Diese Arbeit wurde von OTKA (Hungarian Scientific Research Fund) D45933, T049621, TET (Hungarian Science and Technology Foundation) A4/04, Arg-17/2006 und SIN, Bolyai, Öveges Stipendien und TÁMOP unterstützt 4.2.2-08 / 1 / KMR-2008-0004 und 4.2.1 / B 09/1/KMR-2010-0001. OTKA 83803. Wir möchten William Gesztes für die Bereitstellung der Voice-over danken.

1. Vorbereitung H9c2 cardiomyoblast Zellen H9c2 Ratte cardiomyoblasts wurden von ATCC (Wesel, Deutschland) erhalten und ausgebaut in hoher Glukose (4,5 g / L) DMEM mit 10% fötalem Rinderserum, 4 mM L-Glutamin, 100 U / ml Penicillin und 100 ug / ml Streptomycin. Die H9c2 Myoblasten-Zelllinie aus embryonalen Ratte Herzen abgeleitet wird, wird es als ein in vitro Modell für Skelett-und Herzmuskeln 8,9 verwendet. Bereiten Sie 12 gut Platte H9c2 Zellen: <ol><li> Entfernen Kulturmedium aus Petrischale H9c2 Zellen. </li><li> Wash Zellen zweimal mit 5 ml PBS und verdauen mit 2-3 ml 0,05% Trypsin / EDTA. </li><li> Legen Sie Petrischale für 5 Minuten in die 37 ° C Inkubator. </li><li> Inhibit Trypsin mit 10 ml DMEM Kulturmedium. </li><li> Spin down die Zellen bei 1200 RPM für 8 Minuten. </li><li> Nach Entfernen des Überstands, die Zellen in 1 ml DMEM Kulturmedium und Zählen von Zellen mit Zählkammer mit Trypan-Blau-Färbung.</li><li> Seed 30.000 H9c2 Zellen in 2 ml DMEM pro Well in einer 12-Well-Platte. </li><li> Legen Sie die Platte in die 37 ° C CO 2-Inkubator für 24 Stunden. </li></ol> 2. Simulierte Ischämie / Reperfusion Ischämie / Reperfusion simuliert wurde in vitro, indem Sauerstoff Glukose Deprivation (OGD) auf H9c2 Zellkulturen. Dieses Verfahren wurde auf der Bühne des Zeiss konfokalen Mikroskop mit dem Pecon Zellinkubation System (Erbach, Deutschland) erlaubt die Beobachtung der Zellen während der OGD durchgeführt. <ol><li> Entfernen Medium durch Absaugen von 12-Well-Platte des H9c2 Zellen. </li><li> Wash Zellen zweimal mit PBS. </li><li> Add 3 ml Glucose-freiem Medium pro Vertiefung. </li><li> Legen Sie die Platte in die Zelle Inkubation System. </li><li> Starten Stickstoff, um den Sauerstoff aus der Inkubation System zu spülen. </li><li> Warten Sie, bis der Sauerstoffgehalt erreicht 0,5% dann den Timer zu starten. 0,5% Sauerstoff bedeutet ca. 3 mmHg teilweisen Spannung. Subject Zellen bis 150 Minuten von simulierten Ischämie. </li><li> Am Ende der simulierten Ischämie, entfernen Sie die Glukose-freiem Medium von den Zellen. </li><li> 2 ml normalen Kulturmedium in die Vertiefungen. </li><li> Legen Sie die 12-Well-Platte für 30 Minuten in die 37 ° C CO 2-Inkubator, das ist der &quot;Reperfusion Zeit&quot;. </li></ol> 3. Vorbereitung Rettung mesenchymalen Stammzellen Knochenmark menschlichen Ursprungs wurde in T75 Flaschen genommen und verdünnt mit Dulbecco modifiziertem Eagle-Medium (DMEM) Kultur-Medium mit 10% fötalem Kälberserum (FCS), 100 U / ml Penicillin und 100 ug / ml Streptomycin, 4 mM L-Glutamin und 1 g / l Glukose. Die Kolben wurden bei 37 ° C in einem vollständig befeuchteten Atmosphäre von 5% CO 2 für 3 Tage inkubiert. Nach der Inkubationszeit der BMSCs haftete an der Oberfläche des Flakons und die übrigen Komponenten des Knochenmarks wurden durch Waschen mit PBS entfernt. Die verwendeten BMSCs wurden zwischen 1 und 5 Passagen in den Experimenten. Die IdenTity der Zellen wurde durch die Anwesenheit von Lineage-spezifische Zelloberflächenmarker mit Durchflusszytometrie bestätigt. Hämatopoetische Lineage-spezifische Oberflächenmarker (CD34, CD45) und mesenchymalen Oberflächenmarker (CD73, CD90, CD105 und CD166) wurden untersucht. Während simulierten Ischämie, bereiten Rettung mesenchymale Stammzellen für die Transplantation. <ol><li> Verdünnen Sie die fluoreszierenden Farbstoff Vybrant in 1:200-Verhältnis in DMEM Kulturmedium DiD. </li><li> Absaugen Medium aus dem Rettungs-Zellen und 300 ul DMEM mit Vybrant DiD. </li><li> Legen Sie Petrischale für 30 Minuten in die 37 ° C CO 2-Inkubator. </li><li> Entfernen Kulturmedium aus Petrischale von mesenchymalen Stammzellen. </li><li> Wash Zellen zweimal mit 5 ml PBS und verdauen mit 2-3 ml 0,05% Trypsin / EDTA. </li><li> Legen Sie Petrischale für 5 Minuten in die 37 ° C Inkubator. </li><li> Inhibit Trypsin mit 10 ml DMEM Kulturmedium. </li><li> Spin down die Zellen bei 1200 RPM für 8 Minuten. </li><li> Nach dem Entfernen der Supernatant, die Zellen in 1 ml DMEM Kulturmedium und Zählen von Zellen mit Zählkammer mit Trypan-Blau-Färbung. </li><li> Nach 30 Minuten am Ende der Reperfusion add 20.000 mesenchymalen Stammzellen pro Vertiefung der postischämischen H9c2 kardiale Myoblasten. </li><li> Ort Co-Kultur von Zellen in die 37 ° C CO 2-Inkubator für 24 Stunden. </li></ol> 4. Die Analyse mit der konfokalen Mikroskopie Nach der simulierten Ischämie / Reperfusion, werden die Zellen in normalen Nährmedium für 24 Stunden inkubiert. <ol><li> Nach 24 Stunden waschen Sie die Zellen zweimal mit PBS. </li><li> Stain die Zellen in 500 ul Live / Dead-Farbstoff-Lösung für 30 Minuten (5 nM Calcein und 400 Nm Ethidium-Homodimer-2 in PBS) (5x10 4 Zellen / ml). </li></ol> Die Auswertung der konfokalen Bilder für lebende und tote Zellen durch Morphologie und Fluoreszenz ausgewählt werden können mit dem ImageJ Software (National Institutes of Health, USA) durchgeführt werden. Im Falle von co-Kulturen können MSCs aus H9c2 Zellen aufgrund ihrer Vybrant DiD Zelle gekennzeichnet werden. Das Verhältnis von abgestorbenen Zellen kann in 4 unabhängigen Sehfelder (Objektiv 10x) für jede Kultur in einem blinden fashion 10 ausgewertet werden. H9c2 cardiomyoblasts auf 42 mm Deckgläschen kultiviert kann auch mit Zeitraffer-Video-Mikroskopie, während und nach Ischämie / Reperfusion mithilfe der Zell-Inkubation System der konfokalen Mikroskop untersucht werden. Zell-Zell-Interaktionen wie Zell-Fusion und die Bildung von interzellulären Nanoröhrchen können im Laufe der Zeit untersucht werden. 5. Die Analyse mit Durchflusszytometrie Nach der simulierten Ischämie / Reperfusion, werden die Zellen in normalen Nährmedium für 24 Stunden inkubiert. <ol><li> Nach 24 Stunden ernten die Kulturmedien. Es ist sehr wichtig, um das Kulturmedium sammeln auch, weil einige tote Zellen könnten von der Kulturschale Oberfläche während der 24 Stunden Inkubation abgelöst habenTION. </li><li> Wash Zellen zweimal mit PBS und verdauen mit 0,05% Trypsin-EDTA. </li><li> Inhibit Trypsin mit DMEM Kulturmedium. </li><li> Spin down der Zellen und die geernteten Kulturmedium bei 1200 RPM (ca. 150-200g) für 8 Minuten. </li><li> Überstand verwerfen. </li><li> Suspend die Zellen in 500 ul Live / Dead-Farbstoff-Lösung für 30 Minuten (5 nM Calcein und 400 Nm Ethidium-Homodimer-2 in PBS) (5x10 4 Zellen / ml). </li><li> Transfer-Zellen-Zytometrie Rohre fließen. </li><li> Starten Sie den CellQuest Pro Software. </li><li> So richten Sie das Durchflusszytometer für die Messung, bereiten Kontrollproben von lebenden und toten Zellen. <ol class="lalpha"><li> Platte H9c2 Zellen bei einer Dichte von 3x10 4 in 12-Well-Platten, wie in Punkt 1 beschrieben. </li><li> Bereiten Sie leben Zelle steuert mit einzelnen Trypsinierung wie in Punkt 1 beschrieben. </li><li> Bereiten tote Zelle steuert durch die Behandlung mit 10 uM H 2 O 2 für 1 Stunde. </li><li> Nach Trypsinierung, resuspend lebenden und toten Zellen steuert in 500 ul Live / Dead-Farbstofflösung (5 nM Calcein-AM und 400 Nm Ethidium-Homodimer-2 in PBS). </li></ol></li><li> Das Instrument Einstellungen sollten so eingestellt, die lebenden Zellen Calcein positiven und Ethidium Homodimer negativ sind, während die toten Zellen Calcein negativen und Ethidium Homodimer positive sind. </li><li> Der Anteil dieser Bevölkerungsgruppen ausgedrückt als Balken in einem Diagramm und die statistische Analyse auf diesen Werten durchgeführt werden kann. </li><li> Messen Sie die verschiedenen experimentellen Gruppen. </li></ol> 6. Repräsentative Ergebnisse: Unsere Ergebnisse zeigten, dass hinzugefügt mesenchymalen Stammzellen könnte das Überleben der postischämischen kardialen Zellen zu verbessern. Die konfokale Mikroskopie-Bilder zeigten direkte Zell-Zell-Interaktionen, wie teilweise Membran-Verbindung oder interzelluläre-Nanoröhren, die wahrscheinlich eine wichtige Rolle spielen in den beobachteten Schutz 10. Diese Nanoröhren span über Entfernungen von mehreren Zell-diameters, und ihre Durchmesser wurden zwischen 200 und 500 nm (weiße Pfeile). <img src="/files/ftp_upload/3575/3575fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Nanotube-Bildung zwischen den Zellen. Nanotube-Bildung (weißer Pfeil) wurde zwischen Vybrant DiO gekennzeichnet cardiomyoblasts und Vybrant DiD gekennzeichnet mesenchymalen Stammzellen beobachtet. Durchflusszytometrie-Analyse zeigte die Bevölkerung zu überleben und nekrotische cardiomyoblasts und mesenchymalen Stammzellen. Interessanterweise haben wir auch beobachtet, eine dritte, &#39;verletzt&#39; cardiomyoblast Zellpopulation, die Calcein enthalten, sondern enthielt auch die Anzeige der toten Zellen, Ethidium Homodimer (Abbildung 2). <img src="/files/ftp_upload/3575/3575fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Vertreter dot-plot der verschiedenen Zellpopulationen nach simulierten Ischämie. Die Auswertung mehrerer Versuche showed, dass die Zugabe von mesenchymalen Stammzellen zu postischämischen H9c2 cardiomyoblast signifikant erhöht den Anteil der lebenden Zellen im Vergleich zu den kardialen Myoblasten allein nach Ischämie kultiviert (Abbildung 3). <img src="/files/ftp_upload/3575/3575fig3.jpg" alt="Abbildung 3" /> Abbildung 3. ., Die nach Zugabe erhöht: Einfluss der Stammzell-Therapie am postischämischen cardiomyoblasts Prozentsatz der lebenden Zellen war signifikant nach simulierten Ischämie im Vergleich zur Kontrollgruppe (90,36 ± 2,60 vs 10,52 ± 0,48, *** p&gt; 0,001 Kontrolle vs IR-Modell) reduziert von mesenchymalen Stammzellen (IR-Modell vs IR + BMSC: 10,52 ± 0,48 vs 25,21 ± 2,13, *** p&gt; 0,001). Die Daten repräsentieren Mittelwert ± SEM. Die statistische Analyse der Daten erfolgte mit one-way Varianzanalyse mit mehreren Vergleich Tukey post hoc Test durchgeführt.

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Keine Interessenskonflikte erklärt.

Der Beweis-Protokoll ist ein in vitro Ansatz für die viel mehr komplexe Thema der Stammzellentherapie bei Herzinfarkt, mit allen Vor-und Nachteile eines solchen Modells. Natürlich kann es nicht spiegeln die komplexe (zB immunologische) stattfindenden Veranstaltungen während und nach Myokard-Infarkt, kann aber auf die direkten Auswirkungen der zusätzlichen Zellen auf der postischämischen Zellen konzentrieren. Die Auswirkungen der simulierten Ischämie auf H9c2 cardiomyoblasts in hohem Maße abhängig von der Zeit de...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Calcein-AM </td><td> Molecular Probes </td><td> L3224, C3099 </td><td> <a href="http://www.invitrogen.com">http://www.invitrogen.com</a> </td></tr><tr><td> Ethidium Homodimer-2 </td><td> Molecular Probes </td><td> L3224, E3599 </td><td> <a href="http://www.invitrogen.com">http://www.invitrogen.com</a> </td></tr><tr><td> Vybrant DiD </td><td> Molecular Probes </td><td> V22887 </td><td> <a href="http://www.invitrogen.com">http://www.invitrogen.com</a> </td></tr></tbody></table> Tabelle 1. Reagenzien. <table border="1"><tbody><tr><td> Bezeichnung des Geräts </td><td> Firma </td><td> Kommentare (optional) </td></tr><tr><td> Pecon Zellinkubation System für Zeiss-Mikroskope </td><td> Pecon GmbH </td><td> <a href="http://www.pecon.biz/">www.pecon.biz/</a> </td></tr></tbody></table> Tabelle 2. Equipment.

stem cell transplantation in an in vitro simulated ischemia/reperfusion model

Stammzelltransplantation Protokolle finden ihren Weg in die klinische Praxis 1,2,3. Getting bessere Ergebnisse, so dass die Protokolle mehr robust, und die Suche nach neuen Quellen für implantierbare Zellen stehen im Mittelpunkt der jüngsten Forschung 4,5. Untersuchung der Wirksamkeit von Zelltherapien ist keine leichte Aufgabe, und neue Werkzeuge sind nötig, um die Mechanismen in der Behandlung Verfahren 6 zu erforschen. Wir haben ein experimentelles Protokoll der Ischämie / Reperfusion, um die Beobachtung von zellulären Verbindungen und sogar subzelluläre Mechanismen während der Ischämie / Reperfusion und nach Stammzelltransplantation zu ermöglichen und die Wirksamkeit der Zelltherapie zu bewerten. H9c2 cardiomyoblast Zellen wurden auf Zellkulturplatten 7,8 platziert. Ischämie simuliert wurde mit 150 Minuten in einer Glucose-freiem Medium mit Sauerstoff unter 0,5%. Dann wurden normale Medien-und Sauerstoff wieder zu Reperfusion zu simulieren. Nach Sauerstoff Glukose Deprivation,die geschädigten Zellen wurden mit der Transplantation von markiertem menschlichem Knochenmark abgeleiteten mesenchymalen Stammzellen, indem sie in die Kultur behandelt. Mesenchymale Stammzellen sind in klinischen Studien bevorzugt, weil sie leicht zu erreichen mit minimal-invasiven Chirurgie, leicht erweiterbar und autologe sind. Nach 24-stündiger Kokultivierung wurden die Zellen mit Calcein und Ethidium-Homodimer gefärbt, um zwischen lebenden und toten Zellen zu unterscheiden. Dieses Setup erlaubt uns, die interzellulären Verbindungen mit Hilfe der konfokalen Fluoreszenzmikroskopie zu untersuchen und die Überlebensrate von postischämischen Zellen mittels Durchflusszytometrie zu quantifizieren. Die konfokale Mikroskopie zeigte die Wechselwirkungen der beiden Zellpopulationen wie Zellfusion und die Bildung von interzellulären Nanoröhren. Durchflusszytometrie-Analyse ergab 3 Cluster von beschädigten Zellen, die in einem Diagramm dargestellt und können statistisch ausgewertet werden. Diese Populationen können separat untersucht werden und Rückschlüsse auf diese Daten über die Wirksamkeit der simulierten gezogen werdenTherapieansatz.

Watch this Scientific Journal Video about Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model at JoVE.com

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Wir zeigen, wie die Einrichtung eines<em&gt; In-vitro-</em&gt; Ischämie / Reperfusion-Modell und wie man den Effekt der Stammzelltherapie am postischämischen Herzzellen zu bewerten.

Stem Cell Transplantation in einem In-vitro- Simulierte Ischämie / Reperfusion-Modell

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

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Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

14.0K Aufrufe.  Semmelweis University. Wir zeigen, wie die Einrichtung eines In-vitro- Ischämie / Reperfusion-Modell und wie man den Effekt der Stammzelltherapie am postischämischen Herzzellen zu bewerten.

Serie: Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

Einem Mausmodell der In Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Forschung

Medizin

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Mittels quantitativer real-time PCR zum Donor Cell Engraftment in einem Competitive Murine Bone Marrow Transplantation Modell fest

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischämie-Reperfusion-Modell des akuten Nierenschädigung und nach der Verletzung Fibrose bei Mäusen

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Neural Stem Cell Transplantation in Experimental Gequetscht Modell von Rückenmarksverletzungen

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Sauerstoff-Glukose Deprivation und Reoxygenierung als<em&gt; In-vitro-</em&gt; Ischämie-Reperfusion Injury Modell zum Studium der Blut-Hirnschranke Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

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 ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Murine Modell der intestinalen Ischämie-Reperfusionsschaden

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

Schaffung und Transplantation von einem Fettgewebe-derived Stem Cell (ASC) Blatt in einem diabetischen Wundheilung Modell

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

Zwei-Schiff Okklusion Mausmodell der zerebralen Ischämie-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Ein präklinisches Rattenmodell zur Untersuchung von Ischämie-Reperfusionsverletzungen in der rekonstruktiven Mikrochirurgie

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...