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

Abstract

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.

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

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 s....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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