Name: investigating aortic valve calcification via isolation and culture of t lymphocytes using feeder cells from irradiated buffy coat
Uploaded: 2021-02-04T13:30:00
Description: Watch this Scientific Journal Video about Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat at JoVE.com

All the buffy coat bags used for this protocol were irradiated thanks to availability of Dr. Peter Rosenthal, Dr. Dirk B&#246;hmer and the whole team of the Radiology Department of Charit&#233; Benjamin Franklin. Scholarship Holder/Mary Roxana Christopher, this work is supported by a scholarship from the German Cardiac Society (DGK).

The study was conducted according to the Statute of the Charit&#233; 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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<ol>
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R., Franz, M., Lauten, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+History+of+Transcatheter+Aortic+Valve+Implantation+(TAVI)-A+Personal+View+Over+25+Years+of+development.">The History of Transcatheter Aortic Valve Implantation (TAVI)-A Personal View Over 25 Years of development.</a> Cardiovascular Revascularization Medicine. 21 (3), 398-403 (2020).</li><li>Lauten, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TAVI+for+low-flow,+low-gradient+severe+aortic+stenosis+with+preserved+or+reduced+ejection+fraction:+a+subgroup+analysis+from+the+German+Aortic+Valve+Registry+(GARY).">TAVI for low-flow, low-gradient severe aortic stenosis with preserved or reduced ejection fraction: a subgroup analysis from the German Aortic Valve Registry (GARY).</a> Euro Intervention Journal. 10 (7), 850-859 (2014).</li><li>Mirna, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-biomarker+analysis+in+patients+after+transcatheter+aortic+valve+implantation+(TAVI).">Multi-biomarker analysis in patients after transcatheter aortic valve implantation (TAVI).</a> Biomarkers. 23 (8), 773-780 (2018).</li><li>Wernly, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcatheter+aortic+valve+replacement+for+pure+aortic+valve+regurgitation:+"on-label"+versus+"off-label"+use+of+TAVR+devices.">Transcatheter aortic valve replacement for pure aortic valve regurgitation: "on-label" versus "off-label" use of TAVR devices.</a> Clinical Research in Cardiology. 108 (8), 921-930 (2019).</li><li>Wernly, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcatheter+valve-in-valve+implantation+(VinV-TAVR)+for+failed+surgical+aortic+bioprosthetic+valves.">Transcatheter valve-in-valve implantation (VinV-TAVR) for failed surgical aortic bioprosthetic valves.</a> Clinical Research in Cardiology. 108 (1), 83-92 (2019).</li><li>Mathieu, P., Bouchareb, R., Boulanger, M. 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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Lymphocytic+Infiltration+in+Calcific+Aortic+Stenosis+Predominantly+Consists+of+Clonally+Expanded+T+Cells.">The Lymphocytic Infiltration in Calcific Aortic Stenosis Predominantly Consists of Clonally Expanded T Cells.</a> The Journal of Immunology. 178 (8), 5329-5339 (2007).</li><li>Raddatz, M. A., Madhur, M. S., Merryman, W. 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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...

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 early symptom-onset, unless aortic valve replacement (AVR) is performed2. In patients with severe symptomatic aortic stenosis, the 3-year symptom-free survival was reported as low as 20%3. Transcatheter aortic valve replacement (TAVR) represents a new option, revolutionizing treatment for high-risk patients, especially among the elderly and has dramatically reduced the mortality, which was intrinsically high in this population4,5,6. Despite the promising results of TAVR, further research is necessary to understand CAVD pathophysiology to identify novel early therapeutic targets7,8,9.
Previously thought to be a passive, degenerative process, CAVD is now recognized as an active progressive disease, characterized by an osteoblastic phenotype switch of the aortic valve interstitial cells10. This disease involves progressive mineralization, fibrocalcific changes and reduced motility of the aortic valve leaflets (sclerosis), which ultimately obstruct blood flow leading to narrowing (stenosis) of the aortic valve opening11.
Inflammation is considered a key process in CAVD pathophysiology, similar to the process of vascular atherosclerosis. Endothelial injury enables the deposition and accumulation of lipid species, especially oxidized lipoproteins in the aortic valve12. These oxidized lipoproteins provoke an inflammatory response, as they are cytotoxic, with the inflammatory activity leading to mineralization. The role of innate and adaptive immunity in CAVD development and disease progression has been recently highlighted13. The activation and clonal expansion of specific subsets of memory T cells have been documented in patients with CAVD and mineralized aortic valve leaflets, so that inflammatory processes are assumed to be involved at least in the development of CAVD and presumably in disease progression as well14. In fact, although antigen-presenting cells and macrophages are present in both the healthy and diseased valve, the presence of T lymphocytes is indicative of an aged and diseased aortic valve. This lymphocytic infiltrate along with an increase in neovascularization and metaplasia are characteristic histological signs of CAVD15.
We hypothesize the existence of an interaction between the aortic valve interstitial cells and the activation of the immune system, which potentially triggers the initiation of a chronic inflammatory process in the aortic valve. The assaying of T cells from stenotic aortic valve tissue could be an efficient way to clarify their role in calcification development, as it can provide a close representation of the aortic valve cells in vivo. In the present work, using aortic valve tissue, we isolate T-lymphocytes, culture and clone them, and subsequently characterize them using fluorescence-activated cell sorting (FACS). Fresh aortic valve samples were excised from CAVD patients who received surgical valve replacement for severe aortic stenosis. After the surgical excision, the fresh valve sample was dissected in small pieces and the T cells were cultured, cloned then analyzed using flow cytometry. The staining procedure is simple and the stained tubes can be fixed using 0.5% of paraformaldehyde and analyzed up to 15 days later. The data generated from the staining panel can be used to track changes in T lymphocyte distribution over time in relation to intervention and could easily be further developed to assess activation states of specific T cell subsets of interest.
The extraction of calcified tissue, the isolation of leukocytes from calcified tissue and particularly the use of flow cytometry on this type of tissue can be challenging, due to issues such as autofluorescence. Few publications exist with protocols for this specific purpose16,17,18. Herein we present a protocol designed specifically for the direct isolation and culture of T lymphocyte from human aortic valve samples. Clonal expansion of lymphocytes is a hallmark of adaptive immunity. Studying this process in vitro provides insightful information on the level of lymphocyte heterogeneity19. After a three-week incubation period, the T cell clones are ready to be explanted, as an adequate amount of T cells from each clone was obtained, so as to allow the phenotypic and functional study. Subsequently the phenotype of T clones is studied by cytofluorometry.
This immunological protocol is an adaptation of a method previously developed by Amedei et al. for T cell isolation and characterization from human tissue, especially designed for calcified human tissue, such as in CAVD20,21,22. The protocol here for the isolation of PBMCs (peripheral blood mononuclear cells) using irradiated buffy coat describes an effective way to obtain feeder cells (FC), specifically adjusted for the cloning phase of T lymphocytes isolated from valve interstitial cells. The feeder layer consists of in growth arrested cells, which are still viable and bioactive. The role of feeder cells is important to support in vitro survival and growth of T lymphocytes isolated from valve interstitial cells23. In order to avoid feeder cell proliferation in culture, these cells must undergo a growth arrest. This can be achieved in two ways: through physical methods such as irradiation, or through treatment with cytotoxic chemicals, such as mitomycin C (MMC), an antitumoral antibiotic that can be applied directly to the culture surface24. Here we show feeder cell growth arrest achieved through cell irradiation.
This method presents an efficient, cost-effective way to isolate and characterize T cells from aortic valve tissue, contributing to broadening the spectrum of immunological methods for exploring CAVD pathophysiology.

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	<tbody>
		<tr>
			<td>50 mL plastic syringes</td>
			<td>Fisherbrand</td>
			<td>9000701</td>
			<td></td>
		</tr>
		<tr>
			<td>96- well U- bottom Multiwell plates</td>
			<td>Greiner Bio-One</td>
			<td>10638441</td>
			<td></td>
		</tr>
		<tr>
			<td>Bag Spike (needle free)</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>Dilute to 4% with PBS</td>
		</tr>
		<tr>
			<td>CD14 Brilliant violet 421</td>
			<td>&#160;Biolegend</td>
			<td>560349</td>
			<td></td>
		</tr>
		<tr>
			<td>CD25 PE</td>
			<td>&#160;Biolegend</td>
			<td>302621</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3 PE/Cy7</td>
			<td>&#160;Biolegend</td>
			<td>300316</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4 Alexa Fluor 488</td>
			<td>&#160;Biolegend</td>
			<td>317419</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 Brilliant violet 711</td>
			<td>&#160;Biolegend</td>
			<td>304137</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8 Brilliant violet 510</td>
			<td>&#160;Biolegend</td>
			<td>301047</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tube 1.5 mL</td>
			<td>Eppendorf</td>
			<td>13094697</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tube 0.5 mL</td>
			<td>Thermo Scientific</td>
			<td>AB0533</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15 mL conical centrifuge tube</td>
			<td>Falcon</td>
			<td>10136120</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 mL conical centrifuge tubes</td>
			<td>Falcon</td>
			<td>10788561</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Round-Bottom Polystyrene Tubes</td>
			<td>BD</td>
			<td>2300E</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast read 102 plastic counting chamber</td>
			<td>KOVA INTERNATIONAL</td>
			<td>630-1893</td>
			<td></td>
		</tr>
		<tr>
			<td>Filters for culture medium 250 mL</td>
			<td>NalgeneThermo Fisher Scientific</td>
			<td>168-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>Filters for culture medium 500 mL</td>
			<td>NalgeneThermo Fisher Scientific</td>
			<td>166-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>HB 101 Lyophilized Supplement</td>
			<td>Irvine Scientific</td>
			<td>T151</td>
			<td></td>
		</tr>
		<tr>
			<td>HB Basal Medium</td>
			<td>Irvine Scientific</td>
			<td>T000</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat-Inactivated FBS (Fetal Bovine Serum)</td>
			<td>Euroclone</td>
			<td>ECS0180L</td>
			<td></td>
		</tr>
		<tr>
			<td>HS (Human serum)</td>
			<td>Sigma Aldrich</td>
			<td>H3667</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-2 IS</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-744</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Falcon</td>
			<td>352057</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acids solution</td>
			<td>Sigma</td>
			<td>11082132001</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Thermo Fisher Scientific</td>
			<td>10538931</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (Phosphate-buffered saline)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td>10000 U/mL</td>
		</tr>
		<tr>
			<td>PHA (phytohemagglutinin)</td>
			<td>Stem Cell Technologies</td>
			<td>7811</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Petri dishes</td>
			<td>Thermo Scientific</td>
			<td>R80115TS</td>
			<td>10 0mm x 15 mm</td>
		</tr>
		<tr>
			<td>RPMI 1640 Media</td>
			<td>HyClone</td>
			<td>15-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco by Life technologies</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filters 0,45&#181;l</td>
			<td>Rotilabo-Spritzenfilter</td>
			<td>P667.1</td>
			<td></td>
		</tr>
		<tr>
			<td>T-25 Cell culture flasks</td>
			<td>InvitrogenThermo Fisher Scientific</td>
			<td>AM9625</td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 Cell culture flask</td>
			<td>Thermo Fisher Scientific</td>
			<td>10232771</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;- Mercaptoethanol</td>
			<td>Gibco</td>
			<td>A2916801</td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating aortic valve calcification via isolation and culture of t lymphocytes using feeder cells from irradiated buffy coat

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.

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...

Watch this Scientific Journal Video about Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat at JoVE.com

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

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 ...

This protocol is easy to perform. The cloning phase allows a larger number of T clones to be obtained from fresh samples. Moreover, the phenotype and function of T cells can easily be analyzed.Demonstrating the procedure will be Mary Christopher, a doctorate student from my laboratory. To induce T-cell cloning, use the bag spike with the needle-free valve to open an irradiated buffy coat bag in a laminar flow hood. Transfer up to 50 milliliters of blood from the bag into a T-150 flask, and add 70 milliliters of PBS to the flask.Carefully layer 30 milliliters of the diluted blood onto 20 milliliters of density gradient in each of four 50 milliliter conical tubes, and separate the cells by density gradient centrifugation. Carefully aspirate the supernatant, and transfer the PBMC from each tube into a single new 50 milliliter tube. Use PBS to bring the final volume to 50 milliliters and collect the PBMC by centrifugation.After a second wash under the same conditions, suspend the isolated feeder cells in 10 milliliters of cell culture medium for counting, and add 25 milliliters of a 70 milliliter cell culture medium supplemented with 50 units of IL-2 solution into each of two 50 milliliter tubes. Then add feeder cells to one tube at a 2 by 10 to the 6th cells per milliliter of cell culture medium supplemented with IL-2 concentration. To set up a T lymphocyte culture, pool the T cell clone containing supernatants from stenotic valve sample flask into a single 50 milliliter tube, and collect the cells by centrifugation.Wash the pellet three times in 50 milliliters of PBS per wash, before resuspending the cells in 1 milliliter of cell culture medium for counting. Dilute the cells to a 1 by 10 to the 3rd cells per milliliter concentration, and add 500 microliters of cells to the tube of cell culture medium supplemented with IL-2 without feeder cells. Add the feeder cell suspension to a plastic 100 by 15 milliliter Petri dish, and add 100 microliters of feeder cells to each well of the appropriate number of U-bottom 96-well plates for the experiment.Then add the T-cells to a Petri dish, and add 100 microliters of T-cells to each feeder cell supplemented well for a one week incubation in the cell culture incubator. The PBMC isolation is vital for obtaining feeder cells and enabling the detection and characterization of infiltrating leukocytes in aortic valve samples. After two weeks of incubation, a clone T-cell population can be obtained.Here, the gating scheme for analyzing T-cell subpopulations in patients with calcific aortic valve disease is shown. As observed in this patient, there were more CD4+T-cells than CD8+T-cells. Flow cytometric analysis of uncloned T-cells confirms that the same T-cell markers are present in both native valve and clone samples.Taken together, these data indicate that T-cells are present in calcified aortic valves with CD45+leukocytes, suggesting that calcific aortic valve disease is linked with activation of the immune system and inflammatory activity. This method is very versatile. It can be applied to different protocols for which researcher need to be able to isolate cells from a tissue, for the functional characterization.Keep in mind that it is important to work under sterile conditions to avoid contamination, and to critically observe the cells under the microscope.

Research

JoVE Journal

Immunology and Infection

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

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