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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

Published: July 31st, 2021

DOI:

10.3791/62529

1Mildred Scheel Early Career Centre (MSNZ) for Cancer Research, IZKF/MSNZ, University Hospital Würzburg

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

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.

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 process1,2. 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....

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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 Materi.......

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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.......

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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, .......

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The authors would like to thank Sabine Kranz for assistance. We would like to thank the Core Unit for Confocal Microscopy and Flow Cytometry-based Cell Sorting of the IZKF Würzburg for supporting this study. This work was funded by a grant from the German Cancer Aid (via IZKF/MSNZ Würzburg to K.K.).

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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

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