Authors were funded in part by the intramural program of NIDCR (N.M.M) and supported by a Wellcome Trust Stepping Stones Fellowship (097820/Z/11/B to J.E.K) and by a Manchester Collaborative Centre for Inflammation Research grant (to J.E.K). The authors thank Teresa Wild for critically reviewing the manuscript.

Todos los procedimientos experimentales descritos en este protocolo siguen las pautas requeridas y fueron aprobados por el Cuidado de Animales institucional y el empleo, NIDCR / NIH. 1. Prepárese por adelantado <ol><li> Preparar medio completo: RPMI suplementado con 2 mM L-glutamina, 100 unidades / ml de penicilina, 100 ug / ml de estreptomicina y 10% de FBS. </li><li> Preparar medios DNasa: 50 ml de medio RPMI suplementado con 7,5 mg de DNasa (Haga fresco, mantener en hielo en todo momento). </li><li> Preparar los medios de colagenasa-DNasa: 5 ml de medio DNasa más 3,2 mg / ml de colagenasa tipo IV (Haga fresco, mantener en hielo en todo momento). </li><li> Pre-cool tampón FACS: 0,5% de SFB diluido en PBS. </li><li> centrifugadora pre-enfriamiento a 4 ° C. </li><li> agitador incubadora caliente a 37 ° C. </li></ol> 2. El aislamiento, la disección y aislamiento de células gingivales de los bloques <ol><li> La eutanasia a los ratones mediante la exposición a CO 2 en una cámara apropiada, según las directrices de ARAC. </li> &lt;li&gt; Abra la cavidad torácica y abdominal para exponer el corazón y los órganos internos. Para perfundir, hacer una incisión en la vena cava inferior y perfundir 3 ml de PBS a través del ventrículo izquierdo con una jeringa de 3 ml con una aguja de 27 G. </li><li> Inmovilizar el cuerpo y la cabeza del ratón sobre un cojín con el estómago hacia arriba. </li><li> Cortar cada comisura del labio hacia el cuello con unas tijeras, separando la piel que cubre la región de la mandíbula y el cuello, la exposición de los tejidos y los músculos subyacentes. Diseccionar el labio inferior para descubrir la mandíbula. </li><li> Corte entre los incisivos inferiores para separar ambos lados de la mandíbula e inmovilizar cada lado para acceder a la cavidad oral. </li><li> Visualizar y analizar el paladar lejos de la cavidad nasal. </li><li> Diseccionar áreas utilizando una cuchilla 10 e incluyen áreas molares maxilares y mandibulares y la zona gingival (Figura 1). Hacen incisiones verticales, justo por delante del primer molar y posterior al tercer molar y una incisión horizontal enel borde de la encía (blanco cuello de los dientes adyacentes de tejido). </li><li> Coloque el maxilar y mandibular bloques en un tubo cónico de 50 ml con 5 ml de colagenasa / DNasa (mantener en hielo). </li><li> Incubar en un incubador agitador a 37 ° C durante 1 hora y durante el último 5 min de incubación se añaden 50 l de 0,5 M EDTA. </li><li> Añadir 5 ml de medio de DNasa frío al tubo de 50 ml con los tejidos y agitar suavemente para mezclar. </li><li> La transferencia de los 4 bloques de tejido en una placa de Petri y cubrir con 500 l de DNasa medios. Eliminar encía de cada bloque de tejido, utilizando una hoja de bisturí y apuntando la hoja en las áreas gingivales y interdentales. </li><li> La transferencia de los tejidos y los medios de comunicación de la placa de Petri, así como los medios de comunicación de DNasa anteriores utilizados durante la disección, a un nuevo tubo de 50 ml a través del filtro de células (70 micras de tamaño). Lavar con medios DNasa (3-5 ml) todos los instrumentos utilizados y filtro. </li><li> Usando el émbolo de una jeringa estéril de 3 ml, triturar el tejido contra el colador,filtración con medios DNasa frío (30 a 35 ml). </li><li> Lavar el filtro de células con DNasa medios frío. </li><li> Centrifugar a 4 ° C, 314 xg durante 6 min. </li><li> Descartar el sobrenadante, resuspender en 1 ml de medio completo. </li><li> Recuento de células (la cantidad esperada debe oscilar entre 8 x 10 5 a 1,2 x 10 6 células con una viabilidad de&gt; 80%) usando un contador de células automatizado. </li></ol> 3. Estimulación y FACS La tinción de las células de la encía <ol><li> Estimular la suspensión de células individuales con PMA (50 ng / ml) e ionomicina (2,5 g / ml) en presencia de 1 l / ml de Brefeldina A. </li><li> Incubar durante 3,5 horas a 37 ° C y 5% de CO2. </li><li> Centrifugar y lavar con PBS y centrifugar a 4 ° C; 314 xg durante 6 minutos. </li><li> Tinción de las células utilizando una mancha viabilidad siguiendo las instrucciones del fabricante. </li><li> Mancha de marcadores extracelulares (CD45, TCRγδ, TCR, CD4, CD8, TCRγδ o de hormigasibodies de elección) siguiendo las instrucciones del fabricante. </li><li> Se lavan las células con tampón frío FACS y centrifugar a 4 ° C, 314 xg durante 5 min. </li><li> vórtice suavemente para perturbar el sedimento celular. </li><li> Fijar las células con 2% de paraformaldehído durante 20 min a temperatura ambiente. </li><li> Se lavan las células con tampón frío FACS, se centrifuga a 4 ° C, 314 xg durante 5 min y las manchas para citoquinas intracelulares. <ol><li> Llenar los tubos FACS con una solución de frío saponina 0,5% diluido en tampón de FACS y se centrifuga a 4 ° C, 314 xg durante 5 min. </li><li> Añadir 300 l de solución de saponina 0,5% a los tubos de FACS, se incuba durante 15 min en la oscuridad a 4 ° C y centrifugar a 4 ° C, 314 xg durante 5 min. </li><li> Teñir las células para marcadores intracelulares (IL-17A y IFN o citocinas de elección) siguiendo las instrucciones del fabricante. Lavar las células con solución de saponina 0,5% y centrifugar a 4 ° C, 314 xg durante 5 min. </li><li> Lavar a continuación con FACStampón y se centrifuga a 4 ° C, 314 xg durante 5 min. </li><li> Volver a suspender las células en 300 l de tampón FACS. </li></ol></li><li> Analizar los resultados en un citómetro de flujo. </li></ol> 4. Análisis de Citometría de Flujo <ol><li> visualizar las células a analizar en adelante (FSC) y dispersión lateral (SCC) y la puerta para excluir los desechos. </li><li> Seleccionar celdas individuales con área de dispersión frontal (FSC-A) vs. parámetros de altura (FSC-H). </li><li> Células de puertas que son en vivo (como se indica por la tinción de viabilidad) y positivos para el marcador hematopoyéticas CD45 + para el análisis adicional (Figura 2A y 2C). </li><li> Continuar el análisis de marcadores específicos como se muestra en la Figura 2 y la Figura 3. </li></ol>

<ol>
	<li>Aas, J. A., Paster, B. J., Stokes, L. N., Olsen, I., Dewhirst, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+The+Normal+Bacterial+Flora+Of+The+Oral+Cavity.">Defining The Normal Bacterial Flora Of The Oral Cavity.</a> J Clin Microbiol. 43, 5721-5732 (2005).</li><li>Belkaid, Y., Naik, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalized+And+Systemic+Control+Of+Tissue+Immunity+By+Commensals.">Compartmentalized And Systemic Control Of Tissue Immunity By Commensals.</a> Nat Immunol. 14, 646-653 (2013).</li><li>Darveau, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodontitis:+A+Polymicrobial+Disruption+Of+Host+Homeostasis.">Periodontitis: A Polymicrobial Disruption Of Host Homeostasis.</a> Nat Rev Microbiol. 8, 481-490 (2010).</li><li>Hajishengallis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomicrobial+Pathogenesis+Of+Periodontitis:+Keystones,+Pathobionts,+And+Host+Response.">Immunomicrobial Pathogenesis Of Periodontitis: Keystones, Pathobionts, And Host Response.</a> Trends Immunol. 35, 3-11 (2014).</li><li>Eke, P. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+Of+Periodontitis+In+Adults+In+The+United+States:+2009+And+2010.">Prevalence Of Periodontitis In Adults In The United States: 2009 And 2010.</a> J Dent Res. 91, 914-920 (2012).</li><li>Moutsopoulos, N. M., Lionakis, M. S., Hajishengallis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inborn+Errors+In+Immunity:+Unique+Natural+Models+To+Dissect+Oral+Immunity.">Inborn Errors In Immunity: Unique Natural Models To Dissect Oral Immunity.</a> J Dent Res. , (2015).</li><li>Hajishengallis, G., Lamont, R. J., Graves, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Enduring+Importance+Of+Animal+Modelsin+Understanding+Periodontal+Disease.">The Enduring Importance Of Animal Modelsin Understanding Periodontal Disease.</a> Virulence. 6, 229-235 (2015).</li><li>Sharrow, S. O., Coligan, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+Of+Flow+Cytometry+Data.">Analysis Of Flow Cytometry Data.</a> Current Protocols In Immunology. , (2001).</li><li>Arizon, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Langerhans+Cells+Down-Regulate+Inflammation-Driven+Alveolar+Bone+Loss.">Langerhans Cells Down-Regulate Inflammation-Driven Alveolar Bone Loss.</a> Proc Natl Acad Sci U S A. 109, 7043-7048 (2012).</li><li>Moutsopoulos, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+Neutrophil+Recruitment+In+Leukocyte+Adhesion+Deficiency+Type+I+Disease+Causes+Local+IL-17-Driven+Inflammatory+Bone+Loss.">Defective Neutrophil Recruitment In Leukocyte Adhesion Deficiency Type I Disease Causes Local IL-17-Driven Inflammatory Bone Loss.</a> Sci Transl Med. 6, 229-240 (2014).</li><li>Gaffen, S. L., Jain, R., Garg, A. V., Cua, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+IL-23-IL-17+Immune+Axis:+From+Mechanisms+To+Therapeutic+Testing.">The IL-23-IL-17 Immune Axis: From Mechanisms To Therapeutic Testing.</a> Nat Rev Immunol. 14, 585-600 (2014).</li></ol>

Authors have nothing to disclose

La técnica actual produce con éxito un gran número de células inmunes (de un solo ratón) adecuado, no sólo para la caracterización fenotípica, sino también para estudios funcionales ex vivo. Otro grupo había publicado previamente un protocolo para aislar y caracterizar células inmunes gingivales murino y se introduce el valor de la utilización de la citometría de flujo multicolor en el estudio de la periodontitis en modelos animales 9. Tras la resolución de problemas considerables la mo...

Tejidos gingivales rodean la dentición humana y murina y están constantemente expuestos al complejo biofilm del diente 1. La red de células inmunes la vigilancia de la barrera gingival es vital para mantener la integridad del tejido, asegurando homeostasis con los microbios comensales locales y, al mismo tiempo, proporcionando una inmunidad eficaz contra el desafío patogénico 2. Para lograr la homeostasis, el sistema inmunológico está cuidadosamente adaptada al entorno gingival creación de una red de células inmunes altamente especializado, sin embargo, poco se conoce el detalle de las poblaciones de células inmunes gingivales y su papel en el mantenimiento de la inmunidad tisular 2. Cuando se interrumpe la homeostasis inmune en la encía, ya sea mediante el aumento de la susceptibilidad y / o presencia de comunidades microbianas dysbiotic, una condición inflamatoria del huésped, surge la periodontitis 3 4. La periodontitis es una enfermedad inflamatoria común, lo que lleva a la pérdida del diente sPOYO estructuras. En sus formas severas se observa en aproximadamente el 10% de la población general 5. La disección de los factores clave implicados en la susceptibilidad periodontitis y la progresión ha sido difícil 6. Sin embargo, los modelos animales han sido extremadamente útiles en la comprensión de los mecanismos de iniciación y progresión periodontitis 7. Los modelos pueden ser empleados para definir las poblaciones de células y mediadores moleculares clave que son vitales para el mantenimiento de la homeostasis inmune y el desarrollo de la unidad de la periodontitis. Tal visión va a transformar nuestra comprensión del control-encía específica de la homeostasis inmune y mejorar nuestra comprensión actual de la patogénesis de la enfermedad.

<table><tbody><tr><td>Fine Scissors</td><td>Fine science tools</td><td>14058-11</td><td></td></tr><tr><td>Scalpel Handle #3</td><td>Fine science tools</td><td>10003-12</td><td></td></tr><tr><td>Scalpel Blades #10</td><td>Fine science tools</td><td>10010-00</td><td>Sterile</td></tr><tr><td>Splinter Forceps</td><td>Integra Miltex</td><td>6-304</td><td></td></tr><tr><td>Needles with regular bevel&amp;nbsp;</td><td>BD Medical</td><td>305109</td><td>27 G, 12.7 mm length</td></tr><tr><td>Monoject syringes</td><td>Covidien</td><td>8881513934</td><td>Luer-lock tip, 3 ml</td></tr><tr><td>PBS, pH 7.4</td><td>Life Technologies</td><td>10010-049</td><td>Without Calcium and Magnesium</td></tr><tr><td>RPMI 1640</td><td>Lonza</td><td>12-167F</td><td>Without L-glutamine</td></tr><tr><td>DNase I from bovine pancreas</td><td>Sigma-Aldrich</td><td>DN25-1G</td><td></td></tr><tr><td>Collagenase type IV</td><td>Gibco (by Life technologies)</td><td>17104-019</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gemini Bio-products</td><td>100-106</td><td></td></tr><tr><td>Gentamicin 50 mg/ml</td><td>Quality biological</td><td>120-098-661EA</td><td></td></tr><tr><td>Pen Strep</td><td>Gibco (by Life technologies)</td><td>15140-122</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco (by Life technologies)</td><td>25030-081</td><td></td></tr><tr><td>0.5 M EDTA pH 8.0</td><td>Quality biological</td><td>351-027-721EA</td><td></td></tr><tr><td>50 ml tubes</td><td>Corning</td><td>352070</td><td>Polypropylene, sterile</td></tr><tr><td>70 &amp;mu;M Cell Strainers</td><td>Corning</td><td>352350</td><td></td></tr><tr><td>Petri dishes</td><td>Corning</td><td>351029</td><td>Sterile</td></tr><tr><td>5 ml FACS tubes</td><td>Corning</td><td>352052</td><td>Sterile</td></tr><tr><td>BD GolgiPlug</td><td>BD Biosciences</td><td>555029</td><td>Contains brefeldin A solution</td></tr><tr><td>Phorbol 12-Myristate 13-Acetate (PMA)</td><td>Sigma-Aldrich</td><td>P8139</td><td></td></tr><tr><td>Ionomycin Calcium Salt</td><td>Sigma-Aldrich</td><td>13909</td><td></td></tr><tr><td>Saponin from quillaja bark</td><td>Sigma-Aldrich</td><td>S4521</td><td></td></tr><tr><td>LIVE/DEAD Fixable Aqua Dead Cell Stain Kit</td><td>Life Technologies</td><td>L34957</td><td></td></tr><tr><td>Anti-Mouse CD45 Alexa Fluor 700</td><td>eBioscience</td><td>56-0451-82</td><td></td></tr><tr><td>Anti-Mouse CD4 eFluor 450</td><td>eBioscience</td><td>48-0042-82</td><td></td></tr><tr><td>Anti-Mouse TCR beta APC eFluor 780</td><td>eBioscience</td><td>47-5961-82</td><td></td></tr><tr><td>Anti-Mouse gamma delta TCR FITC</td><td>eBioscience</td><td>11-5711-82</td><td></td></tr><tr><td>Anti-Mouse IL-17A APC</td><td>eBioscience</td><td>12-7311-82</td><td></td></tr><tr><td>Anti-Mouse IFN-&amp;gamma; PE</td><td>eBioscience</td><td>11-5931-82</td><td></td></tr><tr><td>Anti-Mouse NK1.1 PE-Cy7</td><td>eBioscience</td><td>25-5941-82</td><td></td></tr><tr><td>Anti-Mouse CD90.2 APC eFluor 780</td><td>eBioscience</td><td>47-0902-82</td><td></td></tr><tr><td>Anti-Mouse CD3e FITC</td><td>eBioscience</td><td>11-0031-82</td><td></td></tr><tr><td>Anti-Mouse CD19 FITC</td><td>eBioscience</td><td>11-0193-82</td><td></td></tr><tr><td>Anti-Mouse CD11b FITC</td><td>eBioscience</td><td>11-0112-82</td><td></td></tr><tr><td>Anti-Mouse CD11c FITC</td><td>eBioscience</td><td>11-0114-82</td><td></td></tr><tr><td>Anti-Mouse TCR beta FITC</td><td>eBioscience</td><td>11-5961-82</td><td></td></tr><tr><td>Anti-Mouse Ly-6G FITC</td><td>eBioscience</td><td>11-5931-82</td><td></td></tr><tr><td>Anti-Mouse Ly-6C FITC</td><td>BD Pharmingen</td><td>553104</td><td></td></tr></tbody></table>

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.

Para ilustrar la aplicación del protocolo, mostramos los resultados representativos que examinan la red de células inmunes en la encía de los ratones con y sin periodontitis (WT vs. LFA - / -, Figura 2A - C). FACS representativos gráficos muestran células CD45 + hematopoyéticos vivos en la encía (Figura 2A, 2C). El aislamiento y la transformación de las células inmunes con este protocolo da...

Watch this Scientific Journal Video about Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network at JoVE.com

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

Hemos establecido una técnica para el aislamiento, la caracterización fenotípica y análisis funcional de células inmunes de encía murino.

El aislamiento, caracterización y examen funcional de la red celular gingival Inmune

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

isolation-characterization-functional-examination-gingival-immune

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

10.7K vistas.  NIH.  Hemos establecido una técnica para el aislamiento, la caracterización fenotípica y análisis funcional de células inmunes de encía murino. 

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

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

Aislar y inmunoticción Los linfocitos y las células dendríticas de las placas de Peyer murinas de

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Investigación

Inmunología e Infección

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.

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.

El aislamiento, tratamiento y análisis de células gingivales murinos

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

Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental physiopathology of periodontal diseases. Current experimental models of periodontal diseases have been established in various types of animals. However, the physiopathology of animal models is different from that of humans, making it difficult to analyze cellular and molecular mechanisms and evaluate new medicines for periodontal diseases. Here, we present a detailed protocol for reconstructing human inflammatory tissue equivalents of gingiva (iGTE) in vitro. We first build human tissue equivalents of gingiva (GTE) by utilizing two types of human cells, including human gingival fibroblasts (HGF) and human skin epidermal keratinocytes (HaCaT), under three-dimensional conditions. We create a wound model by using a tissue puncher to punch a hole in the GTE. Next, human THP-1 monocytes mixed with collagen gel are injected into the hole in the GTE. By adimistration of 10 ng/mL phorbol 12-myristate 13-acetate (PMA) for 72 h, THP-1 cells differentiated into macrophages to form inflammatory foci in GTE (iGTE) (IGTE also can be stumilated with 2 &#181;g/mL of lipopolysaccharides (LPS) for 48 h to initiate inflammation). IGTE is the first in vitro model of inflammatory gingiva using human cells with a three-dimensional architecture. IGTE reflects major pathological changes (immunocytes activition, intracellular interactions among fibryoblasts, epithelial cells, monocytes and macrophages) in periodontal diseases. GTE, wounded GTE, and iGTE can be used as versatile tools to study wound healing, tissue regeneration, inflammation, cell-cell interaction, and screen potential medicines for periodontal diseases.

The goal of the protocol is to build an inflammatory human gingiva model in vitro. This tissue model co-cultivates three types of human cells, HaCaT keratinocytes, gingival fibroblasts, and THP-1 macrophages, under three-dimensional conditions. This model can be applied to investigating periodontal diseases, such as gingivitis and periodontitis.

Equivalentes tridimensionales tejido humano inflamatorio de la encía

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental ...

Biología del Desarrollo

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Un modelo de ratón para Patógenos inducida inflamación crónica en los sitios locales y sistémicos

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

La detección in situ de las bacterias dentro de los tejidos embebidos en parafina utilizando un ADN sonda digoxina marcada focalización 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis como organismo modelo para la evaluación de la interacción de bacterias anaerobias con células huésped

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

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.

Aislamiento rápido de células individuales de dientes de ratón y humanos

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

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.

Aislamiento y cultivo de células epiteliales gingivales humanas primarias utilizando 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 ...

Bioingeniería

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

Aislamiento de células mononucleares tonitarias para estudiar las respuestas inmunitarias innatas ex vivo en un tejido linfático mucoso humano

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...

Organotypic Tissue Model Systems for Investigating Host-Pathogen Interactions In Vitro

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While in-house grown 2D epithelial monolayers lack true representation of the in vivo situation, commercially available tissue models are often overlooked due to their cost and practicality. However, with careful planning, such models provide reproducible platforms for a vast array of different applications. Here, we report the use of epithelial models that can be utilized for a wide variety of experimental purposes to investigate host-pathogen interactions in various ecological niches, such as the oral cavity, skin, and vaginal mucosa. From simple planktonic cells to complex biofilm co-culture, epithelial models are used to assess microbial adherence and invasion, and to evaluate the host response at a transcriptional and/or protein level, with scope for more detailed profiling using different omics approaches. Furthermore, these biological systems can be used as more accurate test beds for evaluating conventional and novel antimicrobial activity in a complex host-pathogen microenvironment in vitro. The protocols described herein document how models are handled upon arrival and prepared in the laboratory for co-culture stimulation with biofilm communities. The methods detail how experimental outputs are achieved from the model systems, including the processing of tissue, the co-culture setup, and data generation. These experiments include host gene expression through single- and multiplex qPCR analyses and inflammatory protein detection using ELISAs. In conclusion, epithelial models provide useful in vitro systems for preclinical investigatory studies into simple or complex host-pathogen interactions.

The present protocol outlines the application of organotypic tissue models for investigating host-pathogen interactions in vitro. Specifically, the model system described uses a multi-layered oral epithelium exposed to microorganisms associated with biofilms (dental plaque) within the context of oral health versus disease.

Sistemas de modelos de tejidos organotípicos para investigar las interacciones huésped-patógeno in vitro

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While ...