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.

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			<td>R001100</td>
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			<td>0.05% Trypsin/EDTA, phenol red</td>
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			<td>10 mL Serological pipet</td>
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			<td>100 &#181;m Nylon cell strainer</td>
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			<td>15 mL sterile conical tube</td>
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			<td>2 mL Aspirating pipet</td>
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			<td>35 mm Cell culture dish</td>
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			<td>353801</td>
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			<td>50 mL sterile conical tube</td>
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			<td>60 mm Cell culture dish</td>
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			<td>6-well Tissue Culture plate</td>
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			<td>96 Well Culture plate (U bottom)</td>
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			<td>Antibiotic-Antimycotic 100x</td>
			<td>Gibco</td>
			<td>15240062</td>
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			<td>Biocoat Collagen I cellware 60mm dish</td>
			<td>Corning</td>
			<td>356401</td>
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			<td>Blunt Forceps</td>
			<td>AS ONE</td>
			<td>1-8187-03</td>
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			<td>Butorphanol</td>
			<td>Meiji Seika Pharma</td>
			<td></td>
			<td>Vetorphale</td>
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			<td>CoolCell LX</td>
			<td>Corning</td>
			<td>432002</td>
			<td>Cryogenic storage</td>
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			<td>Cotton</td>
			<td>AS ONE</td>
			<td>63-1452-97</td>
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			<td>Cover slips 22 x 22 &#956;m square</td>
			<td>Matsunami Glass Ind.</td>
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			<td>Cryogenic Vial 1.2 mL</td>
			<td>Thermo Scientific</td>
			<td>5000-0012</td>
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			<td>Defined Trypsin Inhibitor (DTI)</td>
			<td>Gibco</td>
			<td>R007100</td>
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			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>276855-100ML</td>
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			<td>Donkey anti-Rabbit IgG (H+L), Alexa Fluor 555</td>
			<td>Invitrogen</td>
			<td>A31572</td>
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		<tr>
			<td>Donkey anti-Rat IgG (H+L), Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A21208</td>
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		<tr>
			<td>Donkey serum</td>
			<td>Sigma-Aldrich</td>
			<td>D9663-10ML</td>
			<td></td>
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			<td>EpiLife Defined Growth Supplement (EDGS)</td>
			<td>Gibco</td>
			<td>S0120</td>
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			<td>EpiLife, with 60 &#181;M calcium</td>
			<td>Gibco</td>
			<td>MEPI500CA</td>
			<td></td>
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			<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 &#945;</td>
			<td>R&#38;D Systems</td>
			<td>AF1062</td>
			<td></td>
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		<tr>
			<td>Goat serum</td>
			<td>Sigma-Aldrich</td>
			<td>G9023-10ML</td>
			<td></td>
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		<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&#228;te</td>
			<td>8100204</td>
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		<tr>
			<td>Hoechst</td>
			<td>Sigma-Aldrich</td>
			<td>B2261</td>
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			<td>Iris Scissors</td>
			<td>Muromachi Kikai</td>
			<td>14090-09</td>
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			<td>Medetomidine</td>
			<td>Nippon Zenyaku Kogyo</td>
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			<td>Domitor</td>
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			<td>Midazolam</td>
			<td>Astellas Pharma</td>
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			<td>Dormicum</td>
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			<td>No.15 Disposable scalpel</td>
			<td>Feather</td>
			<td>219AABZX00136000</td>
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			<td>Paraformaldehyde</td>
			<td>Wako</td>
			<td>162-16065</td>
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			<td>Phosphate Buffered Saline 1x, pH 7.4</td>
			<td>Gibco</td>
			<td>10010049</td>
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		<tr>
			<td>Povidone-iodine</td>
			<td>Y&#39;s Square</td>
			<td>872612</td>
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		<tr>
			<td>Rabbit anti-Cytokeratin 13 antibody</td>
			<td>Abcam</td>
			<td>ab92551</td>
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		<tr>
			<td>Rabbit anti-K14</td>
			<td>BioLegend</td>
			<td>905301</td>
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		</tr>
		<tr>
			<td>Rabbit anti-p63 antibody</td>
			<td>Abcam</td>
			<td>ab124762</td>
			<td></td>
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		<tr>
			<td>Raspatorium #14</td>
			<td>AS ONE</td>
			<td>8-4599-01</td>
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		<tr>
			<td>Rat &#945;6-integrin</td>
			<td>BD Biosciences</td>
			<td>553745</td>
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			<td>Triton X-100</td>
			<td>Wako</td>
			<td>581-81705</td>
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		<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>
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		<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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5.6K 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

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

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 Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

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 ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...