Ce travail a été soutenu par une subvention du National Institute of Arthritis and musculo-squelettiques et les maladies cutanées: 3R01AR054458-02S2.

1. Jour 1 Cellules de la plaque 1) <ol><li> HMSC plaque dans une fiole T75 à un confluence de 80% au moins 18-24 heures avant l&#39;étiquetage. Se reporter au Tableau 1 pour les instructions pour les navires de remplacement. </li></ol> 2. Jour 2 2) Préparer une solution d&#39;étiquetage. Cette préparation d&#39;étiquettes un (1) flacon T75 à 80% de confluence avec une concentration de 400 pg Fe / ml. Se reporter au Tableau 1 pour les instructions pour les navires de remplacement. <ol><li> Créer une solution (solution 1) en mélangeant 1 ml de milieu sans sérum et 93,1 l de ferumoxytol (stock: 30 mg / ml) dans un tube de 15 ml conique. </li><li> Créer une deuxième solution (solution 2) en mélangeant 1 ml de milieu sans sérum et 7 ul de sulfate de protamine (stock: 10 mg / ml) dans un second tube de 15 ml conique. </li><li> Permettre à chaque solution de se reposer pendant 5 minutes. </li><li> Ajouter les solutions 1 et 2 ensemble. Mélanger doucement la solution nouvel étiquetage. Laisser la solution reposer pendant 5 minutes afin de permettre USPIO pro-complexes de sulfate tamine à se former. </li><li> Ajouter 5 ml de milieu sans sérum à l&#39;mixtes de 2 ml de SPIO / solution de sulfate de protamine pour un volume final de 7 ml. </li></ol> 3) Préparer les cellules pour l&#39;étiquetage <ol><li> Aspirer les médias à partir des cellules. </li><li> Lavez délicatement les cellules une fois avec 2-3 ml de pré-chauffé Mg / Ca-libres D-PBS ou un milieu sans sérum pour rincer résiduelle protéines sériques et des médias ou d&#39;autres éléments qui pourraient nuire à l&#39;absorption agent de contraste et l&#39;efficacité d&#39;étiquetage. Aspirer le liquide de rinçage. </li></ol> Cellules label 4) <ol><li> Ajouter le complète de 7 ml de solution d&#39;étiquetage pour les cellules. </li><li> Placez les cellules dans un incubateur (37 ° C / 5% CO 2) et permettre aux cellules à incuber dans la solution d&#39;étiquetage pour les 4 heures. </li><li> Ajouter 700 ul de FCS à la solution d&#39;étiquetage des cellules pour obtenir une concentration finale de sérum de 10%. Le sérum est ajouté à rétablir l&#39;environnement enrichi sur lequel les cellules sont habitués, et pour aider à minimizing mort cellulaire qui pourrait résulter d&#39;un changement brusque d&#39;une exposition de 24 heures à un environnement sans sérum. </li><li> Autoriser les cellules à incuber avec la solution d&#39;étiquetage pour 20 heures supplémentaires. </li></ol> 3. Jour 3 5) Préparer les cellules marquées <ol><li> Aspirer la solution d&#39;étiquetage à partir des cellules. </li><li> Rincer délicatement les cellules avec 2-3 ml de pré-chauffé Mg / Ca-libres D-PBS. Aspirer le PBS. Il est important d&#39;utiliser Mg / Ca PBS sans que le Mg et Ca va nuire à l&#39;efficacité de la trypsine dans l&#39;étape suivante. </li><li> Ajouter 2 ml de pré-chauffé trypsine à 0,05% pour les cellules et incliner le flacon avant et en arrière afin d&#39;assurer toute la surface du flacon est recouverte d&#39;une fine couche de trypsine. Placez les cellules dans un incubateur (37 ° C / 5% CO 2) et permettre aux cellules de se reposer pendant 5-7 minutes ou jusqu&#39;à ce que les cellules commencent à se détacher de la plaque. Il peut être nécessaire pour confirmer le détachement sous un microscope. Tapoter légèrement les côtés du flacon si nécessaire à FAciliter la surface des cellules du détachement. </li><li> Ajouter 4 ml de pré-chauffé média complète dans le ballon pour neutraliser la trypsine. Rincer délicatement le flacon par pipetage la trypsine / media / solution cellulaire de haut en bas plusieurs fois. Recueillir l&#39;ensemble de la solution dans un tube de 15 ml conique. Centrifuger la solution de cellules à 400 FCR pendant 5 minutes. </li><li> Aspirer délicatement le surnageant sans déranger le culot cellulaire. Resuspendre les cellules dans 5 ml de milieu (sérum contenant ou dépourvu de sérum). Centrifuger la solution de cellules à 400 FCR pendant 5 minutes. </li><li> Répéter le lavage de cellules décrite à l&#39;étape 5.5. Au total, les cellules seront rincés trois fois pour éliminer les résidus, sans agent de contraste dans les médias et sur la surface des cellules. </li><li> Compter les cellules à ce stade pour atteindre le nombre de cellules les plus précises, comme les cellules seront perdues lors du lavage précédent. Effectuez un test de viabilité des cellules. Les cellules sont maintenant prêtes pour une analyse ultérieure et ou de l&#39;application expérimentale. IRM peut être réalisée avec T1-w et<html> T2-w paramètres, parce que bien ferumoxytol est un agent de contraste T2-w, ferumoxytol présente également des effets T1. Représentant T2-w paramètres IRM qui peut être utilisé pour l&#39;image ferumoxytol cellules marquées comprennent: des séquences FSE ou SE_Multislice avec un TR de 2000 à 2500 ms et un TE de 60-80 ms. Pour acquérir une image T1-w, diminuer la valeur de TE sur un FSE ou séquence SE_Multislice ou utiliser une séquence GRE avec une haute et basse TR TE. La figure 4 montre un potentiel d&#39;application pour l&#39;imagerie in vivo de cellules souches étiquetées. </li></ol> 4. Les résultats représentatifs: Les cellules marquées montrent un effet de contraste significatif assombrissement ou négatif sur les images IRM pondérées en T2 et un effet de contraste éclaircissant ou positif sur les images IRM pondérées en T1 (figure 2). Longitudinales IRM et des tests de viabilité effectuée cinq jours après l&#39;étiquetage n&#39;a démontré aucun signal significatif MR et aucun impact significatif sur la viabilité par rapport aux contrôles non marqué (données non présentées). jove_content &quot;&gt; cellules souches marquées peuvent ensuite être différenciées en divers types cellulaires ou injecté par voie intraveineuse pour une étude in vivo. <img alt="Figure 1" src="/files/ftp_upload/3482/3482fig1.jpg" /> Figure 1. Schéma ligne de temps pour l&#39;étiquetage des cellules souches avec les nanoparticule d&#39;oxyde de fer, ferumoxytol. <img alt="Figure 2" src="/files/ftp_upload/3482/3482fig2.jpg" /> Figure 2. Images IRM de coupes sagittale tubes eppendorf thrrough avec des cellules en un culot cellulaire. A) Unlabeled contrôles. B) Les cellules marquées montrant un T2 significative ou effet négatif sur les agents de contraste en T2 FSE ou SE_Multislice imagerie et un effet de contraste T1 ou positifs agent sur la séquence pondérée en T1 GRE. <img alt="Figure 3" src="/files/ftp_upload/3482/3482fig3.jpg" /> Figure 3. Sagittale sections de tubes Eppendorf avec des cellules en cellules PELpermet. Aussi peu que 10 000 ferumoxytol cellules marquées peuvent être détectés par l&#39;intermédiaire T2-w IRM. <img alt="Figure 4" src="/files/ftp_upload/3482/3482fig4.jpg" /> Figure 4. Visualisation des MR ferumoxytol marqué les cellules souches injectées dans murin ventricules cérébraux. A) axiale, l&#39;image du MR Barin murin sans injection de USPIO marqué les cellules souches. B) Axial, C) coronale, et D) les images IRM sagittale montrant le T2, effet de contraste négatif de USPIO marqué les cellules souches (flèches blanches) injecté dans le cerveau murin. <img alt="Tableau 1" src="/files/ftp_upload/3482/3482table1.jpg" /> Tableau adaptations Quantité 1. Pour le marquage des cellules dans d&#39;autres bateaux.

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Tous les contributeurs de cette étude offrent aucune divulgation.

Améliorer l&#39;efficacité des cellules souches engraftments est crucial pour l&#39;avancement de la médecine régénérative. Une technique de visualisation non invasive des cellules souches in vivo améliore de façon significative notre capacité à comprendre les mécanismes qui conduisent à des résultats transplantés avec succès. Étiquetage magnétique pour visualiser MR, telles que la procédure, nous avons démontré, permet de suivre in vivo des cellules souches avec l&#39;IRM....

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Société </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> D-MEM glycémie élevée </td><td> Sigma </td><td> D5648 </td><td> Ou moyen autre base pour la ligne de cellules souches souhaitées pour être utilisé </td></tr><tr><td> D-PBS (Ca + +, Mg + + gratuit) </td><td> GIBCO </td><td> 14190-144 </td><td></td></tr><tr><td> Trypsine-EDTA 0,05% </td><td> Invitrogen </td><td> 25300-120 </td><td></td></tr><tr><td> Sérum fœtal bovin (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> Sulfate de protamine </td><td> APP Pharm. </td><td> 22930 </td><td></td></tr>
</tbody>
</table>

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

Thérapies basées sur les cellules souches offrent un potentiel important pour le domaine de la médecine régénérative. Cependant, il reste beaucoup à comprendre sur la cinétique in vivo des cellules transplantées. Une méthode non-invasive pour surveiller de façon répétitive des cellules souches transplantées in vivo permettrait aux enquêteurs de contrôler directement les greffes de cellules souches et de déterminer les résultats greffe réussie ou non. Une large gamme de cellules souches continue à être étudiée pour des applications innombrables. Ce protocole se concentre sur trois différentes populations de cellules souches: embryonnaires de rein humain 293 (HEK293) les cellules, cellules souches mésenchymateuses humaines (hMSC) et souches pluripotentes induites (iPS). Cellules HEK 293 sont issus de cellules rénales embryonnaires humaines mises en culture par l&#39;adénovirus 5 ADN cisaillé. Ces cellules sont largement utilisés dans la recherche parce qu&#39;ils sont faciles à cultiver, se développent rapidement et sont facilement transfectées. hMSCs sont trouvées dans la moelle adulte. Ces cellules can être reproduits comme des cellules indifférenciées, tout en maintenant multipotence ou le potentiel de se différencier en un nombre limité de destins cellulaires. hMSCs peuvent se différencier pour des lignées de tissus mésenchymateux, y compris les ostéoblastes, les adipocytes, chondrocytes, tendons, muscles, et du stroma médullaire. Les cellules iPS sont des cellules adultes reprogrammées génétiquement qui ont été modifiées pour exprimer des gènes et des facteurs semblables à définir les propriétés des cellules souches embryonnaires. Ces cellules sont pluripotentes sens qu&#39;ils ont la capacité de se différencier en tous les lignages cellulaires 1. Les deux hMSCs et les cellules iPS ont démontré la capacité de régénération des tissus in vivo. Par résonance magnétique (IRM) en collaboration avec l&#39;utilisation de l&#39;oxyde de fer superparamagnétiques (SPIO) étiquettes cellulaire des nanoparticules se sont avérées efficaces dans le suivi in vivo des cellules souches en raison de la résolution anatomique microscopiques proximité, une plus longue demi-vie sanguine qui permet d&#39;imagerie longitudinale et la haute SENSIBILITÉy pour la détection de cellules fournies par l&#39;IRM du SPIO nanoparticules 2-4. En outre, l&#39;IRM avec l&#39;utilisation de SPIOs est cliniquement traduisible. SPIOs sont composées d&#39;un noyau d&#39;oxyde de fer avec une couche de surface de dextrane, carboxydextran ou de l&#39;amidon qui sert à contenir le noyau de fer à partir de composants bioréactifs plasma. Ces agents créent locales inhomogénéités de champ magnétique qui conduisent à une diminution du signal sur les images IRM pondérées en T2 5. Malheureusement, SPIOs ne sont plus fabriqués. La deuxième génération, ultrapetites SPIOs (USPIO), cependant, offrir une alternative viable. Ferumoxytol (FerahemeTM) est l&#39;un USPIO composé d&#39;un noyau de magnétite non stœchiométrique entouré d&#39;une couche carboxymethylether sorbitol polyglucose. Le colloïdale, taille des particules de 17-30 nm ferumoxytol est telle que déterminée par diffraction de la lumière. Le poids moléculaire est de 750 kDa, et la relaxivité champ constant à 2T IRM est 58,609 mm-1 sec-1 de force 4. Ferumoxytol a été récemment approuvé par la FDA unes un supplément de fer pour le traitement de la carence en fer chez les patients atteints d&#39;insuffisance rénale 6. Notre groupe a appliqué cet agent dans un &quot;hors étiquette&quot; usage pour les applications de marquage cellulaire. Notre technique démontre l&#39;étiquetage efficace des cellules souches avec ferumoxytol qui mène à d&#39;importants effets de signal IRM de cellules marquées sur les images IRM. Cette technique peut être appliquée pour les non-invasif de surveillance de thérapies par cellules souches dans les paramètres pré-cliniques et cliniques.

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

Nous décrivons une technique d&#39;étiquetage et de suivi avec des cellules souches approuvé par la FDA, le fer superparamagnétiques d&#39;oxyde (SPIO), ferumoxytol (Feraheme). Cette technique d&#39;imagerie cellulaire qui utilise par résonance magnétique (IRM) pour la visualisation, est facilement accessible pour surveillance à long terme et le diagnostic de succès ou non engraftments cellules souches chez les patients.

Étiquetage cellules souches avec Ferumoxytol, une nanoparticule approuvé par la FDA d&#39;oxyde de fer

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

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20.7K vues.  Molecular Imaging Program at Stanford (MIPS). Nous décrivons une technique d'étiquetage et de suivi avec des cellules souches approuvé par la FDA, le fer superparamagnétiques d'oxyde (SPIO), ferumoxytol (Feraheme). Cette technique d'imagerie cellulaire qui utilise par résonance magnétique (IRM) pour la visualisation, est facilement accessible pour surveillance à long terme et le diagnostic de succès ou non engraftments cellules souches chez les patients.

Vidéo: 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.

Différenciation isolement, caractérisation et comparative de l&#39;homme dentaires cellules souches de pulpe Dérivé de dents permanentes à l&#39;aide de deux méthodes différentes

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

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Médecine

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.

Système de co-culture avec un cerveau organotypique et 3D sphéroïde de cellules de carcinome

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.

Etudier le cancer du pancréas Stem Cell Caractéristiques pour le développement de nouvelles stratégies de traitement

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.

Forskoline induite par Gonflement dans Intestinal organites: Un<em&gt; In vitro</em&gt; Essai pour évaluer la réponse des médicaments dans les patients atteints de fibrose kystique

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.

Continu Transfusion Exchange Manuel pour les patients avec la drépanocytose: une méthode efficace pour éviter la surcharge en fer

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.

Synthèse, caractérisation et application de nanosons à oxyde de fer superparamagnetic pour la détection de la tuberculose extrapulmonaire

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.

Nanoparticules de Polyéthylèneimine recouvertes d’oxyde de fer comme un véhicule pour la livraison de petits ARN interférents à Macrophages In Vitro et 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.

Une méthode sans enzyme s'est d'isolement et d'expansion des cellules souches mésenchymales dérivées de l'adipose humaine

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.

Suivi des cellules souches mésenchymales étiquetées à l'oxyde de fer superparamagnetic à l'aide de l'IRM après la livraison intranasale dans un modèle de murine traumatique de lésions cérébrales

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.

Estimation des nanocristaux urinaires chez l’homme à l’aide de l’étiquetage des fluorophores de calcium et de l’analyse de suivi des nanoparticules

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