Ex vivo culture of human atherosclerotic plaques: A model to study immune cells in atherogenesis


      • A new ex vivo model of human atherosclerotic plaques has been established.
      • The cytoarchitecture of the plaques was well preserved for 19 days in culture.
      • The major plaque cell types were preserved for the entire culture period.
      • Plaque tissue continued to release cytokines and chemokines during culture.
      • This model can be used to study complex immunological aspects of atherogenesis.


      Background and aims

      The mechanisms that drive atherosclerotic plaque progression and destabilization in humans remain largely unknown. Laboratory models are needed to study these mechanisms under controlled conditions. The aim of this study was to establish a new ex vivo model of human atherosclerotic plaques that preserves the main cell types in plaques and the extracellular components in the context of native cytoarchitecture.


      Atherosclerotic plaques from carotid arteries of 28 patients undergoing carotid endarterectomy were dissected and cultured. At various time-points, samples were collected and analysed histologically. After enzymatic digestion, single cells were analysed with flow cytometry. Moreover, tissue cytokine production was evaluated.


      We optimised the plaque dissection protocol by cutting plaques into circular segments that we cultured on collagen rafts at the medium–air interface, thus keeping them well oxygenated. With this technique, the relative presence of T and B lymphocytes did not change significantly during culture, and the sizes of lymphocyte subsets remained stable after day 4 of culture. Macrophages, smooth muscle cells, and fibroblasts with collagen fibres, as well as T and B lymphocyte subsets and CD16 natural killer cells, remained largely preserved for 19 days of culture, with a continuous production of inflammatory cytokines and chemokines.


      Our new model of ex vivo human atherosclerotic plaques, which preserves the main subsets of immune cells in the context of tissue cytoarchitecture, may be used to investigate important aspects of atherogenesis, in particular, the functions of immune cells under controlled laboratory conditions.

      Graphical abstract


      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Atherosclerosis
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Shaikh M.
        • Martini S.
        • Quiney J.R.
        • Baskerville P.
        • La Ville A.E.
        • Browse N.L.
        • et al.
        Modified plasma-derived lipoproteins in human atherosclerotic plaques.
        Atherosclerosis. 1988; 69: 165-172
        • Jonasson L.
        • Holm J.
        • Skalli O.
        • Bondjers G.
        • Hansson G.K.
        Regional accumulations of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque.
        Arteriosclerosis. 1986; 6: 131-138
        • Santos-Gallego C.G.
        • Picatoste B.
        • Badimón J.J.
        Pathophysiology of acute coronary syndrome.
        Curr. Atheroscler. Rep. 2014; 16: 401
        • Nikitskaya E.
        • Lebedeva A.
        • Ivanova O.
        • Maryukhnich E.
        • Shpektor A.
        • Grivel J.
        • et al.
        Cytomegalovirus-productive infection is associated with acute coronary syndrome.
        J. Am. Heart Assoc. 2016; 5: e003759
        • Finn A.V.
        • Nakano M.
        • Narula J.
        • Kolodgie F.D.
        • Virmani R.
        Concept of vulnerable/unstable plaque.
        Arterioscler. Thromb. Vasc. Biol. 2010; 30: 1282-1292
        • Taleb S.
        • Tedgui A.
        • Mallat Z.
        Adaptive T cell immune responses and atherogenesis.
        Curr. Opin. Pharmacol. 2010; 10: 197-202
        • Libby P.
        • Okamoto Y.
        • Rocha V.Z.
        • Folco E.
        Inflammation in atherosclerosis: transition from theory to practice.
        Circ. J. 2010; 74: 213-220
        • Zhou X.
        • Nicoletti A.
        • Elhage R.
        • Hansson G.K.
        Transfer of CD4(+) T cells aggravates atherosclerosis in immunodeficient apolipoprotein E knockout mice.
        Circulation. 2000; 102: 2919-2922
        • Ylä-Herttuala S.
        • Palinski W.
        • Butler S.W.
        • Picard S.
        • Steinberg D.
        • Witztum J.L.
        Rabbit and human atherosclerotic lesions contain IgG that recognizes epitopes of oxidized LDL.
        Arterioscler. Thromb. a J. Vasc. Biol. 1994; 14: 32-40
        • Grivel J.-C.
        • Ivanova O.
        • Pinegina N.
        • Blank P.S.
        • Shpektor A.
        • Margolis L.B.
        • et al.
        Activation of T lymphocytes in atherosclerotic plaques.
        Arterioscler. Thromb. Vasc. Biol. 2011; 31: 2929-2937
        • Zhou X.
        • Stemme S.
        • Hansson G.K.
        Evidence for a local immune response in atherosclerosis. CD4+ T cells infiltrate lesions of apolipoprotein-E-deficient mice.
        Am. J. Pathol. 1996; 149: 359-366
        • Getz G.S.
        • Reardon C.A.
        Animal models of atherosclerosis.
        Arterioscler. Thromb. Vasc. Biol. 2012; 32: 1104-1115
        • Dorweiler B.
        • Torzewski M.
        • Dahm M.
        • Ochsenhirt V.
        • Lehr H.-A.
        • Lackner K.J.
        • et al.
        A novel in vitro model for the study of plaque development in atherosclerosis.
        Thromb. Haemost. 2006; 95: 182-189
        • Robert J.
        • Weber B.
        • Frese L.
        • Emmert M.Y.
        • Schmidt D.
        • von Eckardstein A.
        • et al.
        A three-dimensional engineered artery model for in vitro atherosclerosis research.
        PLoS One. 2013; 8: e79821
        • Ross R.
        • Agius L.
        The process of atherogenesis–cellular and molecular interaction: from experimental animal models to humans.
        Diabetologia. 1992; 35: S34-S40
        • Badimon L.
        Atherosclerosis and thrombosis: lessons from animal models.
        Thromb. Haemost. 2001; 86: 356-365
        • Li L.N.
        • Margolis L.B.
        • Hoffman R.M.
        Skin toxicity determined in vitro by three-dimensional, native-state histoculture.
        Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1908-1912
        • Hoffman R.M.
        Three-dimensional histoculture: origins and applications in cancer research.
        Cancer cells. 1991; 3: 86-92
        • Grivel J.-C.
        • Margolis L.
        Use of human tissue explants to study human infectious agents.
        Nat. Protoc. 2009; 4: 256-269
        • Virchov R.L.K.
        Virchow, R.L.K. Cellular Pathology 1859.
        (special ed.) Churchill, John, London, UK1978
        • Thorp E.
        • Cui D.
        • Schrijvers D.M.
        • Kuriakose G.
        • Tabas I.
        Mertk receptor mutation reduces efferocytosis efficiency and promotes apoptotic cell accumulation and plaque necrosis in atherosclerotic lesions of apoe-/- mice.
        Arterioscler. Thromb. Vasc. Biol. 2008; 28: 1421-1428
        • Kruth H.S.
        Sequestration of aggregated low-density lipoproteins by macrophages.
        Curr. Opin. Lipidol. 2002; 13: 483-488
        • Hartvigsen K.
        • Chou M.-Y.
        • Hansen L.F.
        • Shaw P.X.
        • Tsimikas S.
        • Binder C.J.
        • et al.
        The role of innate immunity in atherogenesis.
        J. Lipid Res. 2008; 50: S388-S393
        • Liuzzo G.
        • Biasucci L.M.
        • Trotta G.
        • Brugaletta S.
        • Pinnelli M.
        • Digianuario G.
        • et al.
        Unusual CD4+CD28null T lymphocytes and recurrence of acute coronary events.
        J. Am. Coll. Cardiol. 2007; 50: 1450-1458
        • De Palma R.
        • Del Galdo F.
        • Abbate G.
        • Chiariello M.
        • Calabró R.
        • Forte L.
        • et al.
        Patients with acute coronary syndrome show oligoclonal T-cell recruitment within unstable plaque: evidence for a local, intracoronary immunologic mechanism.
        Circulation. 2006; 113: 640-646
        • Rossmann A.
        • Henderson B.
        • Heidecker B.
        • Seiler R.
        • Fraedrich G.
        • Singh M.
        • et al.
        T-cells from advanced atherosclerotic lesions recognize hHSP60 and have a restricted T-cell receptor repertoire.
        Exp. Gerontol. 2008; 43: 229-237
        • Ait-Oufella H.
        • Sage A.P.
        • Mallat Z.
        • Tedgui A.
        Adaptive (T and B Cells) immunity and control by dendritic cells in atherosclerosis.
        Circ. Res. 2014; 114: 1640-1660
        • Finking G.
        • Hanke H.
        • Anitschkow N.
        • Chalatow S.
        • Anitschkow N.
        • Bocan T.
        • et al.
        Nikolaj Nikolajewitsch Anitschkow (1885-1964) established the cholesterol-fed rabbit as a model for atherosclerosis research.
        Atherosclerosis. 1997; 135: 1-7
        • Hopkins P.N.
        Molecular biology of atherosclerosis.
        Physiol. Rev. 2013; 93: 1317-1542
        • Antonov A.S.
        • Nikolaeva M.A.
        • Klueva T.S.
        • YuA Romanov
        • Babaev V.R.
        • Bystrevskaya V.B.
        • et al.
        Primary culture of endothelial cells from atherosclerotic human aorta. Part 1. Identification, morphological and ultrastructural characteristics of two endothelial cell subpopulations.
        Atherosclerosis. 1986; 59: 1-19
        • MacLeod D.C.
        • Strauss B.H.
        • de Jong M.
        • Escaned J.
        • Umans V.A.
        • van Suylen R.J.
        • et al.
        Proliferation and extracellular matrix synthesis of smooth muscle cells cultured from human coronary atherosclerotic and restenotic lesions.
        J. Am. Coll. Cardiol. 1994; 23: 59-65
        • Tume R.K.
        • Bradley T.R.
        • Day A.J.
        An investigation by tissue culture techniques on the growth of foam cells isolated from rabbit atherosclerotic lesions.
        J. Atheroscler. Res. 1969; 9: 151-157
        • Davies P.F.
        • Truskey G.A.
        • Warren H.B.
        • O'Connor S.E.
        • Eisenhaure B.H.
        Metabolic cooperation between vascular endothelial cells and smooth muscle cells in co-culture: changes in low density lipoprotein metabolism.
        J. Cell Biol. 1985; 101: 871-879
        • Napoleone E.
        • Di Santo A.
        • Lorenzet R.
        Monocytes upregulate endothelial cell expression of tissue factor: a role for cell-cell contact and cross-talk.
        Blood. 1997; 89: 541-549
        • Takaku M.
        • Wada Y.
        • Jinnouchi K.
        • Takeya M.
        • Takahashi K.
        • Usuda H.
        • et al.
        An in vitro coculture model of transmigrant monocytes and foam cell formation.
        Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2330-2339
        • van Buul-Wortelboer M.F.
        • Brinkman H.J.
        • Dingemans K.P.
        • de Groot P.G.
        • van Aken W.G.
        • van Mourik J.A.
        Reconstitution of the vascular wall in vitro. A novel model to study interactions between endothelial and smooth muscle cells.
        Exp. Cell Res. 1986; 162: 151-158
        • Chiu J.-J.
        • Chen L.-J.
        • Lee P.-L.
        • Lee C.-I.
        • Lo L.-W.
        • Usami S.
        • et al.
        Shear stress inhibits adhesion molecule expression in vascular endothelial cells induced by coculture with smooth muscle cells.
        Blood. 2003; 101: 2667-2674
        • Rainger G.E.
        • Nash G.B.
        Cellular pathology of atherosclerosis: smooth muscle cells prime cocultured endothelial cells for enhanced leukocyte adhesion.
        Circ. Res. 2001; 88: 615-622
        • Ni C.-W.
        • Qiu H.
        • Rezvan A.
        • Kwon K.
        • Nam D.
        • Son D.J.
        • et al.
        Discovery of novel mechanosensitive genes in vivo using mouse carotid artery endothelium exposed to disturbed flow.
        Blood. 2010; 116: e66-73
        • Cho Y.-E.
        • Choi J.-E.
        • Alam M.J.
        • Lee M.-H.
        • Sohn H.-Y.
        • Beattie J.H.
        • et al.
        Zinc deficiency decreased cell viability both in endothelial EA.hy926 cells and mouse aortic culture ex vivo and its implication for anti-atherosclerosis.
        Nutr. Res. Pract. 2008; 2: 74-79
        • Huang B.
        • Dreyer T.
        • Heidt M.
        • Yu J.C.M.
        • Philipp M.
        • Hehrlein F.W.
        • et al.
        Insulin and local growth factor PDGF induce intimal hyperplasia in bypass graft culture models of saphenous vein and internal mammary artery.
        Eur. J. Cardiothorac. Surg. 2002; 21: 1002-1008
        • Poppert S.
        • Schlaupitz K.
        • Marre R.
        • Voisard R.
        • Roessler W.
        • Weckermann D.
        • et al.
        Chlamydia pneumoniae in an ex vivo human artery culture model.
        Atherosclerosis. 2006; 187: 50-56
        • Voisard R.
        • von Eicken J.
        • Baur R.
        • Gschwend J.E.
        • Wenderoth U.
        • Kleinschmidt K.
        • et al.
        A human arterial organ culture model of postangioplasty restenosis: results up to 56 days after ballooning.
        Atherosclerosis. 1999; 144: 123-134
        • Wang B.Y.
        • Ho H.K.
        • Lin P.S.
        • Schwarzacher S.P.
        • Pollman M.J.
        • Gibbons G.H.
        • et al.
        Regression of atherosclerosis: role of nitric oxide and apoptosis.
        Circulation. 1999; 99: 1236-1241
        • Moreno J.A.
        • Ortega-Gómez A.
        • Delbosc S.
        • Beaufort N.
        • Sorbets E.
        • Louedec L.
        • et al.
        In vitro and in vivo evidence for the role of elastase shedding of CD163 in human atherothrombosis.
        Eur. Heart J. 2012; 33: 252-263
        • Fortunato G.
        • Di Taranto M.D.
        • Bracale U.M.
        • Del Guercio L.
        • Carbone F.
        • Mazzaccara C.
        • et al.
        Decreased paraoxonase-2 expression in human carotids during the progression of atherosclerosis.
        Arterioscler. Thromb. Vasc. Biol. 2008; 28: 594-600
        • Sukovich D.A.
        • Kauser K.
        • Shirley F.D.
        • DelVecchio V.
        • Halks-Miller M.
        • Rubanyi G.M.
        Expression of interleukin-6 in atherosclerotic lesions of male ApoE-knockout mice: inhibition by 17beta-estradiol.
        Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1498-1505
        • Lebastchi A.H.
        • Qin L.
        • Khan S.F.
        • Zhou J.
        • Geirsson A.
        • Kim R.W.
        • et al.
        Activation of human vascular cells decreases their expression of transforming growth factor-beta.
        Atherosclerosis. 2011; 219: 417-424
        • Erbel C.
        • Okuyucu D.
        • Akhavanpoor M.
        • Zhao L.
        • Wangler S.
        • Hakimi M.
        • et al.
        A human ex vivo atherosclerotic plaque model to study lesion biology.
        J. Vis. Exp. 2014; : 50542
        • Sainz T.
        • Serrano-Villar S.
        • Díaz L.
        • Tomé M.I.G.
        • Gurbindo M.D.
        • de José M.I.
        • et al.
        The CD4/CD8 ratio as a marker T-cell activation, senescence and activation/exhaustion in treated HIV-infected children and young adults.
        AIDS. 2013; 27: 1513-1516