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

Summary

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.

Abstract

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.

Introduction

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 complex^4,5,6. Indeed,....

Protocol

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 Comité de Protection des Personnes (IDRCB/EUDRACT: 2018A0135847). Furthermore, consent of each patient or legal representative is requested to obtain donors' personal data (e.g., sex, age, history of ENT infections) that can help interpret experimental results.

1. Handling of the Human Tonsillar Tissue

1. Put tonsils from every donor in one sterile 50 mL vial containing 25 mL PBS 1x and ....

Results

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.

Discussion

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.......

Materials

List of materials used in this article

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