Source: Beucler, M. J., Miller, W. E. Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers. J. Vis. Exp. (2019)
This video presents the protocol for culturing salispheres, the three-dimensional spheroids derived from salivary glands. The salispheres can be further used to assess the impact of head and neck cancer on saliva production from salivary glands of affected human patients.
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
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.
3. Filtering of Undigested Tissue, RBC Lysis, and Cell Plating
The authors have nothing to disclose.
100 mm culture dishes | Thermo Scientific | 172931 | |
15 mL conical tubes | Thermo Scientific | 339651 | |
50 mL conical tubes | Thermo Scientific | 339653 | |
Bronchial Epithelial Cell Growth Media | Lonza | CC-3171 | Add bullet kit as per manufacturer's instructions. Supplement with 20 mL of charcoal stripped serum. |
Cell strainer 70 µm nylon mesh | Fisher | 22-363-548 | |
Charcoal stripped fetal bovine serum | Gibco | 12676-029 | |
Collagenase type III | Worthington | LS004182 | Store at 4 °C. |
Dispase | Cell Applications | 7923 | Dissolve collagenase to make a 0.15% (w/v) stock. Filter sterilize then store at -20 °C. |
Dissecting scissors | Fisher | 08-940 | |
Dulbecco phosphate buffered saline | Corning | 55-031-PC | |
PathClear Basement membrane extract | Cultrex | 3432-005-01 | Thaw at 4 °C at least 24 hr prior to use. Always handle on ice. |
Six-well culture dishes | Falcon | 353046 | |
Surgical forceps | Fisher | 22-079-742 |