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

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.