000) reached similar conclusions concerning the viability of blood cells labeled with iron.…”

supporting

confidence: 55%

Appearance of iron-labeled blood mononuclear cells in electron microscopy

Horký¹,

Lauschová²,

Klabusay³

et al. 2006

Vet. Med. - Czech

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“…The integral solution to Eq. [6] predicts an exponential reduction in the fractional signal strength for voxels containing multiple magnetic particles as follows:…”

Section: Shell Model For Multiple Magnetic Particlesmentioning

confidence: 99%

Relaxometry model of strong dipolar perturbers for balanced‐SSFP: Application to quantification of SPIO loaded cells

Menon

Bowen

2006

Magnetic Resonance in Med

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Magnetic resonance microscopy using magnetically labeled cells is an emerging discipline offering the potential for nondestructive studies targeting numerous cellular events in medical research. The present work develops a technique to quantify superparamagnetic iron-oxide (SPIO) loaded cells using fully balanced steady state free precession (b-SSFP) imaging. An analytic model based on phase cancellation was derived for a single particle and extended to predict mono-exponential decay versus echo time in the presence of multiple randomly distributed particles. Numerical models verified phase incoherence as the dominant contrast mechanism and evaluated the model using a full range of tissue decay rates, repetition times, and flip angles. Numerical simulations indicated a relaxation rate enhancement (⌬R 2b ‫␥214.0؍‬ ⅐ LMD) proportional to LMD, the local magnetic dose (the additional sample magnetization due to the SPIO particles), a quantity related to the concentration of contrast agent. Key words: steady state free precession; magnetic susceptibility; contrast agent; iron-oxide; relaxometry; magnetic resonance microscopy Magnetic resonance microscopy using magnetically labeled cells has been used to study a variety of cellular events in medical research including stem cell (1-4) and immune cell (5,6) migration, brain ischemia (7,8), and cancer (9 -11). One approach uses high resolution MRI to detect either single (12,13) or groups of cells labeled with paramagnetic contrast agents, such as superparamagnetic iron-oxide (SPIO). Cell labeling using this strategy has been accomplished through both in vitro and in vivo approaches. In vitro labeling involves incubation of a desired cell line in culture with SPIO prior to i.v. (1,5) or tissuespecific injection (2,3), while in vivo (or active) labeling is accomplished through i.v. injection of SPIO particles that are preferentially taken up by a target cell population (14,15). For the vast majority of cell types and with each labeling technique, SPIO agents have been shown to be minimally disruptive to cell function (16) with few exceptions (17), and show great promise for revealing cellular contributions to early stages of pathology (7), as well as the therapeutic effectiveness of stem cell homing in regenerative medicine (2,3).To date, the majority of imaging studies involve a qualitative assessment of the hypo-or hyper-intensities observed in images of tissues containing SPIO-labeled cells. Quantitative measures of cellular migration may allow assessment of the relative effectiveness of pharmacological interventions, stem cell homing, or changes in the intensity of inflammatory response that are typically associated with the progression of pathology. Detection of SPIO labeled cells has been accomplished through T 1 (9,18), T 2 (19,20) and TЈ 2 -weighted (21-23) acquisitions. TЈ 2 -weighted gradient echo acquisitions provide the greatest sensitivity to the presence of SPIO; however, these measurements are also sensitive to background field inhomogeneities induced by im...

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“…However, MR cell tracking techniques are restricted by a limited sensitivity, which requires highly efficient labeling techniques in order to load up the investigated cells with high amounts of contrast agent particles. Such optimized labeling methods have been developed before for various hematopoietic cell populations, including lymphocytes, monocytes and hematopoietc progenitor cells (17,(20)(21)(22)(23)(24)(25)(26). For non-phagocytic cell types, such labeling techniques required transfection, i.e.…”

Section: Introductionmentioning

confidence: 99%

“…For non-phagocytic cell types, such labeling techniques required transfection, i.e. internalization of contrast agents into the cells with the help of liposomes, proteins or viral vectors (17,(20)(21)(22)(23)(24)(25). Genetically engineered lymphocytes already underwent a transfection process in order to shuttle new genetic information into the cells.…”

Section: Introductionmentioning

confidence: 99%