Name: stem cell transplantation in an in vitro simulated ischemia/reperfusion model
Uploaded: 2011-11-05T14:00:00
Description: Watch this Scientific Journal Video about Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model at JoVE.com

Dit werk werd ondersteund door OTKA (Hongaarse Fonds voor Wetenschappelijk Onderzoek) D45933, T049621, TET (Hongaarse Science and Technology Foundation) A4/04, Arg-17/2006 en SIN, Bolyai, Öveges Fellowships en TÁMOP 4.2.2-08 / 1 / KMR-2008-0004 en 4.2.1 / B 09/1/KMR-2010-0001. OTKA 83.803. We willen graag William Gesztes bedanken voor het verstrekken van de voice-over.

1. Voorbereiden H9c2 cardiomyoblast cellen H9c2 rat cardiomyoblasts werden verkregen van ATCC (Wesel, Duitsland) en uitgebreid in hoge glucose (4,5 g / L) DMEM dat 10% foetaal runderserum, 4 mM L-glutamine, 100 U / ml penicilline en 100 ug / ml streptomycine. De H9c2 myoblast cellijn is afkomstig van embryonale rat hart, wordt het gebruikt als een in vitro model voor zowel de skelet-en hartspier 8,9. Bereid 12 well plaat van H9c2 cellen: <ol><li> Ver...

<ol>
	<li>Dimmeler, S., Burchfield, J., Zeiher, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-Based+Therapy+of+Myocardial+Infarction.">Cell-Based Therapy of Myocardial Infarction.</a> Arterioscler Thromb Vasc Biol. 28, (2008).</li><li>Kim, S. U., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-based+cell+therapy+in+neurological+diseases:+A+review.">Stem cell-based cell therapy in neurological diseases: A review.</a> Journal of Neuroscience Research. 87, 2183-21 (2009).</li><li>Lee, K., Chan, C. K., Patil, N., Goodman, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+therapy+for+bone+regeneration-Bench+to+bedside.">Cell therapy for bone regeneration-Bench to bedside.</a> Journal of Biomedical Materials Research Part B: Applied Biomaterials. 89, 252-25 (2009).</li><li>Gaipa, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GMP-based+CD133++cells+isolation+maintains+progenitor+angiogenic+properties+and+enhances+standardization+in+cardiovascular+cell+therapy.">GMP-based CD133+ cells isolation maintains progenitor angiogenic properties and enhances standardization in cardiovascular cell therapy.</a> Journal of Cellular and Molecular Medicine. 14, 1619-1619 (2010).</li><li>Trounson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+perspectives+in+human+stem+cell+therapeutic+research.">New perspectives in human stem cell therapeutic research.</a> BMC medicine. 7, 29-29 (2009).</li><li>Mazhari, R., Hare, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+action+of+mesenchymal+stem+cells+in+cardiac+repair:+potential+influences+on+the+cardiac+stem+cell+niche.">Mechanisms of action of mesenchymal stem cells in cardiac repair: potential influences on the cardiac stem cell niche.</a> Nat Clin Pract Cardiovasc Med. 4, S21-S21 (2007).</li><li>Kimes, B. W., Brandt, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+a+clonal+muscle+cell+line+from+rat+heart.">Properties of a clonal muscle cell line from rat heart.</a> Experimental Cell Research. 98, 367-367 (1976).</li><li>Sardao, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vital+imaging+of+H9c2+myoblasts+exposed+to+tert-butylhydroperoxide--characterization+of+morphological+features+of+cell+death.">Vital imaging of H9c2 myoblasts exposed to tert-butylhydroperoxide--characterization of morphological features of cell death.</a> BMC Cell Biol. 8, 11-11 (2007).</li><li>Hescheler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological,+biochemical,+and+electrophysiological+characterization+of+a+clonal+cell+(H9c2)+line+from+rat+heart.">Morphological, biochemical, and electrophysiological characterization of a clonal cell (H9c2) line from rat heart.</a> Circulation research. 69, 1476-1476 (1991).</li><li>Cselenyak, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+rescue+cardiomyoblasts+from+cell+death+in+an+in+vitro+ischemia+model+via+direct+cell-to-cell+connections.">Mesenchymal stem cells rescue cardiomyoblasts from cell death in an in vitro ischemia model via direct cell-to-cell connections.</a> BMC Cell Biol. 11, 29-29 (2010).</li><li>Sauvant, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementation+of+an+in+vitro+model+system+for+investigation+of+reperfusion+damage+after+renal+ischemia.">Implementation of an in vitro model system for investigation of reperfusion damage after renal ischemia.</a> Cell Physiol Biochem. 24, 567-567 (2009).</li><li>Namas, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-Induced+Overexpression+of+BNIP3+is+Not+Dependent+on+Hypoxia-Inducible+Factor+1alpha+in+Mouse+Hepatocytes.">Hypoxia-Induced Overexpression of BNIP3 is Not Dependent on Hypoxia-Inducible Factor 1alpha in Mouse Hepatocytes.</a> Shock. 36, 196-196 (2011).</li><li>Cao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia/Reoxygenation+Up-Regulated+the+Expression+of+Death+Receptor+5+and+Enhanced+Apoptosis+in+Human+Hepatocyte+Line.">Hypoxia/Reoxygenation Up-Regulated the Expression of Death Receptor 5 and Enhanced Apoptosis in Human Hepatocyte Line.</a> Transplantation Proceedings. 38, 2207-2207 (2006).</li><li>Meloni, B. P., Meade, A. J., Kitikomolsuk, D., Knuckey, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+neuronal+cell+death+in+acute+and+delayed+in+vitro+ischemia+(oxygen-glucose+deprivation)+models.">Characterisation of neuronal cell death in acute and delayed in vitro ischemia (oxygen-glucose deprivation) models.</a> Journal of Neuroscience Methods. 195, 67-67 (2011).</li><li>Mimeault, M., Hauke, R., Batra, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells:+a+revolution+in+therapeutics--recent+advances+in+stem+cell+biology+and+their+therapeutic+applications+in+regenerative+medicine+and+cancer+therapies.">Stem cells: a revolution in therapeutics--recent advances in stem cell biology and their therapeutic applications in regenerative medicine and cancer therapies.</a> Clinical Pharmacology &#38; Therapeutics. 82, 252-252 (2007).</li><li>Angoulvant, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell+conditioned+media+attenuates+in+vitro+and+ex+vivo+myocardial+reperfusion+injury.">Mesenchymal stem cell conditioned media attenuates in vitro and ex vivo myocardial reperfusion injury.</a> J Heart Lung Transplant. 30, 95-95 (2010).</li><li>Lim, Y. J., Zheng, S., Zuo, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphine+Preconditions+Purkinje+Cells+against+Cell+Death+under+In+Vitro+Simulated+Ischemia/Reperfusion+Conditions.">Morphine Preconditions Purkinje Cells against Cell Death under In Vitro Simulated Ischemia/Reperfusion Conditions.</a> Anesthesiology. 100, 562-562 (2004).</li><li>Guo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen-receptor-mediated+protection+of+cerebral+endothelial+cell+viability+and+mitochondrial+function+after+ischemic+insult+in+vitro.">Estrogen-receptor-mediated protection of cerebral endothelial cell viability and mitochondrial function after ischemic insult in vitro.</a> J Cereb Blood Flow Metab. 30, 545-545 (2010).</li></ol>

De aangetoonde protocol is een in vitro benadering van de veel meer complexe kwestie van stamceltherapie in myocard infarct, met alle voordelen en nadelen van een dergelijk model. Uiteraard kan het geen afspiegeling van het complex (bijvoorbeeld immunologische) evenementen die plaatsvinden tijdens en na het hartinfarct, maar kan zich richten op de directe effecten van de toegevoegde cellen op het postischemic cellen. De effecten van gesimuleerde ischemie op H9c2 cardiomyoblasts sterk afhankelijk van het tijdstip van OGD...

<table border="1"><tbody><tr><td> Naam van het reagens </td><td> Vennootschap </td><td> Catalogus nummer </td><td> Reacties </td></tr><tr><td> calceïne-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 homodimeer-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> Tabel 1. Reagentia. <table border="1"><tbody><tr><td> Naam van het apparaat </td><td> Vennootschap </td><td> Commentaar (optioneel) </td></tr><tr><td> Pecon cel incubatie systeem voor Zeiss microscopen </td><td> Pecon GmbH </td><td> <a href="http://www.pecon.biz/">www.pecon.biz/</a> </td></tr></tbody></table> Tabel 2. Equipment.

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

Stamceltransplantatie protocollen vinden hun weg naar de klinische praktijk 1,2,3. Beter resultaat, waardoor de protocollen robuuster, en het vinden van nieuwe bronnen voor de implanteerbare cellen zijn de focus van recent onderzoek 4,5. Onderzoek naar de effectiviteit van de cel therapieën is niet een gemakkelijke taak en zijn nieuwe instrumenten nodig om de mechanismen betrokken bij het ​​behandelingsproces zes onderzoeken. We ontwierpen een experimenteel protocol van ischemie / reperfusie in om het observeren van cellulaire verbindingen en zelfs subcellulaire mechanismen tijdens ischemie / reperfusie schade en na stamceltransplantatie mogelijk te maken en de effectiviteit van celtherapie te evalueren. H9c2 cardiomyoblast cellen werden geplaatst op celcultuur platen 7,8. Ischemie werd gesimuleerd met 150 minuten in een glucose-vrij medium met zuurstof niveau lager dan 0,5%. Daarna werden de normale media en zuurstof niveaus opnieuw naar reperfusie te simuleren. Na zuurstof glucose ontbering,de beschadigde cellen werden behandeld met de transplantatie van gelabelde menselijke beenmerg afgeleide mesenchymale stamcellen toe te voegen aan de cultuur. Mesenchymale stamcellen de voorkeur in klinische studies, omdat ze gemakkelijk te bereiken met minimaal invasieve chirurgie, gemakkelijk uitbreidbaar en autologe. Na 24 uur co-teelt, werden cellen gekleurd met calceïne en ethidium-homodimeer onderscheid te maken tussen levende en dode cellen. Deze opzet liet ons toe om de intercellulaire verbindingen met behulp van confocale fluorescentie microscopie te onderzoeken en om de overleving van postischemic cellen door flowcytometrie kwantificeren. Confocale microscopie toonde de interacties van de twee celpopulaties zoals celfusie en de vorming van intercellulaire nanobuisjes. Flowcytometrie analyse bleek drie clusters van beschadigde cellen die kunnen worden uitgezet in een grafiek en statistisch geanalyseerd. Deze populatie kunnen afzonderlijk worden onderzocht en conclusies kunnen worden getrokken over deze gegevens over de effectiviteit van de gesimuleerdetherapeutische aanpak.

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

Laten we zien hoe het opzetten van een<em&gt; In vitro</em&gt; Ischemie / reperfusie model en hoe het effect van de stamceltherapie op postischemic cardiale cellen te evalueren.

Stamceltransplantatie in een In vitro Gesimuleerde ischemie / reperfusie model

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

Research

JoVE Journal

Medicine

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

A Mouse Model of in Utero Transplantation

A Mouse Model of in Utero Transplantation

Een muis model van de In Utero Transplantatie

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

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

Met behulp van kwantitatieve real-time PCR voor donorcel Engraftment bepalen in een concurrerende Murine beenmergtransplantatie Model

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

Ischemie-reperfusie model van Acute Kidney Injury en Post Letsel Fibrosis in Muizen

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

Neurale stamceltransplantatie bij Experimentele Contusive Model van 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 ...

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

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

Zuurstof-Glucose beroving en reoxygenatie als een<em&gt; In Vitro</em&gt; Ischemie-reperfusie letsel model voor de studie bloed-hersenbarrière 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

Studeren alvleesklier- Stamcel Kenmerken voor het ontwikkelen van nieuwe behandelingsstrategieën

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

Muizenmodel van Intestinale ischemie-reperfusie schade

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

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

Creatie en transplantatie van een blad van de stamcel adipeus afkomstige (ASC) in een diabetische wondgenezing Model

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

Muismodel van twee-schip occlusie van cerebrale ischemie-reperfusie

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

Een pre-klinische rat model voor de studie van ischemie-reperfusie letsel in reconstructieve microchirurgie

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