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 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 or skin epidermis5, the oral epithelia remain poorly understood. However, recent studies uncovered different genes such as Krt14, Lrig1, Sox2, Bmi1, 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/β-catenin target gene Lgr513 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'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 and phenotypically stable they are excellent models for gene editing34,35 to study the gene function36 or tumorigenesis27,37,38,39,40. In addition, organoid cultures can be transplanted into mice37,41 and are used to study host-microbe interactions42 (including pathogenic infections43,44,45). Furthermore, organoid-based co-cultures with cells of the microenvironment such as immune cells46,47,48 and fibroblasts49,50 have been described. In the context of disease, organoids have been used for generations of living tissue biobanks21,22,51 as well as testing drugs27 for efficacy52,53 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 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/β-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.
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
2 Passaging, cryopreservation and thawing of murine oral mucosal organoids
3 Gene expression analysis of murine oral mucosal tissue and organoids
4 Protein expression analysis of murine oral mucosal tissue and organoids
NOTE: Whole-mount staining of tongue epithelium was performed in a 24-well plate, transferring the tissue with forceps from well to well in each step.
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 of cells (about 2-3 cells) using trypsin solution. A complete digestion into single cells is not advisable, as this can negatively impact organoid outgrowth52,53. The resulting cell suspension is seeded into the basement membrane hydrogel (Figure 2A), which is then let to solidify to form a 3D ECM-rich scaffold supporting organoid growth. Within 7 to 14 days, oral mucosal organoids derived from murine tongue epithelium grow into larger compact organoids with a keratinizing inner core (Figure 2A).
Established organoid cultures can be maintained by passaging. Using 0.125% trypsin solution, organoids can be dissociated into smaller cell clumps ideally containing 2-3 cells, which are again embedded into the basement membrane hydrogel (Figure 2B). Over a period of up to 14 days, organoids will regrow into larger organoids with the keratinizing inner core appearing within about 3-5 days (Figure 2B).
Gene expression analysis of murine oral mucosal organoid cultures and reference tongue epithelium demonstrated that organoids do faithfully recapitulate the expression of the key markers of the mucosal epithelium (Figure 3A) such as the proliferation marker Mki67 (Figure 3B), the basal layer markers p63 and Krt5 (Figure 3C,D), the general epithelial marker Cdh1 (encoding E-cadherin) (Figure 3E) as well as the terminal differentiation marker Ivl (encoding Involucrin) (Figure 3F). No statistically significant differences in expression of the marker genes tested were found between the oral mucosal epithelium and the mucosal organoid cultures (Figure 3B–F).
Whole-mount preparations of tongue epithelium and oral mucosal organoids can be used to validate the proper cell composition within established organoid cultures. Figure 4 shows expression of keratin 14 (KRT14) in the basal layer of the tongue epithelium as well as in the outermost layer of the oral mucosal organoids, which corresponds to the epithelial basal layer. In addition, expression of the proliferation marker KI-67 is restricted to the KRT14-positive basal layer in both tissues and organoids (Figure 4A,B). In contrast, all the cells in both tongue epithelium and organoids show expression of the epithelial marker E-cadherin (ECAD; Figure 4A,B), which marks adherens junctions connecting the epithelial cells.
Figure 1: Digestion of the tongue and separation of the epithelium from the lamina propria. (A) A micrograph of the intact tongue before digestion. (B) Micrographs of the tongue epithelium and lamina propria (including the muscle layer) following digestion and separation. Scale bars: 5 mm. Please click here to view a larger version of this figure.
Figure 2: Organoid establishment and passaging. (A) Representative brightfield images of the cell clumps directly following initial plating (left panel) and of the organoids growing in culture between 1 to 10 days following plating (three panels on the right-hand side). (B) Representative brightfield images of the passaged organoids directly after plating (left panel) and growing organoids between 3 to 10 days following passaging (three panels on the right-hand side). Scale bars: 250 μm. Please click here to view a larger version of this figure.
Figure 3: Gene expression analysis of tongue epithelium and oral mucosal organoids. (A) Schematic highlighting the different layers of the stratified and keratinizing tongue epithelium. BL, basal layer; CE, cornified envelope with dead keratinocytes; SL, suprabasal layers with viable keratinocytes (including the spinous layer and granular layer). (B–F) Gene expression analysis of marker genes: proliferation marker Mki67 usually restricted to the basal layer (B), basal layer markers p63 (C) and Krt5 (D), epithelial marker Cdh1 (encoding E-cadherin; E) and differentiation marker Ivl (encoding Involucrin; F). Dark-shaded columns show data from the primary epithelium of the ventral and dorsal tongue and light-shaded columns show data from the organoid cultures established from the respective epithelia. Data were calculated following the ΔCt method and are presented as mean average from 3-4 biological replicates relative to the housekeeping gene Actb. Error bars indicate the standard error of the mean. No statistically significant differences were found (n.s., not significant; p > 0.05) using 2-way ANOVA with Tukey's multiple comparisons test. Please click here to view a larger version of this figure.
Figure 4: Protein expression analysis of tongue epithelium and oral mucosal organoids. Whole-mount staining of ventral tongue epithelium (A) and oral mucosal organoids derived from primary ventral tongue epithelium (B). DAPI in blue marks DNA, keratin 14 (KRT14) expression is shown in green, E-cadherin (ECAD) expression is shown in green and KI-67 expression is shown in red. Scale bars: 100 μm (A) and 50 μm (B). Please click here to view a larger version of this figure.
Medium | Supplement | Original conc. | Dilution | Final conc. |
Basic medium (Advanced DMEM/F12+++ medium) | ||||
GlutaMAX-I | 100x | 1/100 | 1x | |
Penicillin/Streptomycin | 10000 U/mL | 1/100 | 100 U/mL | |
HEPES | 1 M | 1/100 | 10 mM | |
Complete with Advanced DMEM/F12 | ||||
Complete growth medium (murine oral mucosal organoid medium) | ||||
B27 Supplement | 50x | 1/50 | 1x | |
N-acetyl-L-cysteine | 500 mM | 1/400 | 1.25 mM | |
Nicotinamide | 1 M | 1/100 | 10 mM | |
Recombinant human EGF | 0.5 mg/mL | 1/10,000 | 50 ng/mL | |
Recombinant human FGF10 | 0.1 mg/mL | 1/10,000 | 10 ng/mL | |
RSPO3-Fc fusion protein conditioned medium | 1/20 | 4% | ||
ROCK inhibitor Y-27632§ | 100 mM | 1/10,000 | 10 μM | |
Primocin$ | 50 mg/mL | 1/50 | 100 μg/mL | |
Complete with basic medium (Advanced DMEM/F12+++ medium) | ||||
§To be added after initial cell isolation and passaging, $to be added after initial cell isolation |
Table 1: Medium composition.
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 epithelium is very fragile. It is therefore important to take extra care when separating the epithelium to avoid ripping the epithelium apart. When checking for proper separation under the microscope, it should be assessed whether the connective tissue (typically whiteish in appearance) detached from the tongue epithelium. In addition, the specialized epithelium of the dorsal tongue and lining epithelium of the ventral tongue are connected via a transition zone that covers the lateral side of the tongue. When further working with the epithelium, it is therefore important to note that the epithelia of the dorsal and ventral tongue cannot be completely separated.
Organoid appearance and handling
In line with murine skin epidermal organoids25, oral mucosal organoids grow in a compact manner without the cystic spheroid phenotype described for other epithelial organoid cultures11,12,14,15,19,20. When organoids start to attach to the bottom of the cell culture plate, a re-embedding of the organoids will be necessary, as otherwise the cells will start growing as a monolayer sheet at the bottom of the plate. These cells might be difficult to recover and may have changed in characteristics due to 2D culture. Therefore, we strongly recommend using suspension culture plates for murine oral mucosal organoid cultures.
The digestion time for passaging organoids is dependent on the size and compactness of the organoids. We recommend a short digestion time (<5 min) for cultures with mostly smaller organoids. Cultures with larger organoids should be digested for no longer than 10 min, as an overdigestion negatively impacts cell viability and organoid outgrowth. Dispersing the organoids by shaking during the digestion procedure increases the efficiency of the organoid dissociation. Depending on the assay, it may be necessary to have a culture of similarly sized organoids. To achieve a more homogenous culture, we recommend passing the cell clusters through a 70 μm cell strainer upon digestion, as the dissociated cell clusters otherwise clump back together in culture very quickly. As the basal-like cells are located at the very outer layer of the organoids, these cells are likely to be the first ones to be dissociated upon digestion. Thus, big clumps of differentiated and keratinized cells within the newly seeded cell suspension after splitting, might interfere with proper organoid formation. It is of great importance to pre-warm the medium to 37 °C, as the drops might dissociate otherwise.
Gene expression
Using the whole tongue as the input for the RNA isolation may lead to significant differences in marker gene expression between tissue and organoids. In addition, we observed decreased RNA quality when isolating RNA from the whole tongue as the input, which is likely due to the muscle tissue. As was observed in other organoid cultures such as those established from murine skin25, we advise to collect RNA also from later passages (starting at passage 5). Organoid cultures before passaging may still contain non-epithelial cell types such as lymphocytes or fibroblast, causing artefacts in the analysis.25
Protein expression
Depending on the organoid size and tissue thickness, we recommend an additional step for staining the DNA with DAPI for whole-mount staining. Furthermore, the separated tongue epithelium as well as established organoids may also be used for western blot, electron microscopy, or conventional histology based on published studies53.
Possible applications
In line with the already well-established organoid protocols, oral mucosal organoids are amenable to a wide range of downstream applications11. So far, murine, and human tissue-derived oral mucosal organoid cultures have been used for biobanking, testing efficacy52,53, and toxicity54 of anti-cancer drugs27 or radiotherapy52,53. Oral mucosal organoids can also be infected with viruses such as herpes simplex virus and human papilloma virus53. To assess the capacity to form metastasis, organoids derived from oral cancer may also be transplanted into mice53. Based on robust protocols34,35, cultures of oral mucosal organoids may further be used for different gene-editing methods, including CRISPR-Cas9-based approaches.
The authors have nothing to disclose.
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.).
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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