Name: isolation and culture of primary oral keratinocytes from the adult mouse palate
Uploaded: 2021-09-24T15:00:00.000+00:00
Duration: 6 min 28 s
Description: Watch this Scientific Journal Video about Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate at JoVE.com

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.

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
<ol>
	<li>Prepare 40 mL of keratinocyte culture medium containing 60 &#181;M of calcium and 600 &#181;L of antibiotic-antimycotic solution. Prepare 20 mL of 0.025% trypsin and 400 &#181;L of antibiotic-antimycotic solution.
	<ol>
		<li>Thaw the trypsin inhibition solution at room temperature and keep it at 4 &#176;C until use in step 4.1. 
		NOTE: The isolation reagent is prepared for the tissue isolation of five mice.</li>
	</ol>
	</li>
	<li>To prepare the culture media, take 500 mL of medium and add 5 mL of a growth supplement solution (see Table of Materials) (hereafter referred to as complete medium) and 20% calcium-depleted chelexed-fetal bovine serum (FBS)33 (hereafter referred to as chelexed-FBS).</li>
</ol>
2. Dissection of palate tissue from adult mouse
<ol>
	<li>Sacrifice an adult C57BL/6J mouse (either male or female) by cervical dislocation in compliance with the facility&#39;s regulations relating to animal welfare.
	<ol>
		<li>Remove the hair around the mouth with a shaver. Using scissors, cut from the cheek toward the jaw, on both sides. 
		NOTE: The mouse needs to be anesthetized before sacrifice. An anesthetic mixture of medetomidine, midazolam, and butorphanol is used (see Table of Materials).</li>
	</ol>
	</li>
	<li>Use forceps to open the mouth wide and absorb any blood using a cotton swab. To disinfect the palate, wipe the inside of the mouth with a cotton swab containing 10% povidone-iodine.</li>
	<li>To harvest the mouse palate, first, use a surgical scalpel blade to make a full-thickness marginal incision along the palate side of the maxillary teeth. Then, carefully dissect the entire palate using a raspatorium. 
	NOTE: A raspatorium is a tool used to elevate a mucoperiosteal flap (see Table of Materials) (Figure 1B).</li>
	<li>Quickly transfer the palate tissue to a 15 mL tube containing 4 mL of complete medium + antibiotic-antimycotic solution. Keep the tissues on ice until ready for incubation. 
	&#8203;NOTE: For collection from multiple mice, palate tissues may be kept on ice for up to 4 h.</li>
</ol>
3. Pretreatment of palate tissue
<ol>
	<li>In a laminar flow hood, transfer tissues to a 60 mm dish containing 4 mL of complete medium + antibiotic-antimycotic solution.</li>
	<li>Using short, blunt forceps and a scalpel blade, gently remove any blood from the tissues. Wash tissues 10 times in complete medium.</li>
	<li>Transfer tissues to a 35 mm dish containing 4 mL of 0.025% trypsin + antibiotic-antimycotic solution, with the epithelial surface facing down. 
	NOTE: The epithelial surface, which curves inward, should be soaked in the trypsin solution; the lamina propria should face up. The tissue should be flattened as much as possible to be incubated entirely in the trypsin solution.</li>
	<li>Incubate tissues in 0.025% trypsin for ~16 h at room temperature in the culture hood.</li>
</ol>
4. Collection and culture of primary cells
<ol>
	<li>Using one pair of blunt forceps, remove tissue from trypsin solution (in the 35 mm dish) and transfer to trypsin inhibitor solution in a 60 mm dish (4 mL per dish) with the epithelial surface facing up.</li>
	<li>Using forceps to hold onto the edge of the palate, gently scrape the epithelial layer off the underlying lamina propria using a scalpel blade. 
	NOTE: Connective tissue is not digested by trypsin, so it does not peel off during scraping.</li>
	<li>To collect the maximum amount of epithelial cells from tissues, transfer the tissue into another 60 mm dish with 4 mL complete medium and repeat the scraping step. 
	NOTE: Make sure to avoid scraping the tissue with the blade&#39;s tip; use the blade&#39;s edge instead. Scraping is performed for 5-10 min per tissue.</li>
	<li>Place a sterile 100 &#181;m cell strainer on the top of a 50 mL conical tube.</li>
	<li>Using a sterile pipette, transfer 2 mL of trypsin solution (from step 4.1) into the strainer to wet its surface.</li>
	<li>Using a pipette, mix the cell suspension in the 60 mm dish (from steps 4.2-4.3) a few times and filter cells through the 100 &#181;m cell strainer prepared in steps 4.4-4.5.</li>
	<li>Count the number of cells using a hemocytometer. Prepare 15 &#181;L of trypan blue solution and add 15 &#181;L of cell suspension (step 4.6). Transfer 10 &#181;L of the cell-trypan blue mix to a hemocytometer and count the number of cells. 
	NOTE: One piece of mouse palate can yield up to 1 million cells.</li>
	<li>While counting, centrifuge the tube (from step 4.6) at 100 x g for 5 min at room temperature.</li>
	<li>Aspirate the supernatant with a pipette. Add 2 mL of complete medium + chelexed-FBS to the tube. Resuspend the cell pellet by triturating several times using a 5 mL pipette.</li>
	<li>Plate 2-5 x 105 cells from one mouse into one well of a 24-well plate pre-coated with Collagen Type I (see Table of Materials).</li>
	<li>Incubate the cells at 37 &#176;C for 2 days without changing the medium.</li>
	<li>Two days after seeding, replace half of the culture medium with the complete medium + chelexed-FBS. Check the cell morphology under the microscope. Feed cells with complete medium + chelexed-FBS every 2 days. 
	NOTE: Following seeding, cells of different sizes will be observed. Approximately 3-5 days after seeding, keratinocytes with a cobblestone morphology (Figure 2) can be observed. It will take 1-2 weeks of culture before the first passage can be conducted and a subsequent 1-2 weeks is necessary before the cells are ready for the second and third passages. After that, cells will grow faster and may be prepared for cryopreservation. Cells can be re-plated to one well (of a 24-well plate) and a 6-well plate in the first and second passage, respectively. The subsequent passage will be dependent on the cell growth and density. A cell split ratio of 1:2 or 1:3 can be used after the third passage.</li>
</ol>
5. Keratinocyte passage
<ol>
	<li>Collect 2 mL of the supernatant from the culture dish and dispense into a 15 mL conical tube (on ice). Wash the cells with sterile 1x PBS twice. 
	NOTE: When the cells reach approximately 70%-80% confluency, they are ready for passage.</li>
	<li>Add 1 mL of 0.05% trypsin-EDTA to the dish to the one well of a 6-well plate.</li>
	<li>Incubate for 5-15 min at 37 &#176;C; check after 5 min to see whether the cells detach from the dish.&#160;</li>
	<li>Neutralize the reaction using 1 mL of trypsin inhibition solution and 2 mL of complete culture medium + chelexed-FBS by gentle pipetting. Next, transfer the cell suspension into the same 15 mL conical tube as in step 5.1.</li>
	<li>Centrifuge the cell suspension at 100 x g for 5 min at 4 &#176;C. Aspirate the supernatant with a pipette and resuspend the cell pellet in 1 mL of complete culture medium.</li>
	<li>Count the cells using a hemocytometer. Next, plate 1 mL of the cell suspension into a new 24- or 6-well culture plate. 
	&#8203;NOTE: Some of the cells from the early passages can be frozen in a mixture of 70% complete culture medium + 20% chelexed-FBS + 10% DMSO. 1-2 cryovials of cells can be collected from one confluent culture plate.</li>
</ol>
6. Cryopreservation and recovery of keratinocytes
<ol>
	<li>Cell freezing
	<ol>
		<li>Grow keratinocytes to 80%-90% confluency. 
		NOTE: Do not allow cells to overgrow, as this could reduce their proliferation status and viability.</li>
		<li>Treat the keratinocytes in the dish with 0.05% trypsin-EDTA, as described in steps 5.1-5.5.</li>
		<li>Count the keratinocytes using a hemocytometer. Prepare cryovials based on calculated cell numbers to allow for the transfer of 1 x 106 cells/mL to each vial.</li>
		<li>Centrifuge the cell suspension at 100 x g for 5 min at 4 &#176;C. Discard the supernatant and resuspend the cell pellet in a 10 mL solution of 10% DMSO + 20% chelexed-FBS + 70% complete culture medium (9 mL of complete medium + chelexed-FBS and 1 mL of DMSO).</li>
		<li>Dispense the cells into cryovials at 1 mL of suspension per vial. Place the vials in a cryogenic storage container overnight at -80 &#176;C. Transfer the vials to a liquid nitrogen tank the following day.</li>
	</ol>
	</li>
	<li>Cell recovery
	<ol>
		<li>Remove a cryovial from the liquid nitrogen tank and partially thaw at room temperature. In a 15 mL tube, mix 1 mL of the cell suspension with 3 mL of complete culture medium + chelexed-FBS.</li>
		<li>Centrifuge the mixture for 5 min at 100 x g and 4 &#176;C. Discard the supernatant and resuspend the pellet in 1 mL of complete culture medium + chelexed-FBS.</li>
		<li>Plate the cell suspensions into new 60 mm Collagen I-coated culture dishes. Replace the culture medium every 2-3 days and passage the cells once confluent.</li>
	</ol>
	</li>
</ol>
7. Immunofluorescent staining
<ol>
	<li>Culture oral keratinocytes on square coverslips (22 mm x 22 mm) in 6-well plates (5 x 105 cells/well) for 2 days.</li>
	<li>Fix keratinocytes in a solution of 4% paraformaldehyde (PFA) and PBS for 20 min at room temperature before washing three times with 1x PBS.</li>
	<li>Permeabilize cells in a solution of 0.1% Triton in PBS. Incubate cells in blocking reagent (2.5% goat serum, 2.5% donkey serum) for 1 h at room temperature, followed by overnight incubation with primary antibodies (see Table of Materials) at 4 &#176;C. 
	NOTE: Primary antibodies were used at the following dilutions: rabbit anti-K14 (1:1000), rat &#945;6-integrin (1:100), rabbit anti-p63 (1:500), rabbit anti-K13 (1:100), and goat anti-PDGFR&#945; (1:100).</li>
	<li>Wash samples in a solution of 0.1% Triton in PBS, followed by incubation with secondary antibodies for 1 h at room temperature. 
	NOTE: The secondary antibodies (Alexa 488 or 555) were used at a 1:300 dilution.</li>
	<li>Counterstain all samples with Hoechst solution for 10 min and mount cells onto glass slides. 
	NOTE: Hoechst solution is used to stain the nuclei of cells.</li>
	<li>Perform sample imaging using a confocal microscope. 
	NOTE: The brightness and contrast are adjusted to equal intensity using image editing software.</li>
</ol>

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The authors declare no competing interests.

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 &#945;6-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.

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&#39; 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 (&#60; 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.

<table><tbody><tr><td>0.025% Trypsin/EDTA</td><td>Gibco</td><td>R001100</td><td></td></tr><tr><td>0.05% Trypsin/EDTA, phenol red</td><td>Gibco</td><td>25300062</td><td></td></tr><tr><td>0.4w/v% Trypan Blue solution</td><td>Wako</td><td>207-17081</td><td></td></tr><tr><td>10 mL Serological pipet</td><td>Falcon</td><td>357551</td><td></td></tr><tr><td>100 &amp;micro;m Nylon cell strainer</td><td>Falcon</td><td>352360</td><td></td></tr><tr><td>15 mL sterile conical tube</td><td>Falcon</td><td>352096</td><td></td></tr><tr><td>2 mL Aspirating pipet</td><td>Falcon</td><td>357558</td><td></td></tr><tr><td>35 mm Cell culture dish</td><td>Corning</td><td>353801</td><td></td></tr><tr><td>50 mL sterile conical tube</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>60 mm Cell culture dish</td><td>Corning</td><td>353802</td><td></td></tr><tr><td>6-well Tissue Culture plate</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>96 Well Culture plate (U bottom)</td><td>Falcon</td><td>353077</td><td></td></tr><tr><td>Antibiotic-Antimycotic 100x</td><td>Gibco</td><td>15240062</td><td></td></tr><tr><td>Biocoat Collagen I cellware 60mm dish</td><td>Corning</td><td>356401</td><td></td></tr><tr><td>Blunt Forceps</td><td>AS ONE</td><td>1-8187-03</td><td></td></tr><tr><td>Butorphanol</td><td>Meiji Seika Pharma</td><td></td><td>Vetorphale</td></tr><tr><td>CoolCell LX</td><td>Corning</td><td>432002</td><td>Cryogenic storage</td></tr><tr><td>Cotton</td><td>AS ONE</td><td>63-1452-97</td><td></td></tr><tr><td>Cover slips 22 x 22 &amp;mu;m square</td><td>Matsunami Glass Ind.</td><td>C022221</td><td></td></tr><tr><td>Cryogenic Vial 1.2 mL</td><td>Thermo Scientific</td><td>5000-0012</td><td></td></tr><tr><td>Defined Trypsin Inhibitor (DTI)</td><td>Gibco</td><td>R007100</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>276855-100ML</td><td></td></tr><tr><td>Donkey anti-Rabbit IgG (H+L), Alexa Fluor 555</td><td>Invitrogen</td><td>A31572</td><td></td></tr><tr><td>Donkey anti-Rat IgG (H+L), Alexa Fluor 488</td><td>Invitrogen</td><td>A21208</td><td></td></tr><tr><td>Donkey serum</td><td>Sigma-Aldrich</td><td>D9663-10ML</td><td></td></tr><tr><td>EpiLife Defined Growth Supplement (EDGS)</td><td>Gibco</td><td>S0120</td><td></td></tr><tr><td>EpiLife, with 60 &amp;micro;M calcium</td><td>Gibco</td><td>MEPI500CA</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>26140079</td><td></td></tr><tr><td>Fine forceps</td><td>BRC Bio Research Center</td><td>PRI13-3374</td><td></td></tr><tr><td>Goat anti-PDGF Receptor &amp;alpha;</td><td>R&amp;amp;D Systems</td><td>AF1062</td><td></td></tr><tr><td>Goat serum</td><td>Sigma-Aldrich</td><td>G9023-10ML</td><td></td></tr><tr><td>Half-curved forceps</td><td>BRC Bio Research Center</td><td>PRI13-3376</td><td></td></tr><tr><td>Hemocytometer</td><td>Hirschmann Laborger&amp;auml;te</td><td>8100204</td><td></td></tr><tr><td>Hoechst</td><td>Sigma-Aldrich</td><td>B2261</td><td></td></tr><tr><td>Iris Scissors</td><td>Muromachi Kikai</td><td>14090-09</td><td></td></tr><tr><td>Medetomidine</td><td>Nippon Zenyaku Kogyo</td><td></td><td>Domitor</td></tr><tr><td>Midazolam</td><td>Astellas Pharma</td><td></td><td>Dormicum</td></tr><tr><td>No.15 Disposable scalpel</td><td>Feather</td><td>219AABZX00136000</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Wako</td><td>162-16065</td><td></td></tr><tr><td>Phosphate Buffered Saline 1x, pH 7.4</td><td>Gibco</td><td>10010049</td><td></td></tr><tr><td>Povidone-iodine</td><td>Y&#039;s Square</td><td>872612</td><td></td></tr><tr><td>Rabbit anti-Cytokeratin 13 antibody</td><td>Abcam</td><td>ab92551</td><td></td></tr><tr><td>Rabbit anti-K14</td><td>BioLegend</td><td>905301</td><td></td></tr><tr><td>Rabbit anti-p63 antibody</td><td>Abcam</td><td>ab124762</td><td></td></tr><tr><td>Raspatorium #14</td><td>AS ONE</td><td>8-4599-01</td><td></td></tr><tr><td>Rat &amp;alpha;6-integrin</td><td>BD Biosciences</td><td>553745</td><td></td></tr><tr><td>Triton X-100</td><td>Wako</td><td>581-81705</td><td></td></tr><tr><td>Type I Collagen coated 24-well plate</td><td>Corning</td><td>354408</td><td></td></tr><tr><td>Type I Collagen coated 60mm dish</td><td>Corning</td><td>356401</td><td></td></tr><tr><td>Type I Collagen coated 6-well plate</td><td>Corning</td><td>355400</td><td></td></tr></tbody></table>

isolation and culture of primary oral keratinocytes from the adult mouse palate

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 &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

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 &#176;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 &#181;m 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 &#945;6-integrin34. K14 and &#945;6-integrin were expressed in keratinocytes after culturing (Figure 3A,B). The cells were also&#160;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 PDGFR&#945; was performed with the same set of keratinocytes compared with mouse embryonic fibroblast (MEFs). There was no expression of PDGFR&#945; 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62820/62820fig01.jpg" /> 
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 &#181;m. (D) Summary of the protocol. <a href="https://www.jove.com/files/ftp_upload/62820/62820fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62820/62820fig02.jpg" /> 
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 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62820/62820fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62820/62820fig03.jpg" /> 
Figure 3: Isolated mouse oral keratinocytes express basal cell markers. (A-B) Representative images of immunofluorescent staining of K14 (A; red) and &#945;6-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 PDGFR&#945; (red) in passage 4 (I), passage 7 (J), high calcium treatment (K), and MEFs (L). Nuclei are stained with Hoechst (blue). Scale bars: 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62820/62820fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate at JoVE.com

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

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 ...

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5.8K Views.  University of Tsukuba. 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.

Video: Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

Developmental Biology

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

We present a protocol for isolation and culture of primary mouse embryonic palatal mesenchymal cells for time-lapse imaging of two-dimensional (2D) growth and wound-repair assays. We also provide the methodology for analysis of the time-lapse imaging data to determine cell-stream formation and directional motility.

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and ...

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is an important structure of gingival tissue, especially in the repair and regeneration of periodontal tissue. Studying the functions of gingival epithelial cells has crucial scientific value, such as repairing oral defects and detecting the compatibility of biomaterials. As human gingival epithelial cells are highly differentiated keratinized cells, their lifespan is short, and they are difficult to passage. So far, there are only two ways to isolate and culture gingival epithelial cells, a direct explant method and an enzymatic method. However, the time required to obtain epithelial cells using the direct explant method is longer, and the cell survival rate of the enzymatic method is lower. Clinically, the acquisition of gingival tissue is limited, so a stable, efficient, and simple in vitro isolation and culture system is needed. We improved the traditional enzymatic method by adding Y-27632, a Rho-associated kinase (ROCK) inhibitor, which can selectively promote the growth of epithelial cells. Our modified enzymatic method simplifies the steps of the traditional enzymatic method and increases the efficiency of culturing epithelial cells, which has significant advantages over the direct explant method and the enzymatic method.

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is ...

Bioengineering

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately 30 x 106 viable cells per mouse. Primary adult mouse keratinocytes are harvested from the dorsal skin of female mice. Male mice (~6 weeks old) can be used for keratinocyte harvesting depending on the requirements of the experiment. Euthanized mice are shaved and sterilized with serial washes in povidone iodine and ethanol solutions (70% alcohol). After disinfecting the mice, the dorsal skin is removed and the subcutaneous fat and muscle are removed with a scalpel and discarded. The skins are cut into small pieces and treated with a mild, low temperature trypsinization to detach the lower dermis from the epidermis. The scraped epidermises are stirred at low speed, filtered to remove the hairs, counted, and re-suspended in culture medium. This method provides an excellent single cell suspension of highly culturable cells for many downstream applications.

The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes).

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately ...

Dissection of Palate Tissue from Adult Murine Model: A Surgical Procedure to Harvest Hard Palate and Soft Palate from Oral Cavity of Adult Mouse

Source: Xuan Ngo, Y. et al. <a href="https://www.jove.com/video/62820" target="_blank">Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate</a>.&#160;J. Vis. Exp.&#160;(2021)
This video demonstrates a surgical procedure to dissect the palate tissue from a murine model.

Source: Xuan Ngo, Y. et al. Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate.&#160;J. Vis. Exp.&#160;(2021)
This video ...

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Isolation of Circumvallate Papillae (CVP) Epithelium from Mouse Model: An Enzymatic Procedure to Separate CVP Epithelium from Mouse Tongue

Source: Shechtman, L. A. et al. <a href="https://www.jove.com/video/62300" target="_blank">Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells</a>. J. Vis. Exp. (2021)
In this video, we describe the isolation of circumvallate papillae (CVP) epithelium from a murine tongue.

Isolation of Circumvallate Papillae CVP Epithelium from Mouse Model: An Enzymatic Procedure to Separate CVP Epithelium from Mouse Tongue

Source: Shechtman, L. A. et al. Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells. J. Vis. Exp. (2021)
In this video, ...

Isolation and Culture of Murine Tongue Epithelial Cells: A Procedure to Isolate and Culture Tongue Epithelial Cells from Mouse Model

Source: Prasad, M. et al. <a href="https://www.jove.com/video/60410" target="_blank">In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line Using an Adeno-Associated Virus-Cas9 System.</a> J. Vis. Exp. (2020)
In this video, we describe the isolation of epithelial cells from a murine tongue. These cells can be further cultured to give rise to cell colonies.

Source: Prasad, M. et al. In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line Using an Adeno-Associated Virus-Cas9 ...