Атеросклеротические бляшки в системе ex vivo
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Materials and methods. Segments of coronary and carotid arteries with atherosclerotic plaques and normal arterial tissue were studied macroscopically and then dissected into small cubic blocks and large ring-shaped blocks. After that samples were cultured for 12-22 days in original culture medium. On the first day and every three days several samples of cultured tissue were analyzed histologically. For live/dead discrimination, cultured tissue samples were investigated with flow cytometry.
Results. We showed that tissue survival when cultured in small cubic blocks reduced to 8-12 day. According to the flow cytometry data the number of live cells was only 19 in 100 mg of cultured tissue. However, during cultivation of large ring-shaped blocks the survival of tissue was significantly higher. The integrity of endothelial layer and internal elastic membrane, the most sensitive to the changes in culture conditions, maintained for 20-22 days.
Conclusion. We have developed a method by which it became possible to cultivate the atherosclerotic plaque tissue ex vivo with preservation of the explants' architectonics during cultivation.
2. Cho Y. E., Choi J. E., Alam M. J. 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. Practice. 2008. Vol. 2. P. 74–79.
3. Fortunato G., Di Taranto M. D., Bracale U. M. et al. Decreased paraoxonase-2 expression in human carotids during the progression of atherosclerosis // Arterioscler. Thrombos. Vasc. Biol. 2008. Vol. 28. P. 594–600.
4. Grivel J.-C., Margolis L. Use of human tissue explants to study human infectious agents // Nat. Protoc. 2009. Vol. 4. P. 256–269.
5. Harrison R. Observations on the living developing nerve fiber // Anat. Rec. 1907. Vol. 1. P. 5–7.
6. Huang B., Dreyer T., Heidt M. 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. Vol. 21. P. 1002–1008.
7. Lebastchi A. H., Qin L., Khan S. F. et al. Activation of human vascular cells decreases their expression of transforming growth factor-beta // Atherosclerosis. 2011. Vol. 219. P. 417–424.
8. Levin V. A., Panchabhai S., Shen L. et al. Protein and phosphoprotein levels in glioma and adenocarcinoma cell lines grown in normoxia and hypoxia in monolayer and three-dimensional cultures // Proteome science. 2012. Vol. 10. P. 5.
9. Lu H., Searle K., Liu Y. et al. The effect of dimensionality on growth and differentiation of neural in vitro: 3-dimensional spheroid versus 2-dimensional monolayer culture // Cells, tissues, organs. 2012. Vol. 196. P. 48–55.
10. Moreno J. A., Ortega-Gomez A., Delbosc S. et al. In vitro and in vivo evidence for the role of elastase shedding of CD163 in human atherothrombosis // Eur. Heart J. 2011. Vol. 33, № 2. P. 252–263.
11. Ni C. W., Qiu H., Rezvan A. et al. Discovery of novel mechanosensitive genes in vivo using mouse carotid artery endothelium exposed to disturbed flow // Blood. 2010. Vol. 116. P. e66–73.
12. Poppert S., Schlaupitz K., Marre R. et al. Chlamydia pneumoniae in an ex vivo human artery culture model // Atherosclerosis. 2006. Vol. 187. P. 50–56.
13. Proudfoot D., Shanahan C. Human vascular smooth muscle cell culture // Methods Molecular Biol. 2012. Vol. 806. P. 251–263.
14. Rohr S. Cardiac fibroblasts in cell culture systems: myofibroblasts all along? // J. Cardiovasc. Pharmacol. 2011. Vol. 57. P. 389–399.
15. Sukovich D. A., Kauser K., Shirley F. D. et al. Expression of interleukin-6 in atherosclerotic lesions of male ApoE-knockout mice: inhibition by 17beta-estradiol // Arterioscler. Thrombos. Vasc. Biol. 1998. Vol. 18. P. 1498–1505.
16. Voisard R., Krugers T., Reinhardt B. et al. HCMVinfection in a human arterial organ culture model: effects on cell proliferation and neointimal hyperplasia // BMC Microbiol. 2007. Vol. 7. P. 68–74.
17. Voisard R., von Eicken J., Baur R. et al. A human arterial organ culture model of postangioplasty restenosis: results up to 56 days after ballooning // Atherosclerosis. 1999. Vol. 144. P. 123–134.
18. Wang B. Y., Ho H. K., Lin P. S. et al. Regression of atherosclerosis: role of nitric oxide and apoptosis // Circulation. 1999. Vol. 99. P. 1236–1241.
Abstract
Objectives. To develop a model of culturing human atherosclerotic plaques ex vivo.Materials and methods. Segments of coronary and carotid arteries with atherosclerotic plaques and normal arterial tissue were studied macroscopically and then dissected into small cubic blocks and large ring-shaped blocks. After that samples were cultured for 12-22 days in original culture medium. On the first day and every three days several samples of cultured tissue were analyzed histologically. For live/dead discrimination, cultured tissue samples were investigated with flow cytometry.
Results. We showed that tissue survival when cultured in small cubic blocks reduced to 8-12 day. According to the flow cytometry data the number of live cells was only 19 in 100 mg of cultured tissue. However, during cultivation of large ring-shaped blocks the survival of tissue was significantly higher. The integrity of endothelial layer and internal elastic membrane, the most sensitive to the changes in culture conditions, maintained for 20-22 days.
Conclusion. We have developed a method by which it became possible to cultivate the atherosclerotic plaque tissue ex vivo with preservation of the explants' architectonics during cultivation.
References
1. Bachetti T., Morbidelli L. Endothelial cells in culture: a model for studying vascular functions // Pharmacol. Res. 2000. Vol. 42. P. 9–19.2. Cho Y. E., Choi J. E., Alam M. J. 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. Practice. 2008. Vol. 2. P. 74–79.
3. Fortunato G., Di Taranto M. D., Bracale U. M. et al. Decreased paraoxonase-2 expression in human carotids during the progression of atherosclerosis // Arterioscler. Thrombos. Vasc. Biol. 2008. Vol. 28. P. 594–600.
4. Grivel J.-C., Margolis L. Use of human tissue explants to study human infectious agents // Nat. Protoc. 2009. Vol. 4. P. 256–269.
5. Harrison R. Observations on the living developing nerve fiber // Anat. Rec. 1907. Vol. 1. P. 5–7.
6. Huang B., Dreyer T., Heidt M. 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. Vol. 21. P. 1002–1008.
7. Lebastchi A. H., Qin L., Khan S. F. et al. Activation of human vascular cells decreases their expression of transforming growth factor-beta // Atherosclerosis. 2011. Vol. 219. P. 417–424.
8. Levin V. A., Panchabhai S., Shen L. et al. Protein and phosphoprotein levels in glioma and adenocarcinoma cell lines grown in normoxia and hypoxia in monolayer and three-dimensional cultures // Proteome science. 2012. Vol. 10. P. 5.
9. Lu H., Searle K., Liu Y. et al. The effect of dimensionality on growth and differentiation of neural in vitro: 3-dimensional spheroid versus 2-dimensional monolayer culture // Cells, tissues, organs. 2012. Vol. 196. P. 48–55.
10. Moreno J. A., Ortega-Gomez A., Delbosc S. et al. In vitro and in vivo evidence for the role of elastase shedding of CD163 in human atherothrombosis // Eur. Heart J. 2011. Vol. 33, № 2. P. 252–263.
11. Ni C. W., Qiu H., Rezvan A. et al. Discovery of novel mechanosensitive genes in vivo using mouse carotid artery endothelium exposed to disturbed flow // Blood. 2010. Vol. 116. P. e66–73.
12. Poppert S., Schlaupitz K., Marre R. et al. Chlamydia pneumoniae in an ex vivo human artery culture model // Atherosclerosis. 2006. Vol. 187. P. 50–56.
13. Proudfoot D., Shanahan C. Human vascular smooth muscle cell culture // Methods Molecular Biol. 2012. Vol. 806. P. 251–263.
14. Rohr S. Cardiac fibroblasts in cell culture systems: myofibroblasts all along? // J. Cardiovasc. Pharmacol. 2011. Vol. 57. P. 389–399.
15. Sukovich D. A., Kauser K., Shirley F. D. et al. Expression of interleukin-6 in atherosclerotic lesions of male ApoE-knockout mice: inhibition by 17beta-estradiol // Arterioscler. Thrombos. Vasc. Biol. 1998. Vol. 18. P. 1498–1505.
16. Voisard R., Krugers T., Reinhardt B. et al. HCMVinfection in a human arterial organ culture model: effects on cell proliferation and neointimal hyperplasia // BMC Microbiol. 2007. Vol. 7. P. 68–74.
17. Voisard R., von Eicken J., Baur R. et al. A human arterial organ culture model of postangioplasty restenosis: results up to 56 days after ballooning // Atherosclerosis. 1999. Vol. 144. P. 123–134.
18. Wang B. Y., Ho H. K., Lin P. S. et al. Regression of atherosclerosis: role of nitric oxide and apoptosis // Circulation. 1999. Vol. 99. P. 1236–1241.
