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 W&#252;rzburg for supporting this study. This work was funded by a grant from the German Cancer Aid (via IZKF/MSNZ W&#252;rzburg to K.K.).

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 &#176;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&#160;Table of Materi...

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, ...

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&#39;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 tongue and the buccal surface), and the specialized mucosa (covering the dorsal surface of the tongue)2. All oral mucosal tissues consist of two layers: the surface stratified squamous epithelium and the underlying lamina propria1. The oral epithelial keratinocyte is the main cell type of the epithelium, which is also the location of intra-epithelial immune cells such as Langerhans cells1. The stromal compartment, the lamina propria, comprises of the different cell types such as fibroblasts, endothelial cells, neuronal cells, and immune cells1. As in all stratified epithelia, stem and progenitor cells reside in the basal layer of the oral epithelium1. These specialized cells have the ability to replace lost tissue through cell divisions and, therefore, feed the cellular turnover throughout adult life3. In contrast to other epithelia such as the intestinal epithelium4&#160;or skin epidermis5, the oral epithelia remain poorly understood. However, recent studies uncovered different genes such as Krt14, Lrig1, Sox2, Bmi1,&#160;and Gli1 that mark oral epithelial stem and progenitor cells in mice1,6,7,8. As the oral epithelium is the origin of oral carcinomas and a critical player in mucosal inflammation, wounding, and regeneration1, a better understanding of its basic cell biology is paramount for potential new therapeutic approaches and drug discoveries.
Animal models have been widely used for basic studies on the oral mucosal epithelium1. For example, the aforementioned markers of oral epithelial stem and progenitor cells have largely been defined using genetic lineage tracing mouse models1,6,7,8,9. However, ex vivo approaches using cultured cells of human or murine origin have also been broadly used10. Conventionally, such cell culture work has been performed using cell lines derived from oral squamous cell carcinoma (OSCCs) or cell lines generated from (spontaneously or genetically) immortalized primary cells10. These 2D cell culture methods have limitations with critical implications on investigating the adult homoeostasis: (1) cell immortalization is accompanied with a large degree of genetic instability, (2) limited capacity to differentiate, (3) requirement for feeder cells, and (4) a largely undefined growth medium containing serum11. Collectively, these gold standard in vitro methods did not allow long-term cultures of epithelial stem cells without limiting their capacity to proliferate and differentiate as well as transforming their wild-type genome.
Organoid technology has emerged as a tool to establish cultures of near native epithelial tissue in vitro11. In their 2009 study, Sato et al. described the first epithelial organoid culture system12. Their method was based on embedding individual small intestinal stem cells marked by the Wnt/&#946;-catenin target gene Lgr513&#160;into a 3D extra-cellular matrix (ECM)-rich hydrogel12. By providing a defined cocktail of growth factors important for stemness, the seeded adult epithelial stem cells were able proliferate to their capacity in culture12. Eventually, cell clusters formed out of the actively cycling stem cells containing all major intestinal epithelial cell types12, effectively resembling the tissue-of-origin11. In contrast to conventional 2D cultures, organoid technology allowed long-term maintenance of murine intestinal epithelial stem cells under feeder-free conditions with a serum-free and fully defined medium10,11. In addition, the method does not significantly alter the genetic makeup or phenotype of the cultured stem cells11. Furthermore, long-term culture retained the stem cell&#39;s capacity to proliferate and differentiate without a requirement for cell immortalization11. Within just over a decade, this early epithelial organoid culture system was amended to grow adult stem cells from many other epithelial tissues such as colon (large intestine)12,14,15, endometrium16, liver17,18, lungs19,20, mammary glands21, ovaries22, pancreas23,24, skin epidermis25, and stomach26. While most protocols used adult epithelial stem cells derived from mammals such as humans11,27, mice11, cats28, dogs29, and pigs30, it has even been possible to generate epithelial organoids from snake venom glands31. Organoid technology has become a widely used stem cell culture method with a high degree of versatility11. As epithelial organoids remain largely genetically32,33&#160;and phenotypically stable they are excellent models for gene editing34,35&#160;to study the gene function36&#160;or tumorigenesis27,37,38,39,40. In addition, organoid cultures can be transplanted into mice37,41&#160;and are used to study host-microbe interactions42&#160;(including pathogenic infections43,44,45). Furthermore, organoid-based co-cultures with cells of the microenvironment such as immune cells46,47,48&#160;and fibroblasts49,50 have been described. In the context of disease, organoids have been used for generations of living tissue biobanks21,22,51&#160;as well as testing drugs27&#160;for efficacy52,53&#160;and toxicity54.
In this protocol, we describe an optimized methodology for the establishment and maintenance of oral mucosal organoid cultures from murine tongue epithelium. It is based on previous reports describing the isolation of the tongue epithelium using enzymatic digestion55&#160;and the derivation of epithelial organoids from mouse and human oral mucosa52,53. The growth medium for murine oral mucosal organoids contains critical factors maintaining the stem cell state. R-spondin activates the Wnt/&#946;-catenin signaling cascade5, while epidermal growth factor (EGF) and fibroblast growth factor (FGF) 10 are cytokines and ligands of receptor tyrosine kinases that stimulate several signaling pathways such as the MAPK/ERK pathway and the PI3K/AKT/mTOR pathway25. We further describe in detail how the organoid cultures can be characterized by gene and protein expression analysis and compared with the tissue-of-origin.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Media &#38; Media Components</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td>Advanced Dulbecco&#8217;s Modified Eagle Medium (DMEM)/F12</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>12634-028</td>
			<td></td>
		</tr>
		<tr >
			<td >B27 Supplement&#160;</td>
			<td >Thermo Fisher Scientific</td>
			<td >17504-044</td>
			<td></td>
		</tr>
		<tr >
			<td >GlutaMAX-I (100x)</td>
			<td >Thermo Fisher Scientific</td>
			<td >35050-038</td>
			<td></td>
		</tr>
		<tr >
			<td >HEPES</td>
			<td >Thermo Fisher Scientific</td>
			<td >15630-056</td>
			<td></td>
		</tr>
		<tr >
			<td >N-acetyl-L-cysteine</td>
			<td >Sigma Aldrich</td>
			<td >A9165</td>
			<td></td>
		</tr>
		<tr >
			<td >Nicotinamide</td>
			<td >Sigma Aldrich</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin/Streptomycin&#160;</td>
			<td >Thermo Fisher Scientific</td>
			<td >15140-122</td>
			<td></td>
		</tr>
		<tr >
			<td >Primocin</td>
			<td>Invivogen</td>
			<td>ant-pm1</td>
			<td></td>
		</tr>
		<tr >
			<td >RSPO3-Fc fusion protein conditioned medium</td>
			<td >U-Protein Express BV</td>
			<td >R001</td>
			<td></td>
		</tr>
		<tr >
			<td >Recombinant human EGF</td>
			<td >Preprotech</td>
			<td >AF-100-15</td>
			<td></td>
		</tr>
		<tr >
			<td >Recombinant human FGF-10</td>
			<td >Preprotech</td>
			<td >100-26</td>
			<td></td>
		</tr>
		<tr >
			<td >ROCK (Rho kinase) inhibitor Y-27632 dihydrochloride</td>
			<td >H&#246;lzel Biotech</td>
			<td >M1817</td>
			<td></td>
		</tr>
		<tr >
			<td >Antibodies&#160;</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Keratin-14 Polyclonal Antibody 100&#181;l</td>
			<td >Biozol</td>
			<td >BLD-905301</td>
			<td></td>
		</tr>
		<tr >
			<td >E-Cadherin Antibody</td>
			<td >Bio-Techne</td>
			<td >AF748</td>
			<td></td>
		</tr>
		<tr >
			<td >Purified Mouse Anti-Ki-67&#160;Clone B56 &#160;(0.1 mg)</td>
			<td >BD Bioscience</td>
			<td >556003</td>
			<td></td>
		</tr>
		<tr >
			<td >ALEXA FLUOR 594 Donkey Anti Mouse</td>
			<td >Thermo Fisher Scientific</td>
			<td >A21203</td>
			<td></td>
		</tr>
		<tr >
			<td >ALEXA FLUOR 647 Donkey Anti Rabbit</td>
			<td >Thermo Fisher Scientific</td>
			<td >A31573</td>
			<td></td>
		</tr>
		<tr >
			<td >ALEXA FLUOR 488 Donkey Anti Goat</td>
			<td >Thermo Fisher Scientific</td>
			<td >A110555</td>
			<td></td>
		</tr>
		<tr >
			<td >Reagents / Chemicals</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >BME Type 2, RGF Cultrex Pathclear</td>
			<td >Bio-Techne</td>
			<td >3533-005-02</td>
			<td></td>
		</tr>
		<tr >
			<td >Dimethyl sulfoxide (DMSO)</td>
			<td >Sigma Aldrich</td>
			<td >34943-1L-M</td>
			<td></td>
		</tr>
		<tr >
			<td >Collagenase A&#160;</td>
			<td >Roche</td>
			<td >10103578001</td>
			<td></td>
		</tr>
		<tr >
			<td >Donkey Serum</td>
			<td >Sigma Aldrich</td>
			<td >S30-100ML</td>
			<td></td>
		</tr>
		<tr >
			<td >Phosphate Buffered Saline (PBS)</td>
			<td >Thermo Fisher Scientific</td>
			<td >100-100-15</td>
			<td></td>
		</tr>
		<tr >
			<td >EDTA</td>
			<td >Sigma Aldrich</td>
			<td >221465-25G</td>
			<td></td>
		</tr>
		<tr >
			<td >Ethanol, denatured (96 %)</td>
			<td >Carl Roth</td>
			<td >T171.3</td>
			<td></td>
		</tr>
		<tr >
			<td >Formalin Solution, neutral buffered, 10%</td>
			<td >Sigma Aldrich</td>
			<td >HT501128-4L</td>
			<td></td>
		</tr>
		<tr >
			<td >TritonX-100</td>
			<td >Sigma Aldrich</td>
			<td >X100-500ML</td>
			<td></td>
		</tr>
		<tr >
			<td >Tween-20&#160;</td>
			<td >Sigma Aldrich</td>
			<td >P1379-500ML</td>
			<td></td>
		</tr>
		<tr >
			<td >TrypLE Express Enzyme (1&#215;), phenol red</td>
			<td >Thermo Fisher Scientific</td>
			<td >12605-010</td>
			<td></td>
		</tr>
		<tr >
			<td >Xylene</td>
			<td >Sigma Aldrich</td>
			<td >534056-500ML</td>
			<td></td>
		</tr>
		<tr >
			<td >Equipment and Others</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Cell culture 12-Well Multiwell Plates</td>
			<td >Greiner BioOne</td>
			<td >392-0047</td>
			<td></td>
		</tr>
		<tr >
			<td >Cell Strainer: 100 &#181;m</td>
			<td >VWR</td>
			<td >732-2759</td>
			<td></td>
		</tr>
		<tr >
			<td >Cover Slips</td>
			<td >VWR</td>
			<td >631-1569P</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass Bottom Microplates VE=10 4580</td>
			<td >Corning</td>
			<td >13539050</td>
			<td></td>
		</tr>
		<tr >
			<td >Objective Slides: Superfrost Plus</td>
			<td >VWR</td>
			<td >631-0108P</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation and characterization of murine oral mucosal organoid cultures

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.

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...

Read this Scientific Article Protocol about Generation and Characterization of Murine Oral Mucosal Organoid Cultures at JoVE.com

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

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, ...