3R01AR054458-02S2: Diese Arbeit wurde durch ein Stipendium des National Institute of Arthritis and Bewegungsapparates und Hautkrankheiten unterstützt.

1. Tag 1 1) Platte Zellen <ol><li> Platte hMSC in einer T75-Kolben bei einer Konfluenz von 80% mindestens 18-24 Stunden vor der Markierung. Siehe Tabelle 1 für den Unterricht für alternative Fahrzeuge. </li></ol> 2. Tag 2 2) Bereiten Kennzeichnung Lösung. Diese Vorbereitung wird eine (1) T75-Kolben bei 80% Konfluenz mit einer Konzentration von 400 ug Fe / ml Etikett. Siehe Tabelle 1 für den Unterricht für alternative Fahrzeuge. <ol><li> Erstellen Sie eine Lösung (Lösung 1) durch Mischen von 1 ml Serum-freien Medien und 93,1 ul ferumoxytol (Bestand: 30 mg / ml) in einem 15 ml konischen Röhrchen. </li><li> Erstellen Sie eine zweite Lösung (Lösung 2) durch Mischen von 1 ml Serum-freien Medien und 7 pl Protaminsulfat (Bestand: 10 mg / ml) in einem zweiten 15 ml konischen Röhrchen. </li><li> Lassen Sie jede Lösung für 5 Minuten ruhen lassen. </li><li> Add-Lösungen 1 und 2 zusammen. Vorsichtig mischen die neuen Kennzeichnungsvorschriften Lösung. Lassen Sie die Lösung für 5 Minuten ruhen lassen, um USPIO-pro erlaubenHistamin-Sulfat-Komplexe zu bilden. </li><li> 5 ml Serum-freien Medien zu den gemischten 2 ml SPIO / Protaminsulfat-Lösung für ein Endvolumen von 7 ml. </li></ol> 3) Bereiten Zellen für die Etikettierung <ol><li> Saugen Sie die Medien aus den Zellen. </li><li> Vorsichtig waschen Zellen einmal mit 2-3 ml vorgewärmten Mg / Ca-freien D-PBS oder Serum-freien Medien zu abspülen verbleibende Serumproteine ​​und oder anderen Medien-Komponenten, die Kontrastmittelaufnahme und Kennzeichnung Effizienz beeinträchtigen könnten. Saugen Sie die Spülflüssigkeit. </li></ol> 4) Label-Zellen <ol><li> Fügen Sie die komplette 7 ml Lösung zur Kennzeichnung von Zellen. </li><li> Legen Sie Zellen in einem Inkubator (37 ° C / 5% CO 2) und damit die Zellen bei der Kennzeichnung Lösung für 4 Stunden inkubiert. </li><li> Add 700 ul FCS die Etikettierung Lösung der Zellen, um eine endgültige Konzentration im Serum von 10% zu erreichen. Serum zugesetzt wird, um die Wiederherstellung der angereicherten Umwelt, von denen die Zellen sind daran gewöhnt, und in mi unterstützennimizing Zelltod, die von einem abrupten Übergang zu 24 Stunden Exposition gegenüber einem Serum-freien Umgebung führen könnten. </li><li> Lassen Sie die Zellen, die mit der Etikettierung Lösung für weitere 20 Stunden inkubiert. </li></ol> 3. Tag 3 5) Bereiten Sie markierten Zellen <ol><li> Saugen Sie die Kennzeichnung Lösung von Zellen. </li><li> Vorsichtig spülen Zellen mit 2-3 ml vorgewärmten Mg / Ca-freien D-PBS. Saugen Sie die PBS. Es ist wichtig, Mg / Ca-freiem PBS Verwendung als Mg und Ca wird die Effizienz von Trypsin im nächsten Schritt zu beeinträchtigen. </li><li> 2 ml vorgewärmten 0,05% Trypsin zu den Zellen und kippen Sie den Kolben hin und her, um die gesamte Oberfläche des Kolbens zu gewährleisten ist mit einer dünnen Schicht von Trypsin behandelt. Legen Sie die Zellen in einem Inkubator (37 ° C / 5% CO 2) und damit die Zellen für 5-7 Minuten oder ruhen, bis Zellen von der Platte lösen beginnen. Es kann notwendig sein, um Distanz unter einem Mikroskop zu bestätigen. Klopfen Sie leicht den Seiten des Kolbens, wenn notwendig, facilitate Zelloberflächen-Distanz. </li><li> 4 ml vorgewärmten komplette Medien in den Kolben auf die Trypsin zu neutralisieren. Vorsichtig spülen Sie die Flasche mit einer Pipette die Trypsin / media / Zell-Lösung nach oben und unten mehrere Male. Sammeln Sie die gesamte Lösung in einem 15 ml konischen Röhrchen. Zentrifugieren Sie die Zell-Lösung bei 400 RCF für 5 Minuten. </li><li> Vorsichtig absaugen den Überstand ohne die Zellpellet. Resuspendieren der Zellen in 5 ml Medium (Serum mit oder Serum-frei). Zentrifugieren Sie die Zell-Lösung bei 400 RCF für 5 Minuten. </li><li> Wiederholen Sie die Zelle waschen in Schritt 5.5 beschrieben. Insgesamt werden Zellen dreimal gespült werden, um restliche, frei Kontrastmittel in den Medien und auf der Oberfläche der Zellen zu entfernen. </li><li> Zählen von Zellen an dieser Stelle auf das genaueste Zellzahl zu erreichen, wie Zellen während der letzten Waschen verloren geht. Führen Sie eine Viabilitätstest von Zellen. Die Zellen sind nun bereit für die anschließende Analyse und oder experimentelle Anwendung. MR-Bildgebung mit T1-w durchgeführt werden und<html> T2-w-Parameter, denn obwohl ferumoxytol ist ein T2-w-Kontrastmittel, ferumoxytol zeigt auch T1-Effekte. Vertreter T2-w MRT-Parameter, die Bild ferumoxytol markierten Zellen verwendet werden können: FSE oder SE_Multislice Sequenzen mit einer TR von 2000-2500 ms und einer TE von 60-80 ms. Zum Erwerb einer T1-w Bild, verringern Sie die TE-Wert auf einer FSE oder SE_Multislice Sequenz oder verwenden Sie einen GRE-Sequenz mit einer hohen TR und niedrige TE. Abbildung 4 zeigt eine mögliche in vivo-Anwendung für markierte Stammzelle Bildgebung. </li></ol> 4. Repräsentative Ergebnisse: Markierten Zellen zeigen eine deutliche Verdunkelung oder negativen Kontrast auf T2-gewichteten MRT-Bilder und eine Aufhellung oder positiven Kontrast auf T1-gewichteten MRT-Bilder (Abbildung 2). Längs der MR-Bildgebung und die Lebensfähigkeit Tests 5 Tage nach der Kennzeichnung keine signifikanten MR-Signal und keine signifikanten Auswirkungen auf die Lebensfähigkeit im Vergleich zu unmarkiertem Kontrollen (Daten nicht gezeigt) nachgewiesen. jove_content &quot;&gt; markierten Stammzellen können anschließend in verschiedene Zelltypen differenzieren oder intravenös in vivo Untersuchung injiziert. <img alt="Abbildung 1" src="/files/ftp_upload/3482/3482fig1.jpg" /> Abbildung 1. Schematische Zeitplan für die Kennzeichnung Stammzellen mit Eisenoxid-Nanopartikel, ferumoxytol. <img alt="Abbildung 2" src="/files/ftp_upload/3482/3482fig2.jpg" /> Abbildung 2. MR-Bilder des sagittalen Querschnitte thrrough Eppendorf-Röhrchen mit Zellen in einem Zell-Pellet. A) steuert Unlabeled. B) markierten Zellen zeigen eine deutliche T2 oder negative Kontrastmittel auf T2-gewichteten FSE oder SE_Multislice Bildgebung und eine T1-oder positive Kontrastmittel Effekt auf die T1-gewichteten GRE-Sequenz. <img alt="Abbildung 3" src="/files/ftp_upload/3482/3482fig3.jpg" /> Abbildung 3. Sagittal Querschnitte der Eppendorf-Röhrchen mit Zellen in Zell-pelkönnen. So wenig wie 10.000 ferumoxytol markierten Zellen können über T2-w MRT erkannt werden. <img alt="Abbildung 4" src="/files/ftp_upload/3482/3482fig4.jpg" /> Abbildung 4. MR Visualisierung von ferumoxytol-markierten Stammzellen in Mäuse-Hirnventrikel injiziert. A) Axial-, MR-Bild von murinen barin ohne Injektion von USPIO-markierten Stammzellen. B) Axial, C) koronal und D) sagittal MRT Darstellung des T2, negative dagegen Wirkung USPIO-markierten Stammzellen (weiße Pfeile) in einem murinen Gehirn injiziert. <img alt="Tabelle 1" src="/files/ftp_upload/3482/3482table1.jpg" /> Tabelle 1. Menge Anpassungen für die Markierung von Zellen in alternativen Gefäßen.

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Alle Mitwirkenden an dieser Studie bieten keine Meldungen vor.

Verbesserung der Wirksamkeit von Stammzellen engraftments ist entscheidend für die Weiterentwicklung der regenerativen Medizin. Eine nicht-invasive Visualisierung Technik für Stammzellen in vivo signifikant verbessert unsere Fähigkeit, Mechanismen, die eine erfolgreiche Transplantation Ergebnissen führen verstehen. Magnetische Kennzeichnung für MR-Visualisierung, wie das Verfahren, das wir gezeigt haben, können in vivo Verfolgung von Stammzellen mit der MR-Bildgebung. Magnetisch markierte Stammze...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> D-MEM Hohe Glucose </td><td> Sigma </td><td> D5648 </td><td> Oder andere Basismedium für die gewünschte Stammzelllinie verwendet werden </td></tr><tr><td> D-PBS (Ca + +, Mg + + frei) </td><td> GIBCO </td><td> 14190-144 </td><td></td></tr><tr><td> Trypsin-EDTA 0,05% </td><td> Invitrogen </td><td> 25300-120 </td><td></td></tr><tr><td> Fetal Bovine Serum (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> Protaminsulfat </td><td> APP Pharm. </td><td> 22930 </td><td></td></tr>
</tbody>
</table>

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

Stammzell-basierte Therapien bieten erhebliches Potenzial für den Bereich der regenerativen Medizin. Dennoch bleibt noch viel zu verstehen in Bezug auf die in vivo Kinetik der transplantierten Zellen werden. Eine nicht-invasive Methode, um wiederholt zu überwachen transplantierten Stammzellen in vivo erlauben würde Ermittler direkt zu überwachen Stammzell-Transplantationen und identifizieren erfolgreiche oder erfolglose Transplantation Ergebnisse. Eine breite Palette von Stammzellen weiterhin für unzählige Anwendungen untersucht werden. Dieses Protokoll konzentriert sich auf 3 verschiedenen Stammzell-Populationen: menschliche embryonale Nierenzellen 293 (HEK293-Zellen), humanen mesenchymalen Stammzellen (hMSC) und induzierten pluripotenten Stammzellen (iPS-Zellen). HEK 293 Zellen aus menschlichen embryonalen Nierenzellen in Kultur mit geschert Adenovirus 5 DNA gewachsen abgeleitet. Diese Zellen weit verbreitet sind in der Forschung eingesetzt, weil sie leicht kultiviert werden, wachsen schnell und sind leicht transfiziert. hMSCs sind bei erwachsenen Knochenmark gefunden. Diese Zellen ca.n als undifferenzierte Zellen repliziert werden unter Beibehaltung Multipotenz oder das Potential, in einer begrenzten Anzahl von Zellanlagen unterscheiden. hMSCs können Abstammungslinien von mesenchymalen Geweben, einschließlich Osteoblasten, Adipozyten, Chondrozyten, Sehnen, Muskeln und Knochenmarkstroma unterscheiden. iPS-Zellen sind genetisch adulten Zellen, die modifiziert wurden, um Gene und Faktoren ähnlich definierenden Eigenschaften von embryonalen Stammzellen umprogrammiert auszudrücken. Diese Zellen sind pluripotent dh sie haben die Fähigkeit, sich in allen Zelllinien 1 unterscheiden. Beide hMSCs und iPS-Zellen haben Tissue Regenerationsfähigkeit in-vivo demonstriert. Magnetresonanztomographie (MRT) zusammen mit der Verwendung von superparamagnetischen Eisenoxid (SPIO) Nanopartikel-Zell-Labels haben sich für in vivo Verfolgung von Stammzellen aufgrund der Nähe mikroskopischen anatomischen Auflösung effektiv, erlaubt eine längere Blut-Halbwertszeit, die Längs-Bildgebung und die hohe sensitivity für Zell-Erkennung durch MR-Bildgebung von SPIO sofern Nanopartikel 2-4. Darüber hinaus ist der MR-Bildgebung mit dem Einsatz von SPIOs klinisch übersetzbar. SPIOs eines Eisenoxid-Kern mit einer Dextran, Carboxydextran oder Stärke Oberflächenbeschichtung, die den bioreaktiven Eisenkern aus dem Plasma-Komponenten enthalten dient zusammen. Diese Mittel schaffen lokale Magnetfeldinhomogenitäten, die zu einem verringerten Signal auf T2-gewichteten MRT-Bilder zeigen 5 führen. Leider sind SPIOs nicht mehr hergestellt. Zweite Generation, ultrakleinen SPIOs (USPIO), bieten jedoch eine gute Alternative. Ferumoxytol (FerahemeTM) ist eine USPIO einer nicht-stöchiometrischen Magnetit Kern durch eine Polyglucose Sorbit carboxymethylether Mantel umgeben sind. Die kolloidalen, Partikelgröße von ferumoxytol ist 17-30 nm durch Lichtstreuung bestimmt. Das Molekulargewicht beträgt 750 kDa, und die Relaxivität konstant bei 2T MRI Feld ist 58,609 mM-1 sec-1 Stärke 4. Ferumoxytol wurde vor kurzem von der FDA genehmigte eines ein Eisenpräparat zur Behandlung von Eisenmangel bei Patienten mit Nierenversagen 6. Unsere Gruppe hat dieses Mittel in einer &quot;off label&quot; Einsatz für die Zell-Kennzeichnung Anwendungen eingesetzt. Unsere Technik demonstriert eine effiziente Markierung von Stammzellen mit ferumoxytol, die zu erheblichen MR-Signal Auswirkungen der markierten Zellen auf MR-Bildern führt. Diese Technik kann für nicht-invasive Überwachung der Stammzelltherapie in der vorklinischen und klinischen Umgebungen eingesetzt werden.

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

Wir beschreiben eine Technik zur Kennzeichnung und Verfolgung von Stammzellen mit FDA-Zulassung, superparamagnetische Eisenoxid (SPIO), ferumoxytol (Feraheme). Diese zellulären bildgebendes Verfahren, das Magnetresonanz-(MR)-Bildgebung nutzt zur Visualisierung, ist leicht zugänglich für die langfristige Überwachung und Diagnose von Erfolg oder Misserfolg Stammzellen engraftments bei Patienten.

Labeling Stammzellen mit Ferumoxytol, eine FDA-Zulassung Eisenoxid-Nanopartikel

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 Aufrufe.  Molecular Imaging Program at Stanford (MIPS). Wir beschreiben eine Technik zur Kennzeichnung und Verfolgung von Stammzellen mit FDA-Zulassung, superparamagnetische Eisenoxid (SPIO), ferumoxytol (Feraheme). Diese zellulären bildgebendes Verfahren, das Magnetresonanz-(MR)-Bildgebung nutzt zur Visualisierung, ist leicht zugänglich für die langfristige Überwachung und Diagnose von Erfolg oder Misserfolg Stammzellen engraftments bei Patienten.

Serie: 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.

Isolation, Charakterisierung und vergleichende Differenzierung humaner Dental Pulp Stammzellen aus bleibenden Zähne mit zwei verschiedenen Methoden abgeleitet

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

Forschung

Medizin

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.

Co-Kultur-System mit einer Scheibe Organotypische Gehirn-und 3D-Sphäroid von Karzinomzellen

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.

Studieren Bauchspeicheldrüsenkrebs Stammzelleigenschaften für die Entwicklung neuer Behandlungsstrategien

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.

Forskolin-induzierte Quellung in Intestinale Organoide: Ein<em&gt; In Vitro</em&gt; Assay für Fibrosis Patienten in Cystic Drug Response-Beurteilung

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.

Kontinuierliche manuelle Austausch Transfusions für Patienten mit Sichelzellenanämie: Eine effiziente Methode zur Vermeidung von Eisenüberladung

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.

Synthese, Charakterisierung und Anwendung von superparamagnetischen Eisenoxid-Nanosonden zur Erkennung von extrapulmonaler Tuberkulose

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.

Polyethyleneimine beschichteten Eisenoxid-Nanopartikeln als Vehikel für die Lieferung von kleinen interferierende RNA an Makrophagen In Vitro und 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.

Eine enzymfreie Methode zur Isolierung und Expansion von humanen Adsetzen-abgeleiteten mesenchymalen Stammzellen

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.

Verfolgung von superparamagnetischen Eisenoxid-beschrifteten mesenchymalen Stammzellen mit MRT nach intranasaler Lieferung in einem traumatischen Hirnverletzungs-Murine-Modell

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.

Abschätzung von Urin-Nanokristallen beim Menschen mittels Calciumfluorophor-Markierung und Nanopartikel-Tracking-Analyse

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