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.
Protocol
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 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…
Declarações
The authors have nothing to disclose.
Materials
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.
Culturing Salispheres from Salivary Gland Tissue: A Method to Develop Salispheres from Human Salivary Gland Derived Epithelial Cells. J. Vis. Exp. (Pending Publication), e20497, doi: (2023).