Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging

My research delves into the immune system’s role in regeneration and repair. I focus on understanding how macrophages interact with epithelial presenter cells during salivary gland irradiation injury and how this interaction shapes the repair process. Within the fields of immunology and regenerative medicine, recent clinical trials based at the University of Edinburgh have shown that delivery of macrophages as a therapy for liver cirrhosis is effective in reducing liver scarring and is a viable treatment for liver failure.

Using an ex-vivo precision cut slice culture model coupled with live imaging of fluorescently labeled cells allows us to explore cell-to-cell interactions in real time, but in a state that closely resembles tissue in vivo. Understanding the localization of macrophages after injury gives us an idea of what cell types macrophages preferentially interact with after injury. This ultimately allows us to use other methods such as single-cell RNA sequencing analysis to try and unravel the molecular basis of such interactions to inform future therapeutic options.

We will explore the bidirectional signaling between macrophages and epithelial cells and test whether salivary gland homeostasis can be rescued by exogenous replacement of these missing signals. To begin, use forceps to remove any fat and connective tissue from the dissected submandibular salivary gland of a euthanized mouse. Then, place the gland in a collection tube with ice-cold HBSS solution.

Next, use forceps to cut the gland into one-centimeter square pieces. Heat 4%agarose to 50 degrees Celsius and pour the solution into a 35-millimeter dish. Pour a small amount of the agarose solution into a separate dish and add the gland pieces into it.

Then swirl the pieces in the excess agarose to coat it. Next, place four to six pieces of the gland flat in the first dish with agarose. Place the lid on the dish and transfer it to an icebox, covering the plate with ice to cool and set.

To section the gland pieces, first, use a scalpel to carefully cut around the gland-embedded agarose block. Next, apply a drop of super glue to the agarose block and attach it to the stage of a vibratome. Now, fill the vibratome chamber with ice-cold PBS containing 1%penicillin streptomycin.

With a scalpel, trim off any excess agarose and create five-millimeter gaps between each piece of gland. Align the vibratome blade with the agarose block and set the start and end points of the sections. Cut the tissue block into 150-micrometer thick sections at a low speed and high vibration.

Once the sections have been cut, use a paintbrush to pick up the slices and place them in a dish with pre-warmed RPMI media containing the antibiotics. To culture the submandibular slices, first add 1.5 milliliters of RPMI media to the wells of a six-well plate. Place 0.4 micrometer filters into the wells.

Next, with the help of a paintbrush, carefully transfer one to six slices onto each filter. Then incubate the plate at 37 degrees Celsius under 5%carbon dioxide. After irradiating some experimental plates with gamma radiation to induce injury, return the plates to the incubator.

Next, fill the wells of a 24-well plate with 500 microliters of culture media. With a paintbrush, lift the slices from the filter and gently submerge them in the wells. Incubate the slices in the relevant nuclear stains.

Then incubate the slices with antibodies for two hours at 37 degrees Celsius under gentle agitation. Submerge the slices in culture media three times to wash them. Let the slices incubate in each wash solution for 10 minutes at room temperature under gentle agitation.

Next, use forceps to remove the tape from a double-sided imaging spacer. Stick the spacer to the bottom of a glass-bottom six-well plate. Then pipette 50 microliters of media into the gap in the center of the spacer.

Place the slice into the media, ensuring it lays flat. Then with a pipette, carefully remove 20 microliters of the media from the gap. Use forceps to carefully remove the tape from the top side of the spacer and place a 25-millimeter circular cover slip over it.

Press around the spacer edges to ensure firm attachment of the cover slip. Image the slice on a confocal microscope. Non-irradiated slices of the submandibular gland cultured for seven days retained their MT signal and epithelial architecture.

However, at three days post-irradiation, acinar and ductal cell atrophy was observed. Caspase-positive cells were seen at four days post-irradiation. Elevated gamma H2AX was observed in irradiated slices, indicative of in-vivo DNA damage.

Real-time imaging of macrophages confirmed phagocytosis of epithelial cells in the slice culture model. Individual cells can be detected and segmented for subsequent analysis of cell behavior, such as migration.