Name: isolation of tonsillar mononuclear cells to study ex vivo innate immune responses in a human mucosal lymphoid tissue
Uploaded: 2020-06-14T12:30:00
Description: Watch this Scientific Journal Video about Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue at JoVE.com

This work was supported by the Agence National de la Recherche sur le SIDA et les H&#233;patites ANRS (J-P.H) for the experiments and N.B. fellowship (AAP 2017 166). N.S. acknowledges support from the ANRS for fellowship (AAP 2016 1), the European Molecular Biology Organization EMBO for Fellowship (LT 834 2017), the startup funding program &#34;Baustein&#34; of the Medical Faculty of Ulm University (LSBN.0147) and the Deutsche Forschungsgemeinschaft DFG (SM 544/1 1).

The specimens are not collected specifically for research purposes and the study is not considered invasive. However, human tonsils collection requires ethical approval by the local relevant authorities. In our case, it was approved by the Comit&#233; de Protection des Personnes (IDRCB/EUDRACT: 2018A0135847). Furthermore, consent of each patient or legal representative is requested to obtain donors&#39; personal data (e.g., sex, age, history of ENT infections) that can help interpret experimental results.
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Record+of+the+first+physician+to+see+Henrietta+Lacks+at+the+Johns+Hopkins+Hospital:+History+of+the+beginning+of+the+HeLa+cell+line.">Record of the first physician to see Henrietta Lacks at the Johns Hopkins Hospital: History of the beginning of the HeLa cell line.</a> American Journal of Obstetrics and Gynecology. 176 (6), s227-s228 (1997).</li><li>Cummins, J. E., 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=Preclinical+Testing+of+Candidate+Topical+Microbicides+for+Anti-Human+Immunodeficiency+Virus+Type+1+Activity+and+Tissue+Toxicity+in+a+Human+Cervical+Explant+Culture.">Preclinical Testing of Candidate Topical Microbicides for Anti-Human Immunodeficiency Virus Type 1 Activity and Tissue Toxicity in a Human Cervical Explant Culture.</a> Antimicrobial Agents and Chemotherapy. 51 (5), 1770-1779 (2007).</li><li>Abner, S. 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Human tonsils represent an integrative and physiological ex vivo model to study innate immune responses at the mucosal interface, because they mimic the role of a secondary lymphoid organ. Interestingly, the cellular composition of the TMCs is similar to the PBMCs and includes all the major cell populations, although their percentage can be different from PBMCs from blood (Figure 1). Additional populations can also be found, as all immune responses are initiated in tissues (mucosa or seconda...

Studies on human organs are restricted due to accessibility as well as obvious ethical reasons. However, they are essential to fully understand the complexity of human biology. Cultures of isolated cells (primary cultures or cell lines) are a standard system in cell biology studies due to their availability. While isolated cell cultures have allowed outstanding discoveries, the use of cell lines has come upon closer scrutiny because they do not fully mimic in vivo organ biology. However, the culture of three-dimensional cells or tissue explants is highly complex4,5,6. Indeed, a piece of tissue or organ is highly heterogenous because its cell composition differs depending on the localization in the tissue. Thus, using tissue blocks requires the analysis of many technical and biological replicates, leading to the need of a large number of donors or patients.
The mucosa-associated lymphoid tissues (MALT) are structurally similar to the lymph nodes but have unique functions, because their main role is to regulate mucosal immunity7. Unlike the lymph nodes, which are usually located at some distance from the tissues, MALT are generally located immediately below the epithelium of the mucosal tissue. Histologically, they are mainly composed of high concentrations of B and T cells, but also antigen-presenting cells such as macrophages and dendritic cells. MALT constitute about 50% of the lymphoid tissue in the human body. MALT are subdivided into nine groups depending on their location: GALT (gut-), BALT (bronchus-), NALT (nasal-), CALT (conjunctival), LALT (larynx-), SALT (skin-), VALT (vulvo-), O-MALT (organized), and D-MALT (diffused). The O-MALT is mainly composed of the tonsils of Waldeyer&#39;s tonsillar ring and is the most accessible MALT8,9. Indeed, tonsils located in the oropharynx constitute the major barrier protecting the digestive and respiratory tracts from (potential) invasive microorganisms10. In addition, the tonsils are covered by a fine stratified squamous non-keratinizing epithelium, supported by a capsule of connective tissue containing blood vessels, nerves, and lymphatics, providing easy access to the immune cells11,12. Furthermore, tonsillectomy, the surgical act of removing tonsils, is a common procedure performed on children having sleep-disordered breathing, making tonsils an easily available tissue13 in physiological settings.
Tonsils allow the study of immune cell response in pathologies involving mucosal immunity. Indeed, in HIV infection, because tonsils are composed of a high concentration of immune cells, they are the main target of viral replication but also produce a large amount of cytokines that are not detected in the circulation14,15. At steady states, rare populations of innate-like cells are present in various mucosal tissues, including the tonsils, but are essentially absent from blood.
Thus, mononuclear cells from tonsils (TMCs) are a more relevant and complex model than PBMCs and can answer more profound questions. On the other hand, the use of tissue explants can be complex and not always relevant to innate immune studies. Thus, we established a model to study mucosal immune activation using TMCs16. Here, we describe a method for efficient isolation of TMCs from fresh human tonsils. This method allows the recovery of a large number of immune cells while keeping their integrity for ex vivo studies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 meshes steel grid - 1910 &#181;m</td>
			<td>Dutscher</td>
			<td>198586</td>
			<td>To put in the cell strainer Cellector</td>
		</tr>
		<tr>
			<td>60 meshes steel grid - 230 &#181;m</td>
			<td>Dutscher</td>
			<td>198591</td>
			<td>To put in the cell strainer Cellector</td>
		</tr>
		<tr>
			<td>70 &#181;m white ClearLine cell strainers</td>
			<td>Dutscher</td>
			<td>141379C</td>
			<td></td>
		</tr>
		<tr>
			<td>Anios Excell D detergent</td>
			<td>Dutscher</td>
			<td>59852</td>
			<td>Detergent</td>
		</tr>
		<tr>
			<td>Antibiotic solution, 100x</td>
			<td>Thermo Fisher</td>
			<td>15140122</td>
			<td>100 U/mL Penicilium and 100 &#956;g/mL Streptomycin - to add to culture media</td>
		</tr>
		<tr>
			<td>BD FalconTM Round-Bottom Tubes, 5 mL</td>
			<td>BD Biosciences</td>
			<td>352063</td>
			<td>FACS Tubes</td>
		</tr>
		<tr>
			<td>Cell strainer Cellector, 85 mL and 37 mm diameter</td>
			<td>Dutscher</td>
			<td>198585</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter-Glo (CTG) Luminescent Cell Viability Assay</td>
			<td>Promega</td>
			<td>G7572</td>
			<td>Viability assay</td>
		</tr>
		<tr>
			<td>Centrifuge 5810 R</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes Falcon 50 mL</td>
			<td>Dutscher</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved tweezers</td>
			<td>Dutscher</td>
			<td>711200</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td>Without calcium and magnesium</td>
		</tr>
		<tr>
			<td>EnVision</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Measures the luminescence</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td></td>
			<td></td>
			<td>To add to culture media</td>
		</tr>
		<tr>
			<td>Fluorescence labeles antibodies</td>
			<td>See Table 1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pestle</td>
			<td>Dutscher</td>
			<td>198599</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes (1M)</td>
			<td>Thermo Fisher</td>
			<td>15630056</td>
			<td>Use at 20 mM</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LEGENDplex Human Anti-Virus Response Panel</td>
			<td>BioLegend</td>
			<td>740390</td>
			<td>Bead-based immunoassay</td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>StemCell</td>
			<td>7801</td>
			<td>Density gradient medium</td>
		</tr>
		<tr>
			<td>Mr. Frosty container</td>
			<td>Thermo Fisher</td>
			<td>5100-0001</td>
			<td>Slow freezing container</td>
		</tr>
		<tr>
			<td>Pierce 16% Formaldehyde (w/v), Methanol-free</td>
			<td>Thermo Fisher</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>Resiquimod (R848)</td>
			<td>InvivoGen</td>
			<td>tlrl-r848</td>
			<td>TLR7/8 agonist</td>
		</tr>
		<tr>
			<td>RPMI-1640 Medium</td>
			<td>Sigma-Aldrich</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr>
			<td>SPL Cell Culture Dish, 150 x 25 mm (SPL150)</td>
			<td>Dutscher</td>
			<td>330009</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical blade sterile N&#176;23</td>
			<td>Dutscher</td>
			<td>132523</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraComp eBeads Compensation Beads</td>
			<td>Thermo Fisher</td>
			<td>01-2222-41</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 0.5M EDTA, pH 8.0</td>
			<td>Thermo Fisher</td>
			<td>15575020</td>
			<td>To make wash buffer in PBS</td>
		</tr>
	</tbody>
</table>

isolation of tonsillar mononuclear cells to study ex vivo innate immune responses in a human mucosal lymphoid tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

We first characterized the immune profile of cells present in the culture and analyzed the amount of TMCs. We phenotyped the TMCs from tonsils with flow cytometry. As shown in Figure 1, all major immune cell types present in PBMCs from blood were represent in the TMCs from tonsils. However, in TMCs the frequency of all cell types, except B cells, were lower than in PBMCs.
<img alt="Figure 1" class="...

Watch this Scientific Journal Video about Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue at JoVE.com

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...

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