Generation and Characterization of Murine Oral Mucosal Organoid Cultures

Summary

We present a method for the generation and characterization of oral mucosal organoid cultures derived from the tongue epithelium of adult mice.

Abstract

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, gingiva, lips, palate, and tongue. Its uppermost layer, the oral epithelium, is maintained by adult stem cells throughout life. Proliferation and differentiation of adult epithelial stem cells have been intensively studied using in vivo mouse models as well as two-dimensional (2D) feeder-cell based in vitro models. Complementary to these methods is organoid technology, where adult stem cells are embedded into an extracellular matrix (ECM)-rich hydrogel and provided with a culture medium containing a defined cocktail of growth factors. Under these conditions, adult stem cells proliferate and spontaneously form three-dimensional (3D) cell clusters, the so-called organoids. Organoid cultures were initially established from murine small intestinal epithelial stem cells. However, the method has since been adapted for other epithelial stem cell types. Here, we describe a protocol for the generation and characterization of murine oral mucosal organoid cultures. Primary epithelial cells are isolated from murine tongue tissue, embedded into an ECM hydrogel, and cultured in a medium containing: epidermal growth factor (EGF), R-spondin, and fibroblast growth factor (FGF) 10. Within 7 to 14 days of initial seeding, the resulting organoids can be passaged for further expansion and cryopreservation. We additionally present strategies for the characterization of established organoid cultures via 3D whole-mount imaging and gene-expression analysis. This protocol may serve as a tool to investigate oral epithelial stem cell behavior ex vivo in a reductionist manner.

Introduction

The oral mucosa is the mucous lining covering the inside of our mouth. It functions as the entrance of the alimentary tract and is involved in the initiation of the digestive process^1,2. In addition, the oral mucosa acts as our body's barrier to the outer environment providing protection from physical, chemical and biological insults¹. Based on the function and histology, the oral mucosa in mammals can be divided into three types: masticatory mucosa (including the hard palate and gingiva), the lining mucosa (functioning as the surface of the soft palate, the ventral surface of the t....

Protocol

All methods described here were performed in compliance with European Union and German legislation on animal experimentation.

NOTE: Prepare working place, including sterile surgical instruments (fine forceps, fine scissors, and scalpels) and Petri dishes filled with cold PBSO. Thaw BME overnight and keep it at 4 °C or on ice until usage. Pre-warm cell culture plates in an incubator overnight before starting the cell isolation. All materials are provided in the Table of Materials.

1 Establishment of murine oral mucosal organoid culture

1. Dissection of murine tongue

Results

This protocol describes the separation of the tongue epithelium from the underlying lamina propria and muscle using an enzymatic cocktail (Figure 1). The separated epithelium can further be used for organoid generation as well as harvested for different types of gene and protein analyses. Likewise, the digested layer of lamina propria and muscle may be used for procedures of choice.

For organoid cultures, the tongue epithelium is further digested into small clumps.......

Discussion

Tissue digestion
The collagenase digestion helps in separating the epithelium from the underlying lamina propria and muscle tissue. This step allows for a better comparison of the primary tissue with the subsequently generated oral mucosal organoids. As overdigestion with enzymes impacts the organoid-forming capacity of the adult epithelial stem cells, we advise to perform the collagenase incubation for no longer than 1 h and the trypsin digestion no longer than 30 min. Upon collagenase digestion, .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Media & Media Components
Advanced Dulbecco’s Modified Eagle Medium (DMEM)/F12	Thermo Fisher Scientific	12634-028
B27 Supplement	Thermo Fisher Scientific	17504-044
GlutaMAX-I (100x)	Thermo Fisher Scientific	35050-038
HEPES	Thermo Fisher Scientific	15630-056
N-acetyl-L-cysteine	Sigma Aldrich	A9165
Nicotinamide	Sigma Aldrich	N0636
Penicillin/Streptomycin	Thermo Fisher Scientific	15140-122
Primocin	Invivogen	ant-pm1
RSPO3-Fc fusion protein conditioned medium	U-Protein Express BV	R001
Recombinant human EGF	Preprotech	AF-100-15
Recombinant human FGF-10	Preprotech	100-26
ROCK (Rho kinase) inhibitor Y-27632 dihydrochloride	Hölzel Biotech	M1817
Antibodies
Keratin-14 Polyclonal Antibody 100µl	Biozol	BLD-905301
E-Cadherin Antibody	Bio-Techne	AF748
Purified Mouse Anti-Ki-67 Clone B56  (0.1 mg)	BD Bioscience	556003
ALEXA FLUOR 594 Donkey Anti Mouse	Thermo Fisher Scientific	A21203
ALEXA FLUOR 647 Donkey Anti Rabbit	Thermo Fisher Scientific	A31573
ALEXA FLUOR 488 Donkey Anti Goat	Thermo Fisher Scientific	A110555
Reagents / Chemicals
BME Type 2, RGF Cultrex Pathclear	Bio-Techne	3533-005-02
Dimethyl sulfoxide (DMSO)	Sigma Aldrich	34943-1L-M
Collagenase A	Roche	10103578001
Donkey Serum	Sigma Aldrich	S30-100ML
Phosphate Buffered Saline (PBS)	Thermo Fisher Scientific	100-100-15
EDTA	Sigma Aldrich	221465-25G
Ethanol, denatured (96 %)	Carl Roth	T171.3
Formalin Solution, neutral buffered, 10%	Sigma Aldrich	HT501128-4L
TritonX-100	Sigma Aldrich	X100-500ML
Tween-20	Sigma Aldrich	P1379-500ML
TrypLE Express Enzyme (1×), phenol red	Thermo Fisher Scientific	12605-010
Xylene	Sigma Aldrich	534056-500ML
Equipment and Others
Cell culture 12-Well Multiwell Plates	Greiner BioOne	392-0047
Cell Strainer: 100 µm	VWR	732-2759
Cover Slips	VWR	631-1569P
Glass Bottom Microplates VE=10 4580	Corning	13539050
Objective Slides: Superfrost Plus	VWR	631-0108P