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

Este trabalho foi financiado pela OTKA (Hungarian Fundo de Investigação Científica) D45933, T049621, TET (Hungarian Ciência e Tecnologia Foundation) A4/04, Arg-17/2006 e SIN, Bolyai, Bolsas e Öveges TÁMOP 4.2.2-08 / 1 / KMR-2008-0004 e 4.2.1 / B 09/1/KMR-2010-0001. OTKA 83803. Gostaríamos de agradecer Gesztes William para fornecer a voz-over.

1. Preparando H9c2 células cardiomyoblast H9c2 cardiomyoblasts ratos foram obtidos a partir ATCC (Wesel, Alemanha) e ampliado em glicose (4,5 g / L) DMEM contendo 10% de soro fetal bovino, 4 mM L-glutamina, 100 U / ml de penicilina e estreptomicina 100 mg / ml. O H9c2 linha celular de mioblastos é derivada de coração de rato embrionárias, é usada como um modelo in vitro para ambos os músculos esqueléticos e cardíaco 8,9. Prepare 12 placa bem de ...

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O protocolo demonstrado in vitro é uma abordagem para a questão muito mais complexa da terapia com células-tronco em infarto do miocárdio, com todas as vantagens e desvantagens de tal modelo. Obviamente, ele não pode refletir a complexa (imunológica, por exemplo) eventos que ocorrem durante e após infarto do miocárdio, mas pode se concentrar sobre os efeitos diretos das células adicionado nas células pós-isquêmica. Os efeitos da isquemia simulada sobre H9c2 cardiomyoblasts depende muito do tempo de OGD, sobr...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> calceína-AM </td><td> Sondas moleculares </td><td> L3224, C3099 </td><td> <a href="http://www.invitrogen.com">http://www.invitrogen.com</a> </td></tr><tr><td> homodímero de etídio-2 </td><td> Sondas moleculares </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> Sondas moleculares </td><td> V22887 </td><td> <a href="http://www.invitrogen.com">http://www.invitrogen.com</a> </td></tr></tbody></table> Tabela 1. Reagentes. <table border="1"><tbody><tr><td> Nome do equipamento </td><td> Companhia </td><td> Comments (opcional) </td></tr><tr><td> Pecon sistema de incubação de células para microscópios Zeiss </td><td> Pecon GmbH </td><td> <a href="http://www.pecon.biz/">www.pecon.biz/</a> </td></tr></tbody></table> Equipamentos tabela 2..

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

Protocolos de transplante de células-tronco estão encontrando seu caminho na prática clínica 1,2,3. Obter melhores resultados, fazendo com que os protocolos mais robustos, e encontrar novas fontes de células implantáveis ​​são o foco de pesquisas recentes 4,5. Investigar a eficácia de terapias celulares não é uma tarefa fácil e novas ferramentas são necessários para investigar os mecanismos envolvidos no processo de tratamento 6. Nós projetamos um protocolo experimental de isquemia / reperfusão, a fim de permitir a observação de conexões celulares e mecanismos subcelulares mesmo durante a isquemia / reperfusão e após o transplante de células-tronco e para avaliar a eficácia da terapia celular. H9c2 cardiomyoblast células foram colocadas em placas de cultura de células 7,8. Isquemia foi simulado com 150 minutos em um meio de glicose livre com nível de oxigênio abaixo de 0,5%. Então, a mídia normal e os níveis de oxigênio foram reintroduzidos para simular reperfusão. Após a privação de glicose oxigênio,as células danificadas foram tratados com transplante de medula óssea humana rotulados derivados de células-tronco mesenquimais, adicionando-os à cultura. Células-tronco mesenquimais são as preferidas em ensaios clínicos, porque eles são facilmente acessíveis com cirurgia minimamente invasiva, facilmente expansível e autólogo. Após 24 horas de co-cultivo, as células foram coradas com calceína e de etídeo-homodímero de diferenciar entre células vivas e mortas. Esta configuração permitiu investigar as conexões intercelulares por microscopia de fluorescência confocal e quantificar a taxa de sobrevivência de células pós-isquêmica por citometria de fluxo. Microscopia confocal mostrou as interações das duas populações de células, tais como fusão celular e formação de nanotubos intercelular. Citometria de fluxo revelou três grupos de células danificadas que podem ser plotados em um gráfico e analisados ​​estatisticamente. Essas populações podem ser investigados separadamente e conclusões podem ser tiradas destes dados sobre a eficácia do simuladoabordagem terapêutica.

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

Demonstramos como configurar um<em&gt; In vitro</em&gt; Isquemia / reperfusão modelo e como avaliar o efeito da terapia com células-tronco em células cardíacas pós-isquêmica.

Transplante de Células-tronco em um In vitro Modelo de isquemia / reperfusão Simulated

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

Um modelo do rato de No Utero Transplante

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

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

Usando Quantitative PCR em tempo real para determinar enxerto célula dadora num modelo murino de osso Competitiva Marrow Transplantation

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

Isquemia-reperfusão modelo de lesão renal aguda e Pós Lesão fibrose em camundongos

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

Neural Stem Cell Transplantation em Experimental contusão 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 ...

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

Oxigênio-Glucose Privação e reoxigenação como um<em&gt; In Vitro</em&gt; Isquemia e reperfusão Lesão modelo para estudar Disfunção Barreira Hematoencefálica

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

Estudar o cancro do pâncreas Stem características celulares para desenvolver estratégias Novo tratamento

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

Modelo murino de isquemia-reperfusão

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

Criação e transplante de uma folha de células-tronco adiposo-derivado (ASC) em um modelo de cicatrização de feridas diabética

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

Modelo de Mouse de dois vasos oclusão de isquémia-reperfusão Cerebral

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

Um modelo pré-clínico do rato para o estudo da lesão da Isquemia-reperfusion na microcirurgia reconstrutiva

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