Este trabajo fue apoyado por una subvención del Instituto Nacional de Artritis y Enfermedades Musculoesqueléticas y de la piel: 3R01AR054458-02S2.

1. Día 1 1) Las células Plate <ol><li> Placa hMSC en un matraz T75 en una confluencia del 80% por lo menos 18-24 horas antes de la etiqueta. Consulte la Tabla 1 para la instrucción de las embarcaciones alternativa. </li></ol> 2. Día 2 2) Prepare una solución de etiquetado. Esta preparación etiqueta de un (1) T75 matraz a 80% de confluencia con una concentración de 400 mg Fe / ml. Consulte la Tabla 1 para la instrucción de las embarcaciones alternativa. <ol><li> Crear una solución (solución 1) mediante la mezcla de 1 ml de medio libre de suero y 93,1 l de ferumoxytol (stock: 30 mg / ml) en un tubo cónico de 15 ml. </li><li> Crear una segunda solución (solución 2) mediante la mezcla de 1 ml de medio libre de suero y 7 l de sulfato de protamina (stock: 10 mg / ml) en un tubo de 15 ml segunda cónica. </li><li> Permita que cada solución reposar por 5 minutos. </li><li> Agregar las soluciones 1 y 2 juntos. Mezclar cuidadosamente la solución de un nuevo etiquetado. Permita que la solución reposar por 5 minutos para permitir USPIO pro-complejos de sulfato de histamina a la forma. </li><li> Añadir 5 ml de medio libre de suero a la mezcla de 2 ml de SPIO / solución de sulfato de protamina para un volumen final de 7 ml. </li></ol> 3) Preparación de las células de etiquetado <ol><li> Aspirar los medios de comunicación de las células. </li><li> Lave suavemente las células una vez con 3.2 ml de pre-calentado Mg / Ca libre de D-PBS o medio libre de suero para limpiarla de las proteínas del suero residual y otros medios de comunicación o de los componentes que puedan perjudicar la absorción del agente de contraste y etiquetado de eficiencia. Aspirar el líquido de enjuague. </li></ol> 4) las células de etiquetas <ol><li> Agregue el total de 7 ml de solución de etiquetado para las células. </li><li> Células de lugar en una incubadora (37 ° C / 5% CO 2) y permitir que las células se incuban en la solución de etiquetado durante 4 horas. </li><li> Añadir 700 l de FCS a la solución de etiquetado de las células para lograr una concentración final de suero de 10%. Suero se añade a restablecer el ambiente enriquecido de la que las células están acostumbrados, y para ayudar a minimizing muerte de las células que podrían resultar de un cambio abrupto a la exposición de 24 horas a un medio ambiente libre de suero. </li><li> Permitir que las células se incuban con la solución de etiquetado por un período adicional de 20 horas. </li></ol> 3. Día 3 5) Preparar las células marcadas <ol><li> Aspirar la solución de etiquetado de las células. </li><li> Enjuague suavemente las células con 2-3 ml de pre-calentado Mg / Ca libre de D-PBS. Aspirar el PBS. Es importante la utilización de Mg / Ca PBS libre como el Mg y Ca perjudicará a la eficiencia de la tripsina en el siguiente paso. </li><li> Añadir 2 ml de pre-calentado tripsina 0,05% a las células y la inclinación de la botella de ida y vuelta para asegurar toda la superficie del recipiente se cubre con una fina capa de tripsina. Colocar las células en una incubadora (37 ° C / 5% CO 2) y permitir que las células en reposo durante 5-7 minutos o hasta que las células comienzan a desprenderse de la placa. Puede que sea necesario para confirmar la separación bajo el microscopio. Golpee suavemente los lados del frasco si es necesario para facilitar el desprendimiento de la superficie celular. </li><li> Agregar 4 ml de pre-calentado medio completo en el matraz para neutralizar la tripsina. Enjuague suavemente el frasco con la pipeta la tripsina / media / solución de células de arriba a abajo varias veces. Recopilar toda la solución en un tubo cónico de 15 ml. Centrifugar la solución de células a 400 RCF durante 5 minutos. </li><li> Con cuidado aspirar el sobrenadante sin perturbar el sedimento celular. Resuspender las células en 5 ml de los medios de comunicación (que contiene suero o suero libre). Centrifugar la solución de células a 400 RCF durante 5 minutos. </li><li> Repita el lavado de células descritas en el paso 5.5. En total, las células se lavan tres veces para eliminar el agente residual, sin contraste en los medios de comunicación y en la superficie de las células. </li><li> Recuento de células en este punto para alcanzar el recuento de células más precisa, como las células se pierden durante el lavado anterior. Realizar una prueba de viabilidad de las células. Las células están listas para su posterior análisis y la aplicación o experimental. La RM puede llevarse a cabo con T1-W y<html> T2-w parámetros, porque a pesar de ferumoxytol es un agente de contraste T2-w, ferumoxytol también exhibe efectos T1. Representante T2-w parámetros de resonancia magnética que se puede utilizar para la imagen ferumoxytol células marcadas son: secuencias FSE o SE_Multislice con un TR de 2000-2500 ms y un TE de 60 a 80 ms. Para adquirir una imagen T1-w, disminuya el valor de un TE o una secuencia FSE SE_Multislice o utilizar una secuencia GRE con una alta y baja TR TE. La figura 4 muestra un potencial de aplicación in vivo de imágenes etiquetadas de células madre. </li></ol> 4. Los resultados representativos: Células marcadas demostrar un efecto significativo contraste oscuro o negativo en la T2 de RM y un efecto de contraste brillo o positivo en la T1 de RM (Figura 2). Longitudinal de imágenes de RM y las pruebas de viabilidad a cabo 5 días después de etiquetado demostró que no había señal de RM y un impacto significativo sobre la viabilidad en comparación con los controles de etiqueta (datos no mostrados). jove_content &quot;&gt; Etiquetada posteriormente las células madre pueden diferenciarse en varios tipos de células o inyectado por vía intravenosa en la investigación in vivo. <img alt="Figura 1" src="/files/ftp_upload/3482/3482fig1.jpg" /> Figura 1. Esquema de la línea de tiempo para el etiquetado de las células madre con nanopartículas de óxido de hierro, ferumoxytol. <img alt="Figura 2" src="/files/ftp_upload/3482/3482fig2.jpg" /> Figura 2. Imágenes de RM sagital de las secciones transversales tubos eppendorf thrrough con células de un pellet de células. A) Sin etiquetar los controles. B) Las células marcadas T2 demostrando un importante efecto negativo o agente de contraste en T2 FSE o SE_Multislice de imagen y un efecto del agente de contraste T1 o positivo en la secuencia T1 GRE. <img alt="Figura 3" src="/files/ftp_upload/3482/3482fig3.jpg" /> Figura 3. Sagital secciones transversales de los tubos eppendorf con células en células pelpermite. Tan sólo 10.000 células ferumoxytol etiqueta se puede detectar a través de T2-w RM. <img alt="Figura 4" src="/files/ftp_upload/3482/3482fig4.jpg" /> Figura 4. RM de visualización ferumoxytol marcado con células madre inyectadas en murino ventrículos cerebrales. A) Axial, MR imagen de Barin murino sin la inyección de USPIO marcado con células madre. B) axial, C) coronal, y D) imágenes sagitales MR representa el efecto T2, contraste negativo de USPIO marcado con células madre (flechas blancas) se inyecta en el cerebro murino. <img alt="Tabla 1" src="/files/ftp_upload/3482/3482table1.jpg" /> Tabla 1. Cantidad adaptaciones para el etiquetado de las células en los vasos alternativa.

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Todos los colaboradores de este estudio no ofrece revelaciones.

La mejora de la eficacia de engraftments de células madre es fundamental para el avance de la medicina regenerativa. Una técnica de visualización no invasiva de las células madre in vivo aumenta considerablemente nuestra capacidad de entender los mecanismos que conducen a los resultados de éxito del injerto. Etiquetado magnética para la visualización de la RM, tales como el procedimiento que hemos demostrado, permite el seguimiento en vivo de las células madre con la RM. Marcadas magnéticament...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> D-MEM glucosa alta </td><td> Sigma </td><td> D5648 </td><td> O mediano plazo otra base para la línea deseada de células madre para ser utilizado </td></tr><tr><td> D-PBS (Ca + +, Mg + + libre) </td><td> GIBCO </td><td> 14190-144 </td><td></td></tr><tr><td> Tripsina-EDTA al 0,05% </td><td> Invitrogen </td><td> 25300-120 </td><td></td></tr><tr><td> De suero fetal bovino (FBS) </td><td> Hyclone </td><td> SH30071.03 </td><td></td></tr><tr><td> Ferumoxytol (Feraheme) </td><td> AMAG </td><td> 59338-0775-01 </td><td></td></tr><tr><td> Sulfato de protamina </td><td> APP Pharm. </td><td> 22930 </td><td></td></tr>
</tbody>
</table>

labeling stem cells with ferumoxytol, an fda-approved iron oxide nanoparticle

Terapias con células madre basado en ofrecer un potencial significativo para el campo de la medicina regenerativa. Sin embargo, aún queda mucho por entender sobre la cinética en vivo de las células trasplantadas. Un método no invasivo para monitorizar repetidamente trasplantado células madre in vivo que permiten a los investigadores a monitorear directamente los trasplantes de células madre y la identificación de los resultados del injerto con o sin éxito. Una amplia gama de células madre sigue siendo investigado por un sinnúmero de aplicaciones. Este protocolo se centra en tres poblaciones diferentes de células madre: embrionarias de riñón humano 293 (HEK293) las células, las células madre mesenquimales (hMSC) y células madre pluripotentes inducidas (iPS). HEK 293 células derivadas de células embrionarias de riñón en un cultivo con adenovirus 5 esquilada de ADN. Estas células se utilizan ampliamente en la investigación, ya que son de fácil cultivo, crecen rápido y son transfectadas con facilidad. hMSCs se encuentran en la médula ósea de adultos. Estas células can se replican como células indiferenciadas, manteniendo multipotencia o el potencial de diferenciarse en un número limitado de destinos celulares. hMSCs puede diferenciar a los linajes de tejidos mesenquimales, incluyendo los osteoblastos, adipocitos, condrocitos, tendones, músculos, y el estroma de médula ósea. Las células iPS se reprograman genéticamente células adultas que han sido modificadas para expresar los genes y factores similares a las propiedades de la definición de las células madre embrionarias. Estas células son pluripotentes, es decir tienen la capacidad de diferenciarse en todos los linajes de células 1. Ambos hMSCs y las células iPS han demostrado la capacidad regenerativa del tejido in vivo. La resonancia magnética (RM) junto con el uso de óxido de hierro superparamagnético (SPIO) etiquetas de nanopartículas de células han demostrado su eficacia en el seguimiento in vivo de células madre debido a la resolución anatómica microscópica cerca, un análisis de sangre mayor vida media que permite imágenes longitudinal y la alta SENSIBILIDADy para la detección de células proporcionada por imágenes de RM de las nanopartículas SPIO 2-4. Además, la RM con el uso de SPIOs es clínicamente traducible. SPIOs se componen de un núcleo de óxido de hierro con una capa superficial de dextrano, carboxidextrano o almidón que sirve para contener el núcleo de hierro biorreactivo de componentes del plasma. Estos agentes locales crear inhomogeneidades del campo magnético que conducen a una disminución de la señal en imágenes potenciadas en T2 MR 5. Por desgracia, SPIOs ya no se fabrican. De segunda generación, ultra SPIOs (USPIO), sin embargo, ofrecer una alternativa viable. Ferumoxytol (FerahemeTM) es un USPIO compone de un núcleo de magnetita no estequiométrica rodeado por una capa carboxymethylether sorbitol poliglucosa. El tamaño coloidal, las partículas de 17-30 nm ferumoxytol es determinado por la dispersión de luz. El peso molecular es de 750 kDa, y la capacidad de relajación constante en el campo 2T RM es 58,609 MM-1 seg-1 fuerza 4. Ferumoxytol fue recientemente aprobado por la FDA unas un suplemento de hierro para el tratamiento de la deficiencia de hierro en pacientes con insuficiencia renal 6. Nuestro grupo ha aplicado este agente en un &quot;off label&quot; uso para aplicaciones de etiquetado de la célula. Nuestra técnica demuestra el etiquetado eficiente de las células madre con ferumoxytol que conduce a importantes efectos de la señal de RM de las células marcadas en las imágenes de RM. Esta técnica puede ser aplicada para la monitorización no invasiva de las terapias de células madre en la configuración pre-clínicos y clínicos.

Watch this Scientific Journal Video about Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle at JoVE.com

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Se describe una técnica para el etiquetado y el seguimiento de las células madre aprobados por la FDA, superparamagnéticas de óxido de hierro (SPIO), ferumoxytol (Feraheme). Esta técnica de imagen celular que utiliza la resonancia magnética (RM) para la visualización, es fácilmente accesible para su seguimiento a largo plazo y el diagnóstico de engraftments células madre con o sin éxito en los pacientes.

Etiquetado de las células madre con Ferumoxytol, uno de nanopartículas de hierro aprobada por la FDA de óxido

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

labeling-stem-cells-with-ferumoxytol-an-fda-approved-iron-oxide

Research

JoVE Journal

Medicine

20.7K vistas.  Molecular Imaging Program at Stanford (MIPS). Se describe una técnica para el etiquetado y el seguimiento de las células madre aprobados por la FDA, superparamagnéticas de óxido de hierro (SPIO), ferumoxytol (Feraheme). Esta técnica de imagen celular que utiliza la resonancia magnética (RM) para la visualización, es fácilmente accesible para su seguimiento a largo plazo y el diagnóstico de engraftments células madre con o sin éxito en los pacientes.

Video: Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Diferenciación de aislamiento, caracterización y comparativo de las células madre derivadas de la Pulpa Dental de los dientes permanentes mediante dos métodos diferentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Investigación

Medicina

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

Sistema Coculture con un cerebro rebanada organotípicos y 3D esferoide de células de carcinoma

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

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.

El estudio de cáncer de páncreas Tallo Características de las células para el desarrollo de nuevas estrategias de tratamiento

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

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Inducida por la forskolina Hinchazón en organoides intestinal: Una<em&gt; In Vitro</em&gt; Ensayo para evaluar la respuesta de drogas en pacientes con fibrosis quística

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Continuous Manual Exchange Transfusion for Patients with Sickle Cell Disease: An Efficient Method to Avoid Iron Overload

Children with sickle cell anemia (SCA) may be at risk of cerebral vasculopathy and strokes, which can be prevented by chronic transfusion programs. Repeated transfusions of packed red blood cells (PRBCs) is currently the simplest and most used technique for chronic transfusion programs. However, iron overload is one of the major side effects of this therapy. More developed methods exist, notably the apheresis of RBC (erythrapheresis), which is currently the safest and most efficient method. However, it is costly, complicated, and cannot be implemented everywhere, nor is it suitable for all patients. Manual exchange transfusions combine one or more manual phlebotomies with a PRBC transfusion.
At the Reference Center of Sickle Cell Disease, we set up a continuous method of manual exchange transfusion that is feasible for all hospital settings, demands no specific equipment, and is widely applicable. In terms of HbS decrease, stroke prevention, and iron overload prevention, this method showed comparable efficiency to erythrapheresis. In cases where erythrapheresis is not available, this method can be a good alternative for patients and care centers.

We have outlined a method of continuous manual exchange transfusion for the treatment of sickle cell disease in patients. This safe protocol was designed to effectively limit iron overload in patients in need of chronic transfusions and can be used extensively without any special equipment.

Transfusión manual continua para pacientes con enfermedad de células falciformes: un método eficaz para evitar la sobrecarga de hierro

Children with sickle cell anemia (SCA) may be at risk of cerebral vasculopathy and strokes, which can be prevented by chronic transfusion programs. ...

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.

To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.

Síntesis, caracterización y aplicación de nanosondas de óxido de hierro superparamagnético sin empleo para la detección de tuberculosis extrapulmonar

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

Nanopartículas de óxido de hierro recubierto de polietilamina como vehículo para la entrega de ARN interferente pequeño a los macrófagos In Vitro e In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

Un método libre de enzimas para el aislamiento y la expansión de las células madre mesenquimales derivadas de adiposo humano

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical hurdles such as invasive cell delivery and tracking with low transplantation efficiency remain challenges in translational stem-based therapy. This article describes an emerging technique for stem cell labeling and tracking based on the labeling of the mesenchymal stem cells (MSCs) with superparamagnetic iron oxide (SPIO)&#160;nanoparticles, as well as intranasal delivery of the labeled MSCs. These nanoparticles are fluorescein isothiocyanate (FITC)-embedded and safe to label the MSCs, which are subsequently delivered to the brains of TBI-induced mice by the intranasal route. They are then tracked non-invasively in vivo by real-time magnetic resonance imaging (MRI). Important advantages of this technique that combines SPIO for cell labeling and intranasal delivery include (1) non-invasive, in vivo MSC tracking after delivery for long tracking periods, (2) the possibility of multiple dosing regimens due to the non-invasive route of MSC delivery, and (3) possible applications to humans, owing to the safety of SPIO, non-invasive nature of the cell-tracking method by MRI, and route of administration.

Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide&#160;nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

Seguimiento de células madre mesenquimales con etiqueta de óxido de hierro superparamagnético mediante RMN después de la entrega intranasal en un modelo de murino de lesión cerebral traumática

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...

Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) crystals. Crystalluria occurs when urine becomes supersaturated with minerals (e.g., calcium, oxalate, phosphate) and precedes kidney stone formation. Standard methods to assess crystalluria in stone formers include microscopy, filtration, and centrifugation. However, these methods primarily detect microcrystals and not nanocrystals. Nanocrystals have been suggested to be more harmful to kidney epithelial cells than microcrystals in vitro. Here, we describe the ability of Nanoparticle Tracking analysis (NTA) to detect human urinary nanocrystals. Healthy adults were fed a controlled oxalate diet prior to drinking an oxalate load to stimulate urinary nanocrystals. Urine was collected for 24 hours before and after the oxalate load. Samples were processed and washed with ethanol to purify samples. Urinary nanocrystals were stained with the calcium binding fluorophore, Fluo-4 AM. After staining, the size and count of nanocrystals were determined using NTA. The findings from this study show NTA can efficiently detect nanocrystalluria in healthy adults. These findings suggest NTA could be a valuable early detection method of nanocrystalluria in patients with kidney stone disease.

The objective of this study was to determine whether nanoparticle tracking analysis (NTA) could detect and quantify urinary calcium containing nanocrystals from healthy adults. The findings from the current study suggest NTA could be a potential tool to estimate urinary nanocrystals during kidney stone disease.

Estimación De Nanocristales Urinarios En Humanos Usando Etiquetado De Fluoróforo De Calcio Y Análisis De Seguimiento De Nanopartículas

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) ...