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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<ol>
	<li>Taylor, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+History+of+Cell+Culture.">A History of Cell Culture.</a> Viruses and Man: A History of Interactions. , 41-52 (2014).</li><li>Scherer, W. F., Syverton, J. T., Gey, G. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+propagation+in+vitro+of+poliomyelitis+viruses.+IV.+Viral+multiplication+in+a+stable+strain+of+human+malignant+epithelial+cells+(strain+HeLa)+derived+from+an+epidermoid+carcinoma+of+the+cervix.">Studies on the propagation in vitro of poliomyelitis viruses. IV. Viral multiplication in a stable strain of human malignant epithelial cells (strain HeLa) derived from an epidermoid carcinoma of the cervix.</a> The Journal of Experimental Medicine. 97 (5), 695-710 (1953).</li><li>Jones, H. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+Histopathology+of+Mucosa-Associated+Lymphoid+Tissue.">Enhanced Histopathology of Mucosa-Associated Lymphoid Tissue.</a> Toxicologic Pathology. 34 (5), 687 (2006).</li><li>Strioga, M. M., Dobrovolskiene, N. 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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 ...

Tonsillar mononuclear cells are more relevant and complex than blood cells, and their isolation allows the study of immune cell response in pathologies involving mucosal immunity. The technique we have established allows the recovery of a large number of immune cells from a mucosal tissue while keeping their integrity for ex vivo studies. The method is quite straightforward, but the tonsils need to be handled rapidly and with care in order to maximize the yield.Transport the human tonsillar tissue to the laboratory as described in the manuscript text. Next, place the cell strainer onto a 150 by 25 millimeter culture dish. Place all the dissection instruments in another dish to keep them sterile.All your main specimens should be handled with care as they have not been previously qualified and may contain infectious agents. Then pour the PBS from the vial into a strainer, because it will contain some cells that have egressed the tonsils. Using sterile forceps, transfer the tonsils from the vial into the culture dish using sterile forceps.If necessary, add more PBS to immerse the grid. The dissection will take about 45 minutes per tonsil. It is crucial that during this time the tonsils are submerged to ensure good viability and cell yields.Remove cauterized bloody and fibroid tissue using forceps and a scalpel. Using the scalpel and the curved tweezers, cut the tissue into small pieces of less than half a centimeter in diameter. Cut all the tissue so that the small pieces can be immersed at all times.Place a few pieces of tissue in the cell strainer. Scrape them onto the grid with a glass pestle until only a very thin layer of white stroma remains. Remove and discard the stroma to avoid clogging the grid.Then use a 10-milliliter pipette to transfer the cell suspension onto the grid and scrape it one last time. Next, use a 10-milliliter pipette to transfer the cell suspension into a sterile vial. Wash the grid and the cell strainer with PBS once, and transfer the PBS to the vial also.Before proceeding let the cell suspension rest on the bench for five minutes at room temperature. Place a sterile 70-micrometer sieve on top of a new 50-milliliter vial. Gently transfer the cell suspension onto the sieve with a 10-milliliter pipette.If the sieve is clogged, use the back of a sterile one-milliliter pipette tip to scrape the cells through the sieve. Change the sieve as often as necessary. Centrifuge the cells at 250 times g for 10 minutes at four degrees Celsius.After centrifugation, discard the supernatant. Re-suspend the pellet by gently tapping the vial, and then re-suspend the cells in 35 milliliters of PBS. To complete the first phase of cell isolation, place a new 70-micrometer sieve on top of a new vial and transfer the cell suspension onto it with a 10-milliliter pipette.To obtain a clearer cell solution, begin by adding 15 milliliters of a density gradient medium to a new 50-milliliter vial. Pour the TMC solution on top of the density gradient medium, being careful to minimize mixing. Next, centrifuge the solution at 1000 times g for 30 minutes at room temperature with acceleration and brake off.After removing the vial from the centrifuge, use a 10-milliliter pipette to remove and discard the upper layer. Do not disturb the interface between the TMCs and the density gradient medium. Then remove the TMCs with the sterile one-milliliter pipette tip and transfer them to a new 50-milliliter vial.To wash the cells, first add 50 milliliters of PBS containing 2%fetal bovine serum and 2 millimolar EDTA. Then centrifuge the tube at 250 times g for 10 minutes at four degrees Celsius. Wash the cells again, but this time centrifuge the tube at 400 times g for 10 minutes at four degrees Celsius.Prepare R10 culture medium by supplementing RPMI 1640 with 10%heat-inactivated FBS, 2 millimolar L-glutamine, and the antibiotic solution. Re-suspend the TMCs in 10 milliliters of R10. Then count the cells.The cells can either be used immediately or frozen for future use as described in the manuscript. TMCs were stained, incubated with antibodies, and characterized using flow cytometry. TMCs contained all the major immune cell types present in peripheral mononuclear cells from blood, abbreviated PMBC.However, the frequencies of all cell types, except B cells, were lower in TMCs than in PBMCs. To study cell activation in cytokine production, TMCs were stimulated overnight with resiquimod R848. TMCs, plotted in blue, produced all the cytokines tested except for IFN Lambda 2/3 and IL-8, although production was lower than in PBMCs, plotted in green.The fold increases comparing R848 stimulation to no stimulation are noted in red. Purified and isolated TMCs, blue, and tonsillar tissue blocs, orange, were stimulated for 24 hours with Influenza A virus, IAV. IFN production was detected in TMCs but not in the supernatant of the tissue blocs indicating that tissue explants are not the best model for the study of cytokine secretion and cell activation.After pre-incubation with the toxic drug C1 and stimulation with R848, TMCs and PBMCs were assayed for viability. TMCs were more sensitive to C1.This suggests that new drugs in future treatments should be tested in cells from tissues, not only on cell lines, PBMCs, or tissue explants. This procedure allows us to use the TMCs like PBMCs from whole bloods and to perform the same cellular experiments such as purification of a specific cell type, cell culture, flow cytometry or freezing.Isolations of TMCs makes it possible to better characterize, in a relevant model, the immune response in secondary lymphoid organs in pathologies involving mucosal immunity.

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The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow ...