RESUMEN
PURPOSE: Mesenchymal stem cells (MSC) seem to be a promising cell source for cellular cardiomyoplasty. We recently developed a new aptamer-based specific selection of MSC to provide "ready to transplant" cells directly after isolation. We evaluated MRI tracking of newly isolated and freshly transplanted MSC in the heart using one short ex vivo selection step combining specific aptamer-based isolation and labeling of the cells. MATERIALS AND METHODS: Bone marrow (BM) was collected from healthy pigs. The animals were euthanized and the heart was placed in a perfusion model. During cold ischemia, immunomagnetic isolation of MSC from the BM by MSC-specific aptamers labeled with Dynabeads was performed within 2 h. For histological identification the cells were additionally stained with PKH26. Approx. 3 x 10(6) of the freshly aptamer-isolated cells were injected into the ramus interventricularis anterior (RIVA) and 5 x 10(5) cells were injected directly into myocardial tissue after damaging the respective area by freezing (cryo-scar). 3 x 10(6) of the aptamer-isolated cells were kept for further characterization (FACS and differentiation assays). 20 h after cell transplantation, MRI of the heart using a clinical 3.0 Tesla whole body scanner (Magnetom Trio, Siemens, Germany) was performed followed by histological examinations. RESULTS: The average yield of sorted cells from 120 ml BM was 7 x 10(6) cells. The cells were cultured and showed MSC-like properties. MRI showed reproducible artifacts within the RIVA-perfusion area and the cryo-scar with surprisingly excellent quality. The histological examination of the biopsies showed PKH26-positive cells within the areas which were positive in the MRI in contrast to the control biopsies. CONCLUSION: Immunomagnetic separation of MSC by specific aptamers linked to magnetic particles is feasible, effective and combines a specific separation and labeling technique to a "one stop shop" strategy.
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Aptámeros de Nucleótidos , Cardiomioplastia , Imagen por Resonancia Magnética , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Animales , Células de la Médula Ósea , Cardiomioplastia/métodos , Separación Celular , Estudios de Factibilidad , Colorantes Fluorescentes , Separación Inmunomagnética , Células Madre Mesenquimatosas/citología , Isquemia Miocárdica , Compuestos Orgánicos , Coloración y Etiquetado , Porcinos , Factores de TiempoRESUMEN
PURPOSE: To evaluate advantages and limitations of magnetic resonance imaging (MRI) to monitor the migration of superparamagnetic iron oxide (SPIO) labeled cells in the chick embryo. MATERIALS AND METHODS: Labeled human SK-Mel 28 melanoma cells were injected into the E2 chick embryo neural tube. Embryos were examined with a clinical 3 T MRI whole body system using 3D T*(2)-weighted sequences with isotropic spatial resolutions of 0.3-1.0 mm. MR-measurements of embryos were performed 2 - 16 days after cell injection. MRI findings were verified by dissection and histology. RESULTS: After injection, melanoma cells formed aggregations that were detectable in the neural tube as signal voids in MR images from day 2 after injection. Emigrating cells later left MRI detectable tracks. Aggregates that remained in the neural tube left label that was absorbed by glia cells. In E18 chick embryos, signals of haematopoiesis interfered with signals from cell labeling. CONCLUSION: It was shown that SK-Mel 28 cells will resume the neural crest pathways after injection into the embryonic micro-environment. SPIO cell labeling allows monitoring of transplanted melanoma cells during embryonic development. MRI using the standard clinical equipment promises to be valuable for high-sensitive monitoring of ex-vivo labeled cells in the chick embryo.
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Compuestos Férricos , Imagen por Resonancia Magnética , Melanoma Experimental/patología , Imagen de Cuerpo Entero , Animales , Línea Celular Tumoral , Embrión de Pollo , HumanosRESUMEN
In the current study the effect of increasing concentrations of superparamagnetic iron oxide labeled cells on the MRI signal decay at magnetic field strengths of 0.2, 1.5, and 3 T was evaluated. The spin echo and gradient echo cellular transverse relaxivity was systematically studied for various concentrations (N = 1, 5, 10, 20, 40, and 80 cells/microl(gel)) of homogeneously suspended SH U 555A labeled SK-Mel28 human melanoma cells. For all field strengths investigated a linear relationship between cellular transverse relaxation enhancement and cell concentration was found. In the spin echo case, the cellular relaxivities [i.e., d(deltaR2)/dN] were determined to 0.12 s(-1) (cell/microl)(-1) at 0.2 T, 0.16 s(-1) (cell/microl)(-1) at 1.5 T, and 0.17 s(-1) (cell/microl) at 3 T. In the gradient echo case, the calculated cellular relaxivities (i.e., d(deltaR2*)/dN) were 0.51 s(-1) (cell/microl)(-1) at 0.2 T, 0.69 s(-1) (cell/microl)(-1) at 1.5 T, and 0.71 s(-1) (cell/microl)(-1) at 3 T. The proposed preparation technique has proven to be a simple and reliable approach to quantify effects of magnetically labeled cells in vitro. On the basis of this quantification well suited tissue specific models can be derived.
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Imagen Eco-Planar , Procesamiento de Imagen Asistido por Computador , Hierro , Imagen por Resonancia Magnética , Óxidos , Artefactos , Calibración , Células Cultivadas , Medios de Contraste , Dextranos , Compuestos Férricos/química , Óxido Ferrosoférrico , Humanos , Nanopartículas de Magnetita , Melanoma/patología , Coloración y EtiquetadoRESUMEN
UNLABELLED: PURPOSE. This work aims to present a preparation technique for ex-vivo MR examination of SPIO (superparamagnetic iron oxide) containing solutions or SPIO labeled cells. Accumulations of SPIO particles and labeled cells were prepared in different concentrations using agar gel phantoms. Signal extinction around accumulations of magnetic material was examined systematically by gradient echo sequences with variable echo times and spatial resolution. The correlation between local iron concentration and diameter of signal extinction in MR gradient echo images was investigated. METHODS: Resovist, (SHU 555A) was used as superparamagnetic contrast medium. Different concentrations of SPIO-containing solutions (0.75 - 15 mg Fe/10 ml) and magnetically labeled SK-Mel28 cells (25,000-1,000,000 cells/10 ml) were accommodated inside a defined volume in an agar matrix. Diameters of signal void were assessed in dependence on local iron concentration, echo time (5-25 ms) and isotropic spatial resolution (length of voxel 0.25 - 0.60 mm). Measurements were performed on a clinical MR whole body scanner (3 Tesla) using a spoiled gradient echo sequence (FLASH). RESULTS: For the present experimental conditions sensitivity to detect the magnetic label was maximized using TE 25 ms. In contrast, the area of signal cancellation was minimized using TE 5 ms and isotropic resolution of 0.25 mm. In the latter case the image indicated the area of magnetic material most precisely. Diameter of signal cancellation was a logarithmic function on local iron concentration. In the presented set-up detection of concentrations as low as 0.75 mg Fe/10 ml in SPIO-containing solution or 1.25 mg Fe/10 ml in SPIO-labeled SK-Mel28 cells was certainly possible. CONCLUSION: The proposed preparation strategy with a well defined spatial distribution of the magnetic material in an agar gel phantom produced reliable results and appears clearly superior compared to set-ups with randomly distributed material in glass tubes. The diameter of the signal extinction in gradient echo images was significantly affected by the choice of echo time and spatial resolution. The calibration of signal cancellation versus iron concentrations may be valuable to assess SPIO concentrations and possibly numbers of labeled cells under specific conditions in vitro or even in vivo.
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Mezclas Complejas/análisis , Medios de Contraste/química , Aumento de la Imagen/métodos , Hierro/química , Imagen por Resonancia Magnética/métodos , Melanoma/patología , Óxidos/química , Línea Celular Tumoral , Mezclas Complejas/química , Medios de Contraste/análisis , Medios de Contraste/síntesis química , Óxido Ferrosoférrico , Humanos , Hierro/análisis , Ensayo de Materiales , Óxidos/análisis , Óxidos/síntesis química , Soluciones , Coloración y Etiquetado/métodosRESUMEN
PURPOSE: To evaluate the effect of dexamethasone on the growth of cultured human aortic smooth muscle cells in an in-vitro model depending on the dose applied. MATERIALS AND METHODS: Commercially available human aortic smooth muscle cells (haSMC) were incubated with different doses of dexamethasone (10(-6), 10(-8), 10(-10) mol/l). For 20 days, the dose-depending effects of dexamethasone on cell growth were studied by analyzing cell proliferation, clonogenic activity as well as cell cycle distribution. In addition, the migratory ability of haSMC was evaluated using a two compartment in-vitro model. RESULTS: Cell growth was reduced in a dose dependent manner. An applied dose of 10(-6) M dexamethasone effectively inhibited cell growth for the follow-up period of 20 days. Cell cycle analysis revealed a G1-phase block which was dose dependent and significant for a dose of 10(-6) M. Also a reduction of haSMC clonogenic activity could be found in the colony formation assays. Finally, dexamethasone reduced the migratory ability of the treated cells significantly for doses of 10(-6) and 10(-8) M. CONCLUSION: Depending on the dose applied, incubation with dexamethasone results in a significant growth reduction of cultured haSMC, which may be due to a drug induced G1-phase block. Dexamethasone also reduces the clonogenic activity as well as the migratory ability of cultured haSMC.
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Angioplastia de Balón , Antiinflamatorios/farmacología , Aorta/efectos de los fármacos , Constricción Patológica/prevención & control , Dexametasona/farmacología , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Stents , Antiinflamatorios/administración & dosificación , Aorta/citología , Arteriosclerosis/prevención & control , Ciclo Celular , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Medios de Cultivo , Dexametasona/administración & dosificación , Estudios de Seguimiento , Oclusión de Injerto Vascular , Humanos , Hiperplasia , Recurrencia , Factores de Tiempo , Túnica Íntima/patologíaRESUMEN
PURPOSE: Thrombomodulin (TM), an integral endothelial receptor, is known for its anticoagulant functions. Moreover, there is evidence of growth-modulating effects of this cell surface -protein. The aim of our study was to establish by in vitro transfection a stable cell line of vascular smooth muscle cells with overexpression of TM for further investigations concerning the influence of TM on cellular proliferation and its potential role during the formation of restenosis. METHODS: Aortic smooth muscle cells of the rat were transfected with cDNA of mouse TM or one of its three mutants (M1, M2, M4) by a liposome-mediated technique. The expression of mouse TM mRNA in the selected clones was proven with the help of RT-PCR. Changes of cell proliferation were determined by proliferation kinetics over 24 days. The quantification of the total protein TM was made by Western blots. RESULTS: In 44 of 100 cases the RT-PCR confirmed a successful transfection of mouse-TM. The clones with transfected TM, M1 or M2 showed an inhibited cell growth, whereas M4 demonstrated an increased proliferation compared with controls. The comparison of amounts of total TM with cell growth of individual clones resulted in a negative correlation between proliferation and TM-expression (coefficient of correlation for TM -0.87, for M1 -0.59). CONCLUSIONS: It is possible to reproduce stable cell-lines of vascular smooth muscle cells with overexpression of TM by the presented model of in vitro transfection. Thus, a basis exists for detailed examinations of growth-regulating mechanisms by TM.
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Constricción Patológica/prevención & control , Terapia Genética , Músculo Liso Vascular/citología , Trombomodulina/genética , Transfección , Análisis de Varianza , Angioplastia de Balón , Animales , Aorta/citología , Western Blotting , Recuento de Células , División Celular , Línea Celular , Medios de Cultivo , Replicación del ADN , ADN Complementario/genética , Expresión Génica , Cinética , Liposomas , Ratones , ARN Mensajero/análisis , Ratas , Recurrencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
PURPOSE: To evaluate the dose of (188)Re that completely suppresses growth and clonogenic activity of human aortic smooth muscle cells (haSMC) since these cells are mainly responsible for restenosis occurring after PTA. For comparison, growth and clonogenic activity of endothelial cells (EC) were investigated with corresponding doses. MATERIALS AND METHODS: Two days after plating, haSMC and EC were incubated with (188)Re for five days. The doses applied ranged from 4 to 16 Gy. Cell growth was observed for a period of 20 days (EC) or 30 days (haSMC), respectively. Clonogenic activity was monitored over a period of 20 days for both cell lines. RESULTS: Irradiation caused dose-depend-ent inhibition of cell growth and clonogenic activity both in haSMC and in EC. HaSMC growth was completely blocked with 8 Gy, while EC still showed some proliferation even with 16 Gy. The clonal activity of haSMC was also completely blocked with 8 Gy while EC still showed little clonal activity even with 16 Gy. CONCLUSION: Cell growth of both haSMC and EC can be effectively suppressed in a dose-dependent manner. Only haSMC showed a complete growth arrest with 8 Gy while EC were able to proliferate even with 16 Gy. HaSMC colony formation was completely suppressed after application of 8 Gy, while the EC still showed colony formation activity with 16 Gy. (188)Re has some advantageous properties for intravascular irradiation in comparison to other radionuclides making it an interesting radionuclide for stent coating to prevent restenosis.
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Angioplastia de Balón , Células Endoteliales/efectos de la radiación , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de la radiación , Radioisótopos/farmacología , Renio/farmacología , Stents , Aorta/efectos de la radiación , División Celular/efectos de la radiación , Línea Celular , Ensayo de Unidades Formadoras de Colonias , Constricción Patológica/prevención & control , Relación Dosis-Respuesta en la Radiación , Células Endoteliales/citología , Humanos , Músculo Liso Vascular/crecimiento & desarrollo , Dosis de Radiación , Recurrencia , Factores de TiempoRESUMEN
PURPOSE: To evaluate dose-dependent growth-modulating effects of the beta-gamma emitter Rhenium-188 on cultured human aortic smooth muscle cells (haSMC). METHODS AND MATERIALS: HaSMC were plated in 25 cm(2) flasks. Two days after plating, cells were incubated with the Re-188 (beta E(max) 2.12 MeV, tissue range(max) < 10 mm, T(1/2) 17 h) for five days. The doses administered were 0.2 Gy, 1, 4, 6, 8, 16, and 32 Gy. After five days, the radionuclide was removed. Cell growth, cell cycle distribution, and clonogenic activity were analyzed for the following 25 days. RESULTS: The 0.2 and 1 Gy groups did not show relevant growth-inhibiting effects compared to the control groups. The 4 to 32 Gy groups presented dose-dependent growth inhibition, with a complete growth arrest of the 16 and 32 Gy groups. Clonogenic activity of the smooth muscle cell was strongly inhibited from doses > or =8 Gy. Flow cytometry showed a lasting dose-dependent G2/M phase block. CONCLUSION: Smooth muscle cell (SMC) growth can be controlled effectively with Re-188 for at least 25 days after radiation in vitro. As the first four weeks after arterial angioplasty are crucial concerning neointimal formation, Re-188 may be a valuable radionuclide to inhibit restenosis after arterial angioplasty.
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Aorta/efectos de la radiación , División Celular/efectos de la radiación , Músculo Liso Vascular/efectos de la radiación , Radioisótopos/farmacología , Renio/farmacología , Aorta/citología , Relación Dosis-Respuesta a Droga , Humanos , Interfase/efectos de la radiación , Músculo Liso Vascular/citología , RadiobiologíaRESUMEN
PURPOSE: The aim of this study was to evaluate the capability of human aortic smooth musc e cells (HaSMC) and endothelial cells (EC) to recover after incubation with the combined beta/gamma emitter 186rhenium. MATERIALS AND METHODS: Two days after plating, HaSMC and EC were incubated for five days with 186Re (total doses applied 4 Gy-32 Gy). Cell counts were performed for a period of 30 days (haSMC) and 22 days (EC). To detect possible growth recovery, colony formation assays were plated for both cell types on day 5, 10, and 20 (and lay 30 for haSMC). RESULTS: Both cell types presented a dose-dependent growth inhibition which was maximum at a dose of 32 Gy. Human endothelial cells presented with total growth recovery at 4 and 8 Gy, and a partial growth recovery at 16 Gy. Smooth muscle cells only presented partial growth recovery at 4 and 8 Gy. At 16 Gy and more no recovery was detected. CONCLUSION: HaSMC as well as EC growth can be modulated effectively with 186Re over a period of 30 days in vitro. Compared to smooth muscle cells human endothelial cellls seem to possess a higher potential to recover at doses of 8 to 16 Gy. 186Re may be a valuable radionuclide to prevent restenosis.
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Endotelio Vascular/citología , Endotelio Vascular/efectos de la radiación , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de la radiación , Radioisótopos/farmacología , Renio/farmacología , Angioplastia de Balón , Angioplastia Coronaria con Balón , Aorta/citología , Recuento de Células , División Celular , Células Cultivadas , Humanos , Dosis de Radiación , Recurrencia , Factores de TiempoRESUMEN
The purpose of this study was to evaluate the influence on fetal cell growth in vitro of rapidly changing magnetic gradient fields such as those produced by the gradient coils of a typical magnetic resonance (MR) imager. The static magnetic field and the radiofrequency pulses were disabled during all measurements. Human fetal fibroblasts were placed within a specially designed MR-compatible incubation system inside the magnet. Trapezoid-shaped waveforms of 500 and 75 Hz base frequency and an amplitude of 2 mT were applied for 2-24 hours. Proliferation of the cells was monitored for 3 weeks after exposure. Cell cycle analysis was performed until 24 hours after exposure to detect alterations in cell division. Tests were performed under two different conditions of growth to detect increased as well as decreased proliferation effects. None of these tests showed differences in proliferation and cell cycle distribution between exposed and nonexposed cells.
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Anomalías Inducidas por Radiación/etiología , Ciclo Celular/efectos de la radiación , Campos Electromagnéticos/efectos adversos , Fibroblastos/efectos de la radiación , Imagen por Resonancia Magnética/efectos adversos , Muerte Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Estudios de Evaluación como Asunto , Feto , Humanos , Pulmón/citología , Pulmón/embriología , Persona de Mediana Edad , Probabilidad , Sensibilidad y Especificidad , Estadísticas no ParamétricasRESUMEN
PURPOSE: To evaluate whether repetitive exposure to magnetic fields of 0.2, 1.0, and 1.5 T affect the growth of human fetal lung fibroblasts (HFLFs). MATERIALS AND METHODS: Cultured HFLFs were exposed to static magnetic fields of 0.2, 1.0, and 1.5 T for 1 h/d for 5 consecutive days. Control groups were kept under identical environmental conditions, apart from the magnetic field, during the experiments. Cell cycle analysis for synchronously and nonsynchronously growing cells was performed. Population doublings (PDs) were calculated. To rule out midterm effects, proliferation kinetics of the cells were analyzed for 21 days. RESULTS: Cell cycle analysis of synchronized and nonsynchronized cells did not reveal statistically significant differences between the exposed and control cells. The PDs did not indicate any growth modulation during exposure. Proliferation kinetics did not provide any hint of midterm growth modulation effects of repetitive magnetic field exposure. CONCLUSION: Repetitive magnetic field exposure does not exert any growth-modulating effect on overall cell growth and cell cycle distribution of cultured HFLFs. Midterm effects due to magnetic field exposure were not found.
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Pulmón/citología , Magnetismo/efectos adversos , Ciclo Celular , Células Cultivadas , Feto , Fibroblastos/citología , Humanos , Imagen por Resonancia Magnética/instrumentación , Factores de TiempoRESUMEN
The aim of the study was to assess the effects of repetitive exposures to a static magnetic field (1.5 T) on human fetal lung fibroblast (HFL) proliferation. HFL were exposed three times a week for 1 hr to a static magnetic field for 3 weeks. Cells were subcultured every week. Population doublings (PD) and cumulative population doublings (CPD) were calculated weekly. Colony formation assays, bromodeoxyuridine enzyme-linked immunosorbent assay, and cell cycle analysis were performed weekly. After the third week, proliferation kinetics were assessed. Over a period of 3 weeks no statistically significant differences between the PD and CPD of exposed and control cells could be detected. Clonogenic activity, DNA synthesis, cell cycle, and proliferation kinetics were not altered by magnetic field exposure. The data do not provide evidence that repetitive exposures to a static magnetic field (1.5 T) exert effects on HFL proliferation.
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Campos Electromagnéticos/efectos adversos , Fibroblastos/efectos de la radiación , Pulmón/embriología , Imagen por Resonancia Magnética/efectos adversos , División Celular/efectos de la radiación , Células Cultivadas/efectos de la radiación , Feto/efectos de la radiación , Fibroblastos/citología , Humanos , Pulmón/anatomía & histología , Pulmón/citología , Imagen por Resonancia Magnética/métodos , Valores de Referencia , Estadísticas no Paramétricas , Factores de TiempoRESUMEN
PURPOSE: In XeCl-Excimer laser angioplasty, unintended and possibly harmful interaction of the UV-laser light and the contrast media may occur due to the high concentration of contrast medium proximal to the occlusion or subtotal stenosis. METHODS: One ml of three nonionic monomeric contrast agents (iopromide, iomeprol, iopamidol), one nonionic dimeric (jotrolane), and one ionic monomeric (amidotrizoate) X-ray contrast agent were irradiated with a XeCl excimer laser (lambda = 308 nm, pulse duration 120 ns, 50 Hz) using a 9 French multifiber catheter (12 sectors). Up to 20,000 pulses (106 J) were applied. Using high performance liquid chromatography the amount of liberated iodide as well as the fraction of unchanged contrast media were measured. Cytotoxicity of the photoproducts was tested in a colony formation assay of human skin fibroblasts. The contrast agents were irradiated with 2000 pulses/ml (5.3 mJ/pulse; 10.6 J) and then added to the cell cultures for a period of three hours in a concentration of 10%. RESULTS: Excimer laser irradiation induced iodide liberation of up to 3.3 mg iodide/ml. Up to 19% of the contrast agents changed their original molecular structure. Incubation of irradiated contrast agents resulted in a significantly decreased potential for colony formation (p values ranging from 0.0044 to 0.0102) with significantly higher toxicity of amidotrizoate and iomeprol in comparison to iopromide, iotrolan, and iopamidol. DISCUSSION: Due to the cytotoxic photoproducts and the high level of liberated iodide, it is recommended to flush the artery with physiological saline solution before applying a pulsed excimer laser in human arterial obstructions in order to reduce the contrast agent concentration at the site of irradiation.