Culturing Salispheres from Salivary Gland Tissue: A Method to Develop Salispheres from Human Salivary Gland Derived Epithelial Cells

All procedures involving human participants have been performed in compliance with the institutional, national and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Tissue Digestion

1. Using a 100 mm tissue culture plate, mechanically mince the tissue into fine pieces ~1–2 mm in size using autoclave-sterilized dissecting scissors and surgical forceps (Figure 1B, C). The connective tissue between pieces should be cut to ensure proper separation and ease in further steps.
2. Add 6 mL of the dispase/collagenase solution to the minced tissue. Then incubate at 37°C for approximately 30 min to 1 h.
3. Disrupt the tissue by pipetting with a 5 mL serological pipette 15–20 times.
4. Incubate at 37°C for another 30 min to 1 h.
5. Repeat steps 1.3 and 1.4 two-three more times or until the tissue resembles a slurry and can easily pass through the pipette opening (Figure 1D).
   NOTE: The starting size of the tissue specimen and how fineness of the tissue mincing will determine how many times one needs to repeat steps 1.3 and 1.4. Typically, we receive tissue approximately 1 cm x 1 cm in size. Additionally, one can monitor tissue dissociation using a standard inverted tissue culture microscope to track the progress of cell dissociation from the tissue. Small clusters of cells are ideal for the initial outgrowth of salivary cells as salispheres.

2. Coating Wells with Basement Membrane Matrix

NOTE: Basement membrane matrix (BMM) should be thawed overnight at 4°C the day before it will be used. Once thawed, BMM should be constantly maintained on ice or at 4°C as it will rapidly solidify (i.e., form a gel) at warmer temperatures. Additionally, care should be taken if samples have been chilled on ice prior to plating on BMM as cold samples will "melt" the BMM and expose cells to the plastic dish.

1. Slowly pipette BMM (see the Table of Materials) into each well to be coated. Evenly and slowly distribute the BMM over the well.
   NOTE: Generally, we use 500 μL per each well of a 6-well plate, but this volume can be adjusted for use on other variants of plates, such as 12-well or 24-well, or as desired for thicker or thinner hydrogels.
2. Incubate the coated plates at 37°C for at least 15 min prior to use to allow the BMM time to solidify.

3. Filtering of Undigested Tissue, RBC Lysis, and Cell Plating

1. Transfer tissue homogenate from step 1.5 to a 15 mL conical tube. Wash plate once with 6 mL of Dulbecco's phosphate-buffered saline (DPBS) to transfer remaining cells.
2. Filter the homogenate into a 50 mL conical tube through a 70 μm nylon mesh cell strainer to remove undigested tissue. Centrifuge strained cells for 5 min at 500 x g.
3. Dilute 10x RBC lysis buffer in sterile dH₂O to make a 1x working solution.
4. Aspirate the supernatant. Then, resuspend the cell pellet in 10 mL of 1x RBC lysis buffer. Incubate for 5 min at 37°C.
5. Add 20–25 mL of DPBS to neutralize the RBC lysis buffer and minimize the lysis of salivary cells. Centrifuge for 5 min at 500 x g.
   NOTE: After treatment with RBC lysis buffer, the cell pellet should be white. The red color in the pellet indicates that not all RBC have been fully lysed. Repeat steps 3.4 through 3.5 if needed for complete lysis of all RBC.
6. Aspirate the supernatant. Resuspend the pellet in 1–2 mL of BEGM per well, then place resuspended cells onto BMM-coated wells. Incubate at 37°C.
7. Once plated on BMM, cells will form into spherical structures or "salispheres" over a 2–3-day period. Cells can be maintained as salispheres for about 5–7 days before the BMM begins to degrade, allowing the cells to access and adhere to the plastic and grow as a monolayer.