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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

In this study, we describe the process of T lymphocyte isolation from fresh samples of calcified aortic valves and the analytical steps of T cell-cloning for the characterization of the adaptive leukocyte subsets by using flow cytometry analysis.

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.

Calcific aortic valve disease (CAVD) is one of the most common heart valve disorders, with a heavy impact on healthcare. The frequency of aortic valve replacement in the last years has increased dramatically and is expected to increase further, due to the growing elderly population1.

The underlying pathophysiology of CAVD is only partially known and the current therapeutic strategies are limited to conservative measures or aortic valve replacement, either through surgical or percutaneous procedures. To date, no effective medical treatment can hinder or reverse CAVD progression and high mortality is associated with ea....

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The study was conducted according to the Statute of the Charité for Ensuring Good Scientific Practice and the legal guidelines and provisions on privacy and ethics were respected. The Ethics Committee approved all human experiments and the privacy and anonymity of the patients were maintained in accordance with the rules reported on the Ethic Form.

NOTE: For the protocol described below fresh human stenotic valve samples were used.

1. Reagent preparation

    .......

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We used a simple and cost-effective method to characterize the leukocyte population of fresh aortic valve samples derived from human patients with severe aortic valve stenosis (refer to protocol). The method for isolating PBMCs is a vital step in obtaining feeder cells, which are used in every step of the experiment (cloning, refeeding and splitting phases) and enable the detection and characterization of infiltrating leukocytes in aortic valve samples. The key steps of this method are shown in Figur.......

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Here we present a method to characterize T lymphocyte subpopulations isolated from stenotic aortic valve samples, using flow cytometry. This method requires the use of irradiated buffy coat to isolate the PBMCs. The radiation frequency to which the buffy coat bags must be subjected is 9000 Rad/90 Gray (Gy) and it represents a crucial step to halt the proliferation of the feeder cells. The role of the cells isolated from the buffy coat bags is to act only as feeder cells and provide nutrients for the T cells isolated from.......

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All the buffy coat bags used for this protocol were irradiated thanks to availability of Dr. Peter Rosenthal, Dr. Dirk Böhmer and the whole team of the Radiology Department of Charité Benjamin Franklin. Scholarship Holder/Mary Roxana Christopher, this work is supported by a scholarship from the German Cardiac Society (DGK).

....

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Name Company Catalog Number Comments
50 mL plastic syringes Fisherbrand 9000701
96- well U- bottom Multiwell plates Greiner Bio-One 10638441
Bag Spike (needle free) Sigma P6148 Dilute to 4% with PBS
CD14 Brilliant violet 421  Biolegend 560349
CD25 PE  Biolegend 302621
CD3 PE/Cy7  Biolegend 300316
CD4 Alexa Fluor 488  Biolegend 317419
CD45 Brilliant violet 711  Biolegend 304137
CD8 Brilliant violet 510  Biolegend 301047
Eppendorf tube 1.5 mL Eppendorf 13094697
Eppendorf tube 0.5 mL Thermo Scientific AB0533
Falcon 15 mL conical centrifuge tube Falcon 10136120
Falcon 50 mL conical centrifuge tubes Falcon 10788561
Falcon Round-Bottom Polystyrene Tubes BD 2300E
Fast read 102 plastic counting chamber KOVA INTERNATIONAL 630-1893
Filters for culture medium 250 mL NalgeneThermo Fisher Scientific 168-0045
Filters for culture medium 500 mL NalgeneThermo Fisher Scientific 166-0045
HB 101 Lyophilized Supplement Irvine Scientific T151
HB Basal Medium Irvine Scientific T000
Heat-Inactivated FBS (Fetal Bovine Serum) Euroclone ECS0180L
HS (Human serum) Sigma Aldrich H3667
Human IL-2 IS Miltenyi Biotec 130-097-744
L-Glutamine Gibco 11140050
Lymphoprep Falcon 352057
Non-essential amino acids solution Sigma 11082132001
Paraformaldehyde Thermo Fisher Scientific 10538931
PBS (Phosphate-buffered saline) Thermo Fisher Scientific 10010023
Penicillin/Streptomycin Gibco 15070063 10000 U/mL
PHA (phytohemagglutinin) Stem Cell Technologies 7811
Plastic Petri dishes Thermo Scientific R80115TS 10 0mm x 15 mm
RPMI 1640 Media HyClone 15-040-CV
Sodium pyruvate Gibco by Life technologies 11360070
Syringe Filters 0,45µl Rotilabo-Spritzenfilter P667.1
T-25 Cell culture flasks InvitrogenThermo Fisher Scientific AM9625
T-75 Cell culture flask Thermo Fisher Scientific 10232771
β- Mercaptoethanol Gibco A2916801

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