This article details a method for isolating the gingival immune cell network from murine teeth blocks. The process involves enzymatic treatment and mechanical dissociation to extract immune cells for further analysis.
The gingiva, or gum tissue, surrounds the cervical portion of teeth and the jawbone.
Gingival tissue comprises an epithelium layer, separated from the underlying connective tissue by a collagen-IV-rich basement membrane. The gingival connective tissue contains an immune cell network, including lymphocytes, mast cells, and neutrophils that protect the gingiva from infections.
To isolate the gingival immune cell network, take murine teeth blocks containing firmly attached gingival tissue. Treat the blocks with an enzyme cocktail — containing type four collagenases and DNases.
Collagenases enter the tissue, degrade collagen-IV, and disrupt the basement membrane and gingival epithelium, causing the gingiva to loosen from the teeth. Meanwhile, DNases degrade the DNA contaminants. Add a chelating agent to the tube; to remove divalent cations and prevent cell aggregation.
Transfer the digested teeth blocks to a petri dish containing DNase medium. Extract the gingiva tissue from the teeth blocks, and transfer it into a cell strainer.
Using a plunger, mash the tissue. This mechanical dissociation disrupts the gingival connective layer, releasing the immune cells from the tissue. Wash the strainer with media and collect the released cells in the filtrate.
Centrifuge the filtrate, pelletizing the cells, and discard the supernatant containing residual enzymes and debris. Resuspend the cell population carrying immune cells in complete media, ready for future analysis.
Eliminate the excess tissue from the border of the gingiva, and transfer the maxillary and mandibular blocks into a 50-milliliter conical tube containing 5 milliliters of collagenase and DNase on ice. Digest the tissue in a shaker incubator at 37 degrees Celsius for 1 hour, adding 50 microliters of 0.5 molar EDTA during the last five minutes. Then, add 5 milliliters of cold DNase medium to the tissues, and gently mix the tube contents by swirling. Next, transfer the four blocks of tissue into a Petri dish, and cover them with 500 microliters of fresh DNase medium.
Use a scalpel to remove the gingiva from each block of tissue and transfer the entire contents of the dish into a 70-micron cell strainer on top of a 50-milliliter tube. Wash the Petri dish and strainer with 3 to 5 milliliters of fresh DNase medium, and then, use the plunger of a sterile 3-milliliter syringe to mash the gingival tissue against the mesh of the strainer. Rinse the strainer with 30 to 35 milliliters of cold DNase medium, and centrifuge the filtrate. Then, resuspend the pellet in one milliliter of complete medium, and determine the number of viable cells.
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