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Abstract

Immunology and Infection

Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat

Published: February 4th, 2021

DOI:

10.3791/62059

1Department of Cardiology, Campus Benjamin Franklin, Charité Universitätsmedizin Berlin and German Centre for Cardiovascular Research (DZHK), 2Department of Experimental and Clinical Medicine, University of Florence, 3Berlin Institute of Health, 4Department of Cardiology, German Heart Centre Berlin (DHZB), 5Sod of Interdisciplinary Internal Medicine, Azienda Ospedaliera Universitaria Careggi (AOUC), 6Department of General and Interventional Cardiology, Helios Klinikum Erfurt

* These authors contributed equally

Abstract

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high mortality, despite new therapeutic options such as transcatheter aortic valve replacement (TAVR).

The complete pathways that start with valve calcification and lead to severe aortic stenosis remain only partly understood. By providing a close representation of the aortic valve cells in vivo, the assaying of T lymphocytes from stenotic valve tissue could be an efficient way to clarify their role in the development of calcification. After surgical excision, the fresh aortic valve sample is dissected in small pieces and the T lymphocytes are cultured, cloned then analyzed using fluorescence activated cell sorting (FACS).

The staining procedure is simple and the stained tubes can also be fixed using 0.5% of paraformaldehyde and analyzed up to 15 days later. The results generated from the staining panel can be used to track changes in T cell concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific T cell subtypes of interest. In this study, we show the isolation of T cells, performed on fresh calcified aortic valve samples and the steps of analyzing T cell clones using flow cytometry to further understand the role of adaptive immunity in CAVD pathophysiology.

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Keywords T Lymphocytes

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