Aislamiento y cultivo primario de células endoteliales aórticas de ratón
Summary
The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.
Abstract
The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.
Introduction
El endotelio vascular es no sólo una capa de barrera que separa la sangre y el tejido, se considera una amplia glándula endocrina que se extiende sobre todo el árbol vascular con una superficie de 400 metros cuadrados 1. El bienestar del endotelio es esencial para la homeostasis vascular. El endotelio disfuncional participa en diversos trastornos cardiovasculares, incluyendo la aterosclerosis, vasculitis y las lesiones por isquemia / reperfusión, etc. 2-4. Hasta la fecha, los mecanismos celulares y moleculares específicos implicados en estos ajustes de la enfermedad no se comprenden bien debido a la naturaleza anatómica difusa de endotelio.
El ratón es un modelo importante para la investigación porque las técnicas de manipulación genética están más desarrollados en ratones que en cualquier otra especie de mamífero. Sin embargo, el aislamiento de células endoteliales aórticas primaria murino se considera particularmente difícil debido a que el pequeño tamaño de la aorta hace enzymatdigestión ic de endotelio poco práctico. Algunos informaron procedimientos para aislar y purificar las CE exijan 5-7.
El objetivo de este protocolo es el uso de un método simple para aislar y expandir las células endoteliales de la aorta de ratón sin utilizar ningún equipo especial. En este protocolo, la aorta recién aisladas se corta en pequeños segmentos y se sembró sobre una matriz con el endotelio hacia abajo para permitir la germinación endotelial. Después de segmentos se eliminan, las células endoteliales se expandieron en medio del endotelio favorecida y están listas para experimentos después de dos o tres pasajes. Las ventajas del método descrito son que: 1) considerablemente alto número de células endoteliales se obtienen de una sola aorta; 2) la viabilidad celular está bien conservada; y 3) no se necesita ningún equipo o técnica especial. Se proporciona un medio eficaz para la identificación de los mecanismos celulares y moleculares específicos en la fisiopatología de las células endoteliales. Para aquellos que estén interesados en el estudio de prLas células endoteliales cultivadas imary de cualquiera de los genes de ratones knock-out, genes knock-en ratones, o un modelo murino de la enfermedad, este protocolo es muy útil y fácil de practicar.
Protocol
Representative Results
Discussion
Este estudio demuestra un método simple para aislar y endotelial cultura células de una aorta de ratón sin ningún equipo especial. La tinción de inmunofluorescencia indicó que la mayoría de las células eran células endoteliales después de la segunda pasaje. Se sugiere que las células en segundo-tercio paso son apropiados para in vitro e in vivo para estudiar la biología endotelial.
Las Notas clave de la presente Protocolo
<p class="jove_cont…Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This study is supported by American Heart Association Scientist Development Grant 13SDG16930098 and the National Science Foundation of China Youth Award 81300240 (PI: Wang). We thank Roberto Mendez from Wayne State University for assisting in the preparation of the manuscript.
Materials
| 4- or 6-week-old mice (Jackson Laboratory, #000664). |
| Sterile 1X phosphate-buffered saline (PBS, Gibco, #10010-023). |
| Sterile 1X PBS containing 1,000 U/ml of heparin (Sigma Aldrich, H3149). |
| Endothelial cell growth medium (Dulbeccos’ Modified Eagle’s Medium[DMEM] with 25mM HEPES[Gibco, #12320-032 ], supplemented with 100μg/ml endothelial cell growth supplement from bovine neural tissue [ECGS, Sigma,#2759], 10% fetal bovine serum [FBS, Gibco, #10082-147], 1,000 U/ml heparin [Sigma Aldrich, H3149], 10,000U/ml penicillin and 10mg/ml streptomycin [Gibco, #15140-122]). |
| Growth factor reduced matrix (BD Biosciences, #356231). |
| Neutral proteinase (Dispase, 1U/ml, Fisher Scientific #CB-40235) and D-Val medium (D-Valine, 0.034g/L[Sigma, #1255], in Dulbeccos’ Modified Eagle’s Medium, low glucose[Gibco, #12320-032 ]). |
| Gelatin (100‑200 μg/cm2, Sigma, #G1393). |
| 1,1`-dioctadecyl-3,3,3`,3`- tetramethylindo- carbocyanine perchlorate-labeled acetylated LDL (Dil-ac-LDL, Life Technologies, #L3484), FITC-labeled Ulex europeus agglutinin (Ulex-Lectin, Sigma, #L9006), Anti-mouse CD31-FITC conjugated antibody (BD Biosciences, # 553372). |
| Anti-mouse vascular endothelial growth factor receptor 2 antibody (Cell signaling, #9698), anti-mouse endothelial nitric oxide synthase (Abcam, #ab5589), anti-mouse vascular endothelium-cadherin (Abcam, #33168), anti-mouse calponin (Abcam, #700), FITC-conjugated anti-rabbit IgG (Sigma, #F6005) |
| One ml syringe fitted with 25-G needle (Fisher Scientific, #50-900-04222). |
| 100mm Peri dishes (Fisher Scientific, #07-202-516). |
| Six-well cell culture plates (Fisher Scientific, #08-772-1B). |
| T12.5 cultuer flask (Fisher Scientific, #50-202-076) |
| Scissors, forceps, microdissection scissors and forceps, Scalpel blade (Fine Science Tools, Inc) |
| Anesthesia machine with isoflurane (Webster Veterianary Supply, #07-806-3204), heating lamp |
| Centrifuge machine. |
| Inverted phase-contrast microscope. |
| inverted fluorescence microscope. |
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