Isolation of valvular endothelial cells.
Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (α-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.
Department of Biomedical Engineering, Cornell University.
Russell A
RA
Gould
Jonathan T
JT
Butcher
D000818:Animals; D003584:Cytological Techniques / Q000379:methods*; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D006351:Heart Valves / Q000166:cytology* / Q000378:metabolism; D006801:Humans
Circ Res. 2005 Apr 15;96(7):792-9
15761200
Am J Pathol. 2001 Oct;159(4):1335-43
11583961
Arterioscler Thromb Vasc Biol. 2004 Aug;24(8):1429-34
15117733
Am J Anat. 1979 Apr;154(4):545-56
433796
Lab Invest. 1983 Nov;49(5):589-98
6314039
J Mol Cell Cardiol. 1988 Feb;20(2):103-18
2840511
Arterioscler Thromb Vasc Biol. 2006 Jan;26(1):69-77
16293796
Blood. 1998 Jul 15;92(2):362-7
9657732
Circulation. 2003 Mar 4;107(8):1164-9
12615796
J Heart Valve Dis. 2008 Nov;17(6):674-81
19137801
Circ Res. 2006 Oct 13;99(8):861-9
16973908