Name: isolation and culture of primary endothelial cells from canine arteries and veins
Uploaded: 2016-11-18T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins at JoVE.com

The authors would like to acknowledge Hans de Graaf and Tomas Veenendaal for their technical assistance in culturing the ECs.

Ética declaração: Os vasos sanguíneos utilizados neste estudo foram colhidas como material excedente obtido a partir de cadáveres caninos frescos (n = 4) a partir de cães saudáveis ​​sacrificados para outras pesquisas independentes (a política Universidade 3R). vasos sanguíneos anormais (desvios portossistêmicos intra e extra-hepáticos, n = 1 cada) foram colhidas post-mortem após consentimento informado dos proprietários de cães apresentados com a Clínica Universitária de Animais de Companhia da Univ...

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Em estudos com foco em ECs canino a linha primária CnAOEC é usado para modelar as linhagens endoteliais do cão 3, 12, 13. Em estudos humanos, a cultura HUVEC ainda é considerada o padrão-ouro. Claramente, concentrando-se apenas em ECs derivadas do cordão umbilical é uma restrição firme na investigação cardiovascular. As células endoteliais têm um padrão de expressão de genes específicos de determinar especificação arteriovenosa. A fim de levar em conta essas diferenças nos vasos pós-natal ...

Cães são usados como modelo animal grande para a pesquisa da doença cardiovascular e também podem sofrer de anormalidades vasculares (genéticas) inatos 1, 2. Para estudar estas doenças linhas de células endoteliais comerciais são muitas vezes utilizados para avaliar a funcionalidade das células endoteliais (CE). Para os cães que há uma linha de células endoteliais comercial disponível (CnAOEC), derivadas da aorta canino. Esta linha celular é usado principalmente em estudos como controlo normal ECs 3-5. Na pesquisa cardiovascular humano as linhas de células endoteliais mais utilizados são células endoteliais da veia umbilical humana (HUVECs) e umbilical humano, Artéria células endoteliais (HUAECs) derivados da veia do cordão umbilical humano e artéria, respectivamente. HUVECs foram usados como o padrão de ouro na pesquisa vascular desde a década de 1980 6. Eles são considerados como sendo o sistema de modelo clássico para estudar a função endotelial e doença de adaptação. As células endoteliais isoladas a partir de diferentes vasos sanguíneos variam em appearance e funcionalidade devido às características genéticas e exposição ao microambiente 7. Além disso, células HUVEC e HUAECs são derivados a partir do cordão umbilical, uma estrutura vascular do desenvolvimento que não pode vasos sanguíneos adulto totalmente mímica com respeito às condições a que estão expostos e resposta à doença. Assim, traduzindo resultados encontrados em HUVECs e HUAECs a doenças cardiovasculares, em geral, é inadequada. Ao estudar a adaptação e comportamento do adulto ECs, ECs primários do vaso de interesse deve ser usado como uma abordagem mais direta. Para isolar estas células, vários métodos têm sido relatados. Um método amplamente descrito, que também é usada para HUVEC, é a lavagem do recipiente com uma solução de digestão enzimática 8. Isto muitas vezes resulta na contaminação com não-CEs, tais como células de músculo liso e de fibroblastos 9. Outro método frequentemente utilizado para o isolamento é a digestão enzimática do tecido navio picada seguido por fluorescence-de células activadas (FACS) com base no marcador de células endoteliais de Cluster de diferenciação (CD) 31 7, 8. separação por FACS e cultura de células subsequente requer quantidades relativamente grandes de células e, portanto, não é apropriado para o isolamento do endotélio de pequenos vasos sanguíneos. Por conseguinte, objectivo desenvolver um novo método robusto para isolamento de uma população pura de células endoteliais a partir de vários vasos sanguíneos caninos com elevado grau de pureza. Para testar a eficiência do novo método de isolamento, foram isoladas e obteve culturas puras Canine primária de células endoteliais (Capec) de diferentes artérias caninas e veias, grandes e pequenas. Este método também permite a cultura de células endoteliais provenientes do doente e / ou vasos aberrantes, tais como desvios portossistêmicos intra ou extra-hepáticas inatos, uma doença comum em cães 2. O método permite o isolamento de tipos de células relevantes adicionais do mesmo recipiente, tais como as células do músculo liso vascular, uma vez mais da embarcação permanece intagir durante o procedimento.

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isolation and culture of primary endothelial cells from canine arteries and veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Vasos sanguíneos diferentes foram submetidas com sucesso para o protocolo de isolamento descrito (Figura 2). Foi possível dissecar e inverter aorta, veia cava, veia porta e artéria coronária em cães saudáveis ​​(todos os vasos de cada cão, n = 4). Com os mesmos ECs abordagem foram isolados a partir de dois desvios portossistêmicos congênitos (extra-hepáticas e intra-hepática, n = 1 cada). Embora aorta foi facilmente invertida, segmentos da aorta torácica ...

Watch this Scientific Journal Video about Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins at JoVE.com

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

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