This research was supported by grants from the Israel Science Foundation (No. 1418/11) to AHH and (No. 1933/12) to AW, the German Israeli Foundation for young investigators (GIF Young) to AHH, and the Dr. I. Cabakoff Research Endowment Fund at the Hebrew University-Hadassah School of Dental Medicine to AHH and AW.

Prepare in advance:
<ul>
	<li>PBS + 2% FCS</li>
	<li>PBS + 2% FCS with 2 mg/ml of Collagenase Type II and 1 mg/ml of DNAse Type I (1 ml per sample)</li>
	<li>Sterilized surgical instruments</li>
	<li>0.5 M EDTA solution</li>
</ul>
1. Upper Gingival Excision Technique
<ol>
	<li>Euthanize mice using approved IACUC guidance.</li>
	<li>Cut both sides of the oral cavity including the cheeks and the mandible ramus with a sharp/blunt straight scissors.</li>
	<li>Pull down the mandible.</li>
	<li>Cut the soft and hard tissues in an imaginary line 1 mm behind the thirds molars with standard scissors. Direct the scissors perpendicular to the plane of the palatal bone (Figure 2A, blue lines).</li>
	<li>Incise the soft and hard tissues 2 mm behind the incisors (Figure 2A, blue lines).</li>
	<li>Pull down the anterior part of the mouth. The nasal cavity is observable after this step.</li>
	<li>Place the scissors parallel to the palate, put one blade into the nasal cavity, the other blade should incise at the vestibule. Cut until the posterior incision (Figure 2A, green lines). Repeat this step on both sides of the maxilla. At the end the maxilla is detached from the rest of the skull (Figure 2B).</li>
	<li>Remove the maxilla, cut it in the middle suture (Figure 2, black dashed line) and trim the palatal tissue of each hemi-maxilla until reaching the alveolar bone.</li>
	<li>Peel the gingival tissues (both hemi-maxillae) from their anterior border with Adson forceps (without teeth).</li>
	<li>Place the excised gingiva in a plate with PBS + 2% FCS.</li>
</ol>
2. Gingival Processing
<ol>
	<li>Put the excised gingiva in a tissue culture dish (35 x 10 mm) with 1 ml PBS + 2% FCS, 2 mg/ml of Collagenase Type II and 1 mg/ml of DNAse Type I.</li>
	<li>Mince well the tissue with an N&deg;15 sterile surgical blade.</li>
	<li>Transfer the tissue from the plate to a 15 ml conical test tube.</li>
	<li>Wash the tissue/cells remaining on the plate with 1 ml PBS + 2% FCS and transfer to the same test tube.</li>
	<li>Incubate in a shaker incubator for 20 min at 37 &deg;C, 200 rpm (recommendation: preheat the incubator).</li>
	<li>Add 20 &mu;l of EDTA 0.5 M and incubate for another 10 min at 37 &deg;C, 200 rpm.</li>
	<li>Fill the test tube up to 12 ml with PBS + 2% FCS and centrifuge at 4 &deg;C, 400 x g for 8 min (recommendation: pre-chill the centrifuge).</li>
	<li>Remove supernatant and resuspend cells with 2 ml PBS + 2% FCS.</li>
	<li>Filter the sample with a 70 &mu;m cell-strainer and save flow through.</li>
	<li>Centrifuge at 4 &deg;C, 320 x g for 5 min.</li>
	<li>Remove supernatant and resuspend cells with 300 &mu;l PBS + 2% FCS.</li>
	<li>Count the cells (approximately 5-10 x 105 cells are expected from a single maxilla).</li>
</ol>
3. Extracellular and Intracellular Antibody Staining for Flow Cytometry Analysis
It is recommended to work inside a hood, with lights off. It is critical to keep the cells in constant cold environment, as well as all the materials needed.
<ol>
	<li>Stain cells against extracellular molecules by adding 0.2-0.5 &mu;g of each selected antibody per 1 x 106 cells in a total volume of 100 &mu;l.</li>
	<li>Vortex briefly.</li>
	<li>Incubate the tubes for 15 min in the dark at 4 &deg;C.</li>
	<li>Wash cells with 2 ml PBS + 2% FCS.</li>
	<li>Centrifuge at 4 &deg;C, 320 x g for 5 min.</li>
	<li>Aspirate supernatant and resuspend cells by adding, drop wise, 500 &mu;l cold BD Cytoperm/Cytofix while vortexing.</li>
	<li>Incubate the tubes for 40 min at 4 &deg;C in the dark.</li>
	<li>Vortex briefly.</li>
	<li>Wash the cells twice with 1 ml cold BD Perm/Wash Buffer and spin at 800 x g for 7 min at 4 &deg;C.</li>
	<li>Aspirate supernatant except for 300 &mu;l.</li>
	<li>Stain cells intracellularly by adding 1 &mu;g of each selected antibody per 1 x 106 cells in a total volume of 100 &mu;l.</li>
	<li>Vortex briefly.</li>
	<li>Incubate for 30 min in the dark at 4 &deg;C.</li>
	<li>Wash the cells twice with 1 ml cold BD Perm/Wash Buffer and spin at 800 x g for 7 min at 4 &deg;C.</li>
	<li>Aspirate supernatant and resuspend cells by adding 300 &mu;l cold BD Perm/Wash Buffer.</li>
	<li>Vortex briefly.</li>
	<li>Transfer the suspension while filtering to FACS tubes.</li>
	<li>Keep the tubes in a dark and cold environment until analysis.</li>
</ol>

<ol><li>Arizon, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Langerhans+cells+down-regulate+inflammation-driven+alveolar+bone+loss%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Langerhans cells down-regulate inflammation-driven alveolar bone loss.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 7043-7048 (2012).</li>
<li>Listgarten, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathogenesis+of+periodontitis%5BTitle%5D)+AND+%22J.+Clin.+Periodontol%22%5BJournal%5D)">Pathogenesis of periodontitis.</a> J. Clin. Periodontol. 13, 418-430 (1986).</li>
<li>Kinane, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Causation+and+pathogenesis+of+periodontal+disease%5BTitle%5D)+AND+%22Periodontol%22%5BJournal%5D)">Causation and pathogenesis of periodontal disease.</a> Periodontol. 25, 8-20 (2000).</li>
<li>Jotwani, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mature+dendritic+cells+infiltrate+the+T+cell-rich+region+of+oral+mucosa+in+chronic+periodontitis%3A+in+situ%2C+in+vivo%2C+and+in+vitro+studies%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">Mature dendritic cells infiltrate the T cell-rich region of oral mucosa in chronic periodontitis: in situ, in vivo, and in vitro studies.</a> J. Immunol. 167, 4693-4700 (2001).</li>
<li>Graves, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interleukin-1+and+tumor+necrosis+factor+antagonists+inhibit+the+progression+of+inflammatory+cell+infiltration+toward+alveolar+bone+in+experimental+periodontitis%5BTitle%5D)+AND+%22J.+Periodontol%22%5BJournal%5D)">Interleukin-1 and tumor necrosis factor antagonists inhibit the progression of inflammatory cell infiltration toward alveolar bone in experimental periodontitis.</a> J. Periodontol. 69, 1419-1425 (1998).</li>
<li>Kabashima, H., Yoneda, M., Nagata, K., Hirofuji, T., Maeda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+presence+of+chemokine+%28MCP-1%2C+MIP-1alpha%2C+MIP-1beta%2C+IP-10%2C+RANTES%29-positive+cells+and+chemokine+receptor+%28CCR5%2C+CXCR3%29-positive+cells+in+inflamed+human+gingival+tissues%5BTitle%5D)+AND+%22Cytokine%22%5BJournal%5D)">The presence of chemokine (MCP-1, MIP-1alpha, MIP-1beta, IP-10, RANTES)-positive cells and chemokine receptor (CCR5, CXCR3)-positive cells in inflamed human gingival tissues.</a> Cytokine. 20, 70-77 (2002).</li>
<li>Moughal, N. A., Adonogianaki, E., Kinane, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Langerhans+cell+dynamics+in+human+gingiva+during+experimentally+induced+inflammation%5BTitle%5D)+AND+%22J.+Biol.+Buccale%22%5BJournal%5D)">Langerhans cell dynamics in human gingiva during experimentally induced inflammation.</a> J. Biol. Buccale. 20, 163-167 (1992).</li>
<li>Cochran, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inflammation+and+bone+loss+in+periodontal+disease%5BTitle%5D)+AND+%22J.+Periodontol%22%5BJournal%5D)">Inflammation and bone loss in periodontal disease.</a> J. Periodontol. 79, 1569-1576 (2008).</li>
<li>Taubman, M. A., Valverde, P., Han, X., Kawai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immune+response%3A+the+key+to+bone+resorption+in+periodontal+disease%5BTitle%5D)+AND+%22J.+Periodontol%22%5BJournal%5D)">Immune response: the key to bone resorption in periodontal disease.</a> J. Periodontol. 76, 2033-2041 (2005).</li>
<li>Graves, D. T., Kang, J., Andriankaja, O., Wada, K., Rossa, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+to+study+host-bacteria+interactions+involved+in+periodontitis%5BTitle%5D)+AND+%22Front+Oral+Biol%22%5BJournal%5D)">Animal models to study host-bacteria interactions involved in periodontitis.</a> Front Oral Biol. 15, 117-132 (2012).</li>
</ol>

Authors have nothing to disclose.

Maxilla gingival tissues obtained from a single mouse are sufficient for analyzing sub-populations of T and B lymphocytes, as well as their capability to express extracellular and intracellular molecules as we previously described 1. Nevertheless, if rare cell populations are of interest (for example DCs), it is recommended to pool tissues from 2-3 mice. Of note, if preferable, it is possible to peel both the palatal and gingival tissues and then to excise the gingiva (a modification of step 1.8-1.9). It should be also mentioned that mandibular gingiva can be collected as well; still, its isolation is more complicated and may include other tissues that might interfere with the analysis.
Common techniques to study immune cells in the gingiva are immunohistochemistry and immunofluorescence. These techniques enable visualization of the distribution of a target molecule/cell through the gingiva, and screening of numerous fields is required in order to study a certain area. Whereas the physiological localization of certain cells cannot be determined by flow cytometry due to gingival processing, this approach provides several other advantages: (1) flow cytometry is a rapid and simple method to analyze large numbers of cells (2) it reduces analysis errors originate from tissue heterogeneity (3) it enables analysis of rare cell populations (4) it allows simultaneous analysis of various variables (5) it allows discrimination between live and dead cells (6) it permits functional analysis of the resulting cells.
The ability to perform flow cytometry analysis on gingival tissues during experimental periodontal disease enhances our ability to reveal the immunological mechanisms involved in this process. Longitudinal studying of cellular changes that occurs in the gingiva following exposure to oral pathogens should provide more relevant information rather than measuring immune responses systemically. This aspect is of particular importance when analyzing the phenotype and kinetics of innate and adaptive inflammatory cells in inflamed gingiva.

Gingiva is the soft tissue surrounding the cervical portion of the teeth and covers the alveolar process (Figure 1). The gingiva is a type of masticatory mucosa that can be further separated into mucosal epithelium and connective tissue (also known as submucosa or lamina propria). The anatomical structure of the gingiva and adjacent teeth allows bacteria to reside and develop plaque (biofilm) that constantly challenges the local immune system. As a result, inflammatory response develops in the gingiva, which in certain circumstances becomes destructive - a condition termed periodontal diseases 2. Basically, plaque-induced periodontal pathologies can be divided into gingivitis and periodontitis. Gingivitis represents a reversible condition of local inflammatory response that is confined to the gingiva. Periodontitis, on the other hand, is an irreversible destructive process in which the attachment apparatus (alveolar bone, periodontal ligament, cementum and gingiva) is destroyed 3.
The gingiva was proposed to serve both as effector and inductive sites during periodontal diseases 4. Human studies have suggested that in response to dental plaque, immune effector cells and molecules dynamically infiltrated or departure the gingiva 5-7. This activity was shown to play a major role in periodontal destruction 8,9. Whereas data generated by those studies provided valuable information regarding this pathological process, working with human tissues possess major ethical, technical and experimental limitations. The development of experimental models allowed cause-effect experimentations via employing transgenic mice and in vivo interventions 10. As a result, our knowledge on the mechanisms involved in periodontal disease increased considerably during the last two decades. Even so, due to the complexity of periodontal diseases, there is an ongoing debate regarding the nature of local immune response facilitating tissue destruction. There is also a lack in our understanding on the function of central immune cells in the gingiva during periodontal diseases. It is thus essential to study pathological inflammatory events occurring in the target tissue of the disease, the gingiva.

<table><tbody><tr><td>Collagenase Type II</td><td>Worthington Biochemical Corp.</td><td>CLS-2</td><td></td></tr><tr><td>DNAse I</td><td>SIGMA</td><td>DN25-1G</td><td></td></tr><tr><td>EDTA</td><td>SIGMA</td><td>E6758-100G</td><td></td></tr><tr><td>Dulbecco&#039;s PBS</td><td>SIGMA</td><td>D8537</td><td></td></tr><tr><td>Fetal Bovine Serum </td><td>Biological Industries</td><td>04-121-1</td><td>Heat Inactivated</td></tr><tr><td>Vacuum-Driven Filter</td><td>Jet Biofil</td><td>FPE-204-500</td><td></td></tr><tr><td>5 ml Polystyrene Round-Bottom Tube</td><td>BD Falcon</td><td>352052</td><td></td></tr><tr><td>Cell Strainer 70 &amp;mu;m</td><td>SPL Lifesciences</td><td>93070</td><td></td></tr><tr><td>Tissue Culture Dish 35&amp;#215;10 mm</td><td>NUNC</td><td>153066</td><td></td></tr><tr><td>BD Cytofix/Cytoperm</td><td>BD</td><td>554714</td><td></td></tr><tr><td>Anti-mouse CD11c antibody</td><td>Biolegend</td><td>117305</td><td>Clone N418</td></tr><tr><td>Anti-mouse CD45.2 antibody</td><td>Biolegend</td><td>109819</td><td>Clone 104</td></tr><tr><td>Anti-mouse CD4 antibody</td><td>Biolegend</td><td>100413</td><td>Clone GK1.5</td></tr><tr><td>Anti-mouse CD8a antibody</td><td>Biolegend</td><td>100733</td><td>Clone 53-6.7</td></tr><tr><td>Anti-mouse CD3 antibody</td><td>Biolegend</td><td>100214</td><td>Clone 17A2</td></tr><tr><td>Anti-mouse CD326 (Ep-CAM) antibody</td><td>Biolegend</td><td>118219</td><td>Clone G8.8</td></tr><tr><td>Anti-mouse CD207 (Langerin) antibody</td><td>Imgenex</td><td>DDX0362D</td><td>Clone 929F3.01</td></tr><tr><td>Anti-mouse I-A&lt;sup&gt;d&lt;/sup&gt; (MHC-II) antobody</td><td>Biolegend</td><td>115010</td><td>Clone 39-10-8</td></tr><tr><td>LSR II Flow Cytometer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>FlowJo Software v 7.6.5</td><td>Tree Star</td><td></td><td></td></tr></tbody></table>

isolation, processing and analysis of murine gingival cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

Examples of flow cytometry analysis on gingival cells are presented. Gingival cells pooled from 2 mice were run in an LSR II flow cytometer and analyzed using FlowJo software. Figure 3A demonstrated the distribution of gingival cells from naïve mice in a side scatter (SSC) versus forward scatter (FSC) plot. Gating strategy to identify (i) lymphocytes (ii) monocytes / dendritic cells and (iii) granulocytes is indicated. For comparison purpose, we also present a FACS plot illustrating gingival cells purified from P. gingivalis infected mice 21 days after oral gavage in a setting of experimental periodontitis as we previously described (Figure 3B). An increase in the various immune cell populations can be easily detected in the infected mice as compared to naïve mice. Next, we identify immune cells in the processed gingiva by gating on cells expressing the hematopoietic marker CD45 (Figure 4A). These cells further segregated into CD3-positive cells representing T cells (Figure 4B). We also identified gingival neutrophils according to expression of the Ly6G and CD11b molecules (Figure 4C). Finally, langerhans cells, an epithelial subset of dendritic cells (DCs), were identified by gating on MHC class II+CD11c+ cells (a marker of murine DCs) and expression of Ep-CAM and langerin/CD207 (intracellular staining) (Figure 4D).
<img alt="Dental anatomy diagram showing tooth structure: crown, root, alveolar bone, and gingiva layers." fo:content-width="4.5in" fo:src="/files/ftp_upload/50388/50388fig1highres.jpg" src="/files/ftp_upload/50388/50388fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Schematic diagram showing the anatomical landmarks of the gingiva.
<img alt="Rodent oral anatomy diagram and photo; excised gingiva tissue sample for histological analysis." fo:content-width="6in" fo:src="/files/ftp_upload/50388/50388fig2highres.jpg" src="/files/ftp_upload/50388/50388fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Schematic view of mouse oral cavity resulting after completing step 1.3. Soft tissues from the back to the front: masseter muscle (MM), soft palate (SP), hard palate (HP), gingiva (G). Hard tissue: molars and incisors. The main goal of this technique is to isolate the gingival tissue that surrounds the three molars (inside the black oval).
<img alt="Naïve vs. inflamed gingiva flow cytometry diagrams; FSC vs. SSC scatter plots for cellular analysis." fo:content-width="5in" fo:src="/files/ftp_upload/50388/50388fig3highres.jpg" src="/files/ftp_upload/50388/50388fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Representative FACS plots demonstrating cellular distribution of gingival leukocytes. Processed gingival samples were run on the LSR II flow cytometer and analyzed using FlowJo software. Side scatter (SSC) versus forward scatter (FSC) plots of the gingival cells from (A) naïve mice or (B) inflamed gingiva are presented. The location of (i) lymphocytes (ii) monocytes/dendritic cells and (iii) granulocytes populations is indicated.
<img alt="Flow cytometry diagram: cell population analysis of T cells, neutrophils, dendritic cells using SSC/CD markers." fo:content-width="6in" fo:src="/files/ftp_upload/50388/50388fig4highres.jpg" src="/files/ftp_upload/50388/50388fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4. Representative FACS plots demonstrating analysis of immune gingival cells. (A) Immune cells were identified according to CD45 expression. Following gating on CD45-positive cells and lymphocytes population, expression of CD3 enabled targeting T-cell subset. (B) Neutrophils were identified in the gingiva according to ability of CD45+ cells to express also Ly6G and CD11b. (C) Dendritic cells were identified by gating on CD45+ cells from the monocyte/dendritic population [Figure 3, gate (ii)], and then on MHC class II and CD11c positive cells. (D) Langerhans cells were further segregated from other dendritic cells based on expression of Ep-CAM and the intracellular langerin molecule. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50388/50388fig4large.jpg" target="_blank"> Click here to view larger figure</a>.

Watch this Scientific Journal Video about Isolation, Processing and Analysis of Murine Gingival Cells at JoVE.com

Isolation, Processing and Analysis of Murine Gingival Cells

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

isolation-processing-and-analysis-of-murine-gingival-cells

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

18.9K Views.  Hebrew University - Hadassah Medical Center. Deze studie beschrijft een efficiënte techniek voor het isoleren en verwerken tandvleesweefsels van de muis mondholte om een-celkweek produceren. De resulterende cellen kunnen verder worden gebruikt voor flowcytometrie-analyse en moleculaire studies.

Video: Isolation, Processing and Analysis of Murine Gingival Cells

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Rapid Isolation of Single Cells from Mouse and Human Teeth

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental pulp tissue, rich in the extracellular matrix, requires a long and tedious dissociation process that is typically beyond the reasonable time for single-cell transcriptomics. For avoiding artificial changes in gene expression, the time elapsed from euthanizing an animal until the analysis of single cells needs to be minimized. This work presents a fast protocol enabling to obtain single-cell suspension from mouse and human teeth in an excellent quality suitable for scRNA-seq (single-cell RNA-sequencing). This protocol is based on accelerated tissue isolation steps, enzymatic digestion, and subsequent preparation of final single-cell suspension. This enables fast and gentle processing of tissues and allows using more animal or human samples for obtaining cell suspensions with high viability and minimal transcriptional changes. It is anticipated that this protocol might guide researchers interested in performing the scRNA-seq not only on the mouse or human teeth but also on other extracellular matrix-rich tissues, including cartilage, dense connective tissue, and dermis.

The current protocol presents a fast, efficient, and gentle method for isolating single cells suitable for single-cell RNA-seq analysis from a continuously growing mouse incisor, mouse molar, and human teeth.

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental ...

Developmental Biology

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Biology

Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically modified strains, the vast number of analyses that can be performed on harvested soft and hard tissues. However, many of these systems are subject to procedural criticisms. As an alternative, the ligature-induced model of periodontal disease, driven by the localized development and retention of a dysbiotic oral microbiome, can be employed, which is rapidly induced and relatively reliable. Unfortunately, the variants of ligature-induced murine periodontitis protocol are isolated to focal regions of the periodontium and subject to premature avulsion of the installed ligature. This minimizes the amount of tissue available for subsequent analyses and increases the number of animals required for study. This protocol describes the precise manipulations required to place extended molar ligatures with improved retention and usage of a novel rinse technique to recover oral neutrophils in mice with an alternative approach that mitigates the aforementioned technical challenges.

This article presents a protocol for establishing a ligature-induced model of murine periodontitis involving multiple maxillary molars, resulting in larger areas of the involved gingival tissue and bone for subsequent analysis as well as reduced animal usage. A technique to assess oral neutrophils in a manner analogous to human subjects is also described.

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically ...

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

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

Neuroscience

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

Isolation of Splenic Microvesicles in a Murine Model of Intraperitoneal Bacterial Infection

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular effectors. In rats, spleen MVs (SMVs) are surrogate markers of pathophysiological conditions. Previous in vitro studies demonstrated that Porphyromonas gingivalis (P. gingivalis), a major periodontal pathogen, enables&#160;the endothelial shedding and apoptosis while lipopolysaccharide (LPS) favors the shedding of splenocyte-derived microvesicles (SMVs). In vivo studies showed the feasibility of pharmacological control of SMV shedding. The present protocol establishes a standardized procedure for isolating splenic SMVs from the P. gingivalis acute murine infection model.&#160;P. gingivalis infection was induced in young C57BL/6 mice by intraperitoneal injection (three injections of 5 x 107 bacteria/week). After two weeks, the spleens were collected, weighed, and the splenocytes were counted. SMVs were isolated and quantified by protein, RNA, and prothrombinase assays. Cell viability was assessed by either propidium iodide or trypan blue exclusion dyes. Following splenocyte extraction, neutrophil counts were obtained by flow cytometry after 24 h of splenocyte culture. In P. gingivalis-injected mice, a 2.5-fold increase in spleen weight and a 2.3-fold rise in the splenocyte count were observed, while the neutrophils count was enhanced by 40-folds. The cell viability of splenocytes from P. gingivalis-injected mice ranged from 75%-96% and was decreased by 50% after 24 h of culture without any significant difference compared to unexposed controls. However, splenocytes from injected mice shed higher amounts of MVs by prothrombinase assay or protein measurements. The data demonstrate that the procoagulant SMVs are reliable tools to assess an early spleen response to intraperitoneal&#160;P. gingivalis infection.

Microvesicles shed from the plasma membrane act as cellular effectors. Spleen microvesicles (SMVs) are surrogate markers of pathophysiological conditions. In rats and mice, SMV content and properties characterize aging or inflammation and are altered by cytoprotective treatments, making them a valuable yet abundant monitoring tool for preclinical research.

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular ...

A Bacterial Infection Assay for Studying Anaerobic Bacteria-Host Cell Interactions

Source: Wunsch, C. M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53408">Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells.</a> J. Vis. Exp. (2015)
This video demonstrates a technique for assessing the ability of Porphyromonas gingivalis, an anaerobic pathogenic bacterium, to adhere to and invade human umbilical vein endothelial cells (HUVECs). After infecting the host cells with the bacteria, the host cells are lysed to release both adhered and internalized bacteria. When the lysate is plated in an appropriate medium, the presence of colonies indicates successful bacteria-host cell interactions.

The following protocols will describe methods for culturing and studying the invasion by the anaerobic species, P. gingivalis; however, these protocols ...

Isolation, Processing and Analysis of Murine Gingival Cells

Samenvatting

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