The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.
For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and α6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFRα. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.
The oral epithelium serves as a first barrier in protecting the body from environmental stresses, including chemical or physical damage and bacterial and viral infections1,2. The oral mucosa comprises an outer layer of stratified squamous epithelium that consists of keratinocytes and underlying connective tissue called the lamina propria, which mainly consists of fibroblasts and the extracellular matrix. The mouse oral mucosa can be broadly divided into three subtypes: masticatory (hard palate and gingiva), specialized (dorsal tongue), and lining (buccal mucosa, ventral tongue, soft palate, lips) mucosa2,3 (Figure 1A). The oral epithelium is keratinized in the masticatory and specialized mucosa and non-keratinized in the lining mucosa. Despite its anatomical location, the oral epithelium is similar to the skin epidermis in that it consists of tightly packed epithelial cells with varying degrees of differentiation: basal layers containing undifferentiated cells; spinous, granular, and cornified layers that form keratinized epithelium, or intermediate and superficial layers that form non-keratinized epithelium4. Transgenic mouse models have facilitated the study of oral epithelial stem cells' cellular and molecular features in the palate, buccal mucosa, tongue, and gingiva5,6,7,8,9,10,11. However, most of these studies primarily used in vivo mouse experiments. Cell culture systems were not typically employed owing to a lack of established and efficient protocols.
An in vitro culture system can be used for the molecular and biochemical analysis of stem cell regulators, cell-based assays, and drug screening. Currently, protocols for the culture of primary keratinocytes of the skin epidermis have been developed, in which basal keratinocytes can be successfully isolated and cultured for clinical and research purposes12,13,14,15. In 1980, Hennings et al. showed that a low calcium concentration (< 0.09 mM Ca2+) in the culture medium facilitated proliferation and maintained cells in an undifferentiated state. A higher level of calcium promoted cell differentiation and reduced proliferation16. Subsequently, culture methodologies for neonatal and adult murine epidermal keratinocytes have been established and widely applied to numerous mouse models with different genetic backgrounds for in vitro studies17,18,19. Although skin and oral epithelia share common characteristics, they also show intrinsic differences, e.g., in their keratinization status, turnover rate, gene expression, and wound healing ability3,11,20,21,22,23,24,25,26.
Although human oral keratinocyte culture has been successfully performed27,28,29, publications on mouse oral keratinocyte culture30,31,32 are limited due to the small size of the target tissue and the distinct characteristics of the cells compared to skin epidermal keratinocytes. This protocol describes the isolation and long-term culture techniques of mouse primary oral keratinocytes.
All animal experiments were performed according to the Institutional Animal Experiment Committee guidelines at Kumamoto University and the University of Tsukuba.
1. Preparation of reagents and culture media
2. Dissection of palate tissue from adult mouse
3. Pretreatment of palate tissue
4. Collection and culture of primary cells
5. Keratinocyte passage
6. Cryopreservation and recovery of keratinocytes
7. Immunofluorescent staining
Overview of the dissection process and isolation of oral keratinocytes from the adult mouse palate
Dissociated oral keratinocytes were collected from the adult mouse palate and cultured in a customized 20% chelexed-FBS. The mouse palate consists of the hard palate and the soft palate (Figure 1C). The procedure for the isolation of mouse oral keratinocytes is summarized in Figure 1D. The palate tissue is dissected and transferred to a media containing an antibiotic-antimycotic solution before being incubated in 0.025% trypsin solution at 4 °C overnight. The following day, the palate tissue is treated with trypsin inhibitor solution and complete culture medium in equal volumes. Subsequently, the tissues are scraped using a surgical scalpel blade to collect oral keratinocytes. The cell suspension is filtered through a 100 µm cell strainer and centrifuged. The cells are then seeded in Collagen I-coated 24-well plates containing 2 mL of complete culture medium + chelexed-FBS.
Representative results of the successful isolation of mouse oral keratinocytes
Primary oral keratinocytes grew as a monolayer and displayed a cobblestone morphology (Figure 2). Small keratinocyte colonies were visible at 3-5 days (Figure 2A,B); these grew larger and formed tight colonies at 1 week of incubation (Figure 2C). Keratinocyte colonies displayed the typical morphological features of basal keratinocytes, indicating their healthy conditions. Human oral keratinocytes remained undifferentiated for several passages in the complete culture medium containing 0.06 mM Ca2+16,28. The first passage was performed approximately 2 weeks from the initial plating (Figure 2D). At later passages, keratinocytes exhibited stable growth with a shorter period of culture (Figure 2E-G). Keratinocytes stopped growing if significant fibroblasts contamination occurred during the isolation process (Figure 2H).
Isolated mouse oral keratinocytes express basal cell markers
To confirm the status of primary oral keratinocytes, immunostaining was performed using the basal cell markers Keratin 14 (K14) and α6-integrin34. K14 and α6-integrin were expressed in keratinocytes after culturing (Figure 3A,B). The cells were also stained with stem cell marker p63 to confirm their stemness. Early passage (passage 4) and late passage (passage 7) cells showed uniform expression of p63 (Figure 3C,D). In contrast, keratinocytes treated with high calcium (1.2 mM induction for 2 days) exhibited decreased p63 expression (Figure 3E), indicating that high calcium treatment suppresses stem cell-related genes in primary keratinocytes as previously reported16. The differentiation marker Keratin 13 (K13) showed rare or no expression in both early and late passages and significant expression under high calcium treatment (Figure 3F-H). To test the possibility of fibroblast contamination in the keratinocyte culture, staining using the fibroblast marker PDGFRα was performed with the same set of keratinocytes compared with mouse embryonic fibroblast (MEFs). There was no expression of PDGFRα in the keratinocyte culture, compared with the high expression observed in MEF cells (Figure 3I-3L). These results indicated that this protocol could successfully isolate basal keratinocytes and maintain these cells in the undifferentiated state.
Figure 1: Overview of the dissection procedure and isolation of mouse oral keratinocytes. (A) Schematic representation of the mouse oral cavity. (B) Instruments used to dissect the palate and isolate mouse oral keratinocytes. (C) Brightfield image of the mouse palate. Scale bar: 100 µm. (D) Summary of the protocol. Please click here to view a larger version of this figure.
Figure 2: Representative results of the successful isolation of mouse oral keratinocytes. (A-G) Time-course images of cultured primary oral keratinocytes at 3 days (A), 5 days (B), 1 week (C), and 2 weeks (D) of culture after isolation. Morphologies of mouse oral keratinocytes after the first (E), second (F), and third (G) passages are shown. (H) Example of fibroblast contamination in mouse oral keratinocyte culture. Scale bar: 400 µm. Please click here to view a larger version of this figure.
Figure 3: Isolated mouse oral keratinocytes express basal cell markers. (A-B) Representative images of immunofluorescent staining of K14 (A; red) and α6-integrin (B; green) in passage 4. (C-E) Representative images of immunofluorescent staining of p63 (green) in passage 4 (C), passage 7 (D), and high calcium treatment (E). (F-H) Immunostaining images of K13 (green) in passage 4 (F), passage 7 (G), and high calcium treatment (H). (I-L) Immunostaining images of PDGFRα (red) in passage 4 (I), passage 7 (J), high calcium treatment (K), and MEFs (L). Nuclei are stained with Hoechst (blue). Scale bars: 100 µm. Please click here to view a larger version of this figure.
Primary keratinocytes isolated from human or mouse skin epidermis have been utilized for many years in research and clinical applications12,13,15,18,27,28,29. By contrast, few protocols have been established to isolate and culture primary oral keratinocytes from adult mice30,31,32. The present study used a commercial complete culture medium and chelexed-FBS to maintain keratinocytes in a proliferative or stem cell-like state. This culture system can be employed in molecular and biochemical assays to further understand the features of oral epithelial stem cells and their related diseases.
Several critical steps are included in this protocol. Firstly, the trypsin concentration and the incubation time are essential in producing viable cells for subsequent cultures. We consistently used 0.025% trypsin solutions and 16 h incubation periods in the chamber hood at room temperature. If not incubated for a sufficient length of time, keratinocytes would not properly dissociate from the tissue, resulting in a lower final cell yield. Secondly, gentle pipetting of the cell suspension on the second day notably affects cell viability. Scraping should gently start from the epithelial side and not exceed 10 min per tissue sample. Finally, the first isolated cell suspension contains fibroblasts and other cell types; these unwanted cells will usually begin to disappear in subsequent cultures.
Potential limitations were identified during cell isolation and culture. In rare cases, fibroblasts may be contaminated during the isolation process, and the fibroblasts may inhibit the growth of keratinocytes in subsequent passages (Figure 2H). It is necessary to select a commercial medium that contains a fibroblast growth inhibitor to eliminate such contamination in the culture. Because the mouse palate and other oral mucosa have relatively small sizes, the initial cell yield from one mouse may be low. Therefore, the entire culture period of this protocol-until the cryopreservation stage-is longer than that for primary skin keratinocytes. Isolated oral keratinocytes are best used within 10 passages, as more extended culture periods could change the cell properties and lower the number of stem cells.
The current method showed that mouse oral keratinocytes exhibited a tightly packed, cobblestone morphology and formed monolayer colonies under proliferative conditions. They also showed high expression of the basal markers α6-integrin, K14, and stem cell marker p63. In future studies, in addition to immunofluorescence staining, RNA-sequencing, RT-PCR, and western blot analyses will be used to verify the cellular heterogeneity and purity of oral keratinocytes, which will further enhance our understanding of the nature of these cells.
After 2-3 passages, oral keratinocytes were stable enough to be used in further functional experiments. Importantly, this culture protocol can be combined with transgenic mouse lines, including gene knockout, Cre-loxP, and tet-inducible systems, and can also be used in cellular and molecular assays. Thus, the present protocol provides researchers with a fundamental and efficient method that could be used to understand oral keratinocyte stem cell biology further.
This work was supported by the Grant-in-Aid for Scientific Research (B) (20H03266) (to A.S.), Grant-in-Aid for Early-Career Scientists (18K14709) (to A.S.), AMED under Grant Number JP21gm6110016 and 21bm0704067 (to A.S.), and research grants from the Takeda Science Foundation (to A.S.). We thank the Center for Animal Resources and Development at Kumamoto University and the Animal Resource Center at the University of Tsukuba for their excellent mouse care. We thank the IRCMS core facility at Kumamoto University for its support in confocal imaging.
Name | Company | Catalog Number | Comments |
0.025% Trypsin/EDTA | Gibco | R001100 | |
0.05% Trypsin/EDTA, phenol red | Gibco | 25300062 | |
0.4w/v% Trypan Blue solution | Wako | 207-17081 | |
10 mL Serological pipet | Falcon | 357551 | |
100 µm Nylon cell strainer | Falcon | 352360 | |
15 mL sterile conical tube | Falcon | 352096 | |
2 mL Aspirating pipet | Falcon | 357558 | |
35 mm Cell culture dish | Corning | 353801 | |
50 mL sterile conical tube | Falcon | 352070 | |
60 mm Cell culture dish | Corning | 353802 | |
6-well Tissue Culture plate | Falcon | 353046 | |
96 Well Culture plate (U bottom) | Falcon | 353077 | |
Antibiotic-Antimycotic 100x | Gibco | 15240062 | |
Biocoat Collagen I cellware 60mm dish | Corning | 356401 | |
Blunt Forceps | AS ONE | 1-8187-03 | |
Butorphanol | Meiji Seika Pharma | Vetorphale | |
CoolCell LX | Corning | 432002 | Cryogenic storage |
Cotton | AS ONE | 63-1452-97 | |
Cover slips 22 x 22 μm square | Matsunami Glass Ind. | C022221 | |
Cryogenic Vial 1.2 mL | Thermo Scientific | 5000-0012 | |
Defined Trypsin Inhibitor (DTI) | Gibco | R007100 | |
Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | 276855-100ML | |
Donkey anti-Rabbit IgG (H+L), Alexa Fluor 555 | Invitrogen | A31572 | |
Donkey anti-Rat IgG (H+L), Alexa Fluor 488 | Invitrogen | A21208 | |
Donkey serum | Sigma-Aldrich | D9663-10ML | |
EpiLife Defined Growth Supplement (EDGS) | Gibco | S0120 | |
EpiLife, with 60 µM calcium | Gibco | MEPI500CA | |
Fetal Bovine Serum (FBS) | Gibco | 26140079 | |
Fine forceps | BRC Bio Research Center | PRI13-3374 | |
Goat anti-PDGF Receptor α | R&D Systems | AF1062 | |
Goat serum | Sigma-Aldrich | G9023-10ML | |
Half-curved forceps | BRC Bio Research Center | PRI13-3376 | |
Hemocytometer | Hirschmann Laborgeräte | 8100204 | |
Hoechst | Sigma-Aldrich | B2261 | |
Iris Scissors | Muromachi Kikai | 14090-09 | |
Medetomidine | Nippon Zenyaku Kogyo | Domitor | |
Midazolam | Astellas Pharma | Dormicum | |
No.15 Disposable scalpel | Feather | 219AABZX00136000 | |
Paraformaldehyde | Wako | 162-16065 | |
Phosphate Buffered Saline 1x, pH 7.4 | Gibco | 10010049 | |
Povidone-iodine | Y's Square | 872612 | |
Rabbit anti-Cytokeratin 13 antibody | Abcam | ab92551 | |
Rabbit anti-K14 | BioLegend | 905301 | |
Rabbit anti-p63 antibody | Abcam | ab124762 | |
Raspatorium #14 | AS ONE | 8-4599-01 | |
Rat α6-integrin | BD Biosciences | 553745 | |
Triton X-100 | Wako | 581-81705 | |
Type I Collagen coated 24-well plate | Corning | 354408 | |
Type I Collagen coated 60mm dish | Corning | 356401 | |
Type I Collagen coated 6-well plate | Corning | 355400 |
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