1. Buffer: 1 % (w/v) bovine serum albumin in Hank's balanced salt solution
2. Reconstitute enzymes. Hyaluronidase enzyme: 40mg / mL, dissolved in buffer. Collagenase II: 23mg / mL, dissolved in buffer. Use freshly prepared enzyme solutions fresh for each isolation. When dissolved, store at 4 °C until use for digestion.
3. 50 mM calcium chloride in distilled water. Filter sterilize through a 0.2 uM pore size filter.
4. Mouse salivary gland (MSG) culture medium: DMEM:F12 with penicillin (100 I.U. / mL), streptomycin (100 μg / mL), glutamax (2 mM), epidermal growth factor-2 (20 ng / mL), fibroblast growth factor-2 (20 ng / mL), N2 supplement (1 %), insulin (10 μg / mL) and dexamethasone (1 μM).

1. Weigh the dissected salivary glands.
2. Chop glands into a homogenous pulp using sterile curved dissection scissors in a small petri dish.
3. Collect minced tissue in 14 mL tubes, using 1mL of buffer per 80mg submandibular tissue. Rinse the petri dishes clean of tissue using some of the buffer.
4. Add another 1 mL of buffer per 80 mg tissue, followed by 25 μL collagenase II enzyme solution, 25 μL hyaluronidase enzyme solution and 250 μL calcium chloride solution per 80 mg tissue. If working with large amounts of tissue, steps 2.4 - 2.9 can be performed in T25 tissue culture flasks for convenience.
5. Incubate in a shaking waterbath set at 37 °C for 20 minutes. Remove tubes and triturate by pipette to mix enzyme thoroughly through tissue again.
6. Replace in waterbath for another 20 mins.
7. Collect tissue by centrifugation at 400 x g, for 8 minutes. Discard supernatant.
8. Resuspend in 2 mL buffer for each 80 mg tissue, and repeat enzyme and calcium chloride addition as above. Incubate 20 minutes in shaking water bath. Remove tubes and triturate by pipette to mix enzymes thoroughly.
9. Incubate for final 20 min in shaking water bath. Collect cells by centrifugation as above, discard supernatant.

1. Resuspend each 80 mg of tissue in 2 mL buffer and pipette to wash tissue free of enzymes.
2. Centrifuge as previously to collect. Discard supernatant.
3. Repeat wash using 1 mL buffer per 80 mg tissue. Centrifuge to collect, discard supernatant.

1. Resuspend tissue solution in 1 mL buffer per 80 mg tissue.
2. Add solution to 100 μm pore-size filter placed over 50 mL Falcon tube. Do not apply more than 3 mLs of minced tissue solution per column, as filters may become blocked. Allow to seep through. Remove filtered material hanging on underside of filter by pipette, and add to filtrate.
3. Use syringe with 26 gauge needle to take filtrate from 50mL tubes and apply to 50 μm pore size filters on 5 mL tubes. Allow to filter through, assisting by loosening lids if necessary.
4. Centrifuge tubes as previously to collect. Discard supernatant.

1. Combine all pellets into one volume. Count using automated cell counter or haemocytometer.
2. Plate cells at density of 0.4 x 10⁶ cells per well of 12-well plate, or 2.67 x 10⁶ cells per T25 tissue culture flask. Add 1 mL MSG medium to each well or 6 mL to each T25 flask.
3. Incubate at 37 °C. Spheres should be clearly visible by day 2.

After two to three days in culture, small aggregates of cells (salispheres) will be apparent in the cultures. Salispheres will continue to grow in size over a period of ten days in culture. Representative phase contrast microscopy images of salispheres are shown in Figure 1. Proliferating cells expressing stem cell-associated marker proteins can be isolated from these spheres, optimally between days 3-5 post isolation, and are capable of differentiation into functional, saliva producing acinar cells.

Figure 1. Salisphere formation in vitro. Following mechanical and enzymatic digestion using the present protocol, spheres of increasing size can be found in the floating cultures. Panels are representative phase contrast microscopy images of spheres from days 0 (A), 4 (B), 7 (C) and 10 (D). Scale bar = 50 μm.