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.

このプロトコルに記載されている全ての実験手順は、必要なガイドラインに従い、施設内動物管理使用委員会、NIDCR / NIHによって承認されました。 1.事前に準備<ol><li>完全培地を調製する：RPMIは2mM L-グルタミン、100単位/ mlのペニシリン、100μgの/ mlストレプトマイシン、10％FBSを補充しました。 </li><li> 7.5μgのDNA分解酵素を添加したRPMI培地50ミリリットル（、新鮮なメイク常に氷上に保つ）：DNアーゼメディアを準備します。 </li><li> DNアーゼメディアプラス3.2ミリグラム/コラゲナーゼIV型のミリリットルの5ミリリットル（、新鮮なメイク常に氷上に保つ）：コラゲナーゼ-DNアーゼメディアを準備します。 </li><li>前クールFACS緩衝液：PBSで希釈した0.5％FBS。 </li><li> 4℃に予冷遠心分離機。 </li><li> 37°Cまでのウォームシェーカーインキュベーター。 </li></ol>歯肉ブロックから2単離、解剖、および細胞単離<ol><li> ARACガイドラインに従って、適切なチャンバ内のCO 2にそれらを暴露することによって、マウスを安楽死させます。 </li> &lt;李&gt;は、心臓や内臓を露出するために胸部と腹腔を開きます。灌流するために、下大静脈内の切開を行い、27 G針で3ミリリットル注射器を用いて左心室を経てPBSの3ミリリットルを灌流。 </li><li>上を向いて胃とパッド上でマウスの体と頭を固定化します。 </li><li>下にある組織と筋肉を露出させ、下顎と首の領域を覆う皮膚を分離し、はさみで首に向かって唇の各交連をカット。下顎を発見するために下唇を解剖。 </li><li>下顎の両側を分離し、口腔にアクセスするためのそれぞれの側を固定する下側切歯間で切断。 </li><li>離れて鼻腔から口蓋を視覚化し、解剖。 </li><li> 10ブレードを使用して領域を解剖し、周囲の歯肉（ 図1）で上顎と下顎臼歯の領域を含みます。縦切開はちょうど第一大臼歯と後部第三大臼歯にとの水平切開部に前方ください歯肉の境界線（組織周囲の歯のホワイトカラー）。 </li><li> 5ミリリットルコラゲナーゼ/ DNアーゼ（氷上で保持）で50ミリリットルコニカルチューブに上顎と下顎のブロックを配置します。 </li><li> 37℃で1時間振とうインキュベーター中でインキュベートし、インキュベーションの最後の5分間に0.5 M EDTA50μlのを追加します。 </li><li>組織と50ミリリットルチューブに冷たいアーゼメディアの5ミリリットルを加え、穏やかに混合することが渦巻きます。 </li><li>シャーレ上の組織の4ブロックを転送し、DNアーゼメディア500μlのでカバー。手術用メスの刃を使用して、組織の各ブロックから歯肉を削除し、歯肉と歯間の領域に刃を向けます。 </li><li>セルストレーナー（70μmの大きさ）を介して新しい50mlチューブに、ペトリ皿だけでなく、解剖時に使用される前のDNアーゼメディアから組織やメディアを転送します。使用される機器のすべてとフィルタDNアーゼメディア（3-5 ml）で洗浄します。 </li><li>無菌の3ミリリットルシリンジのプランジャーを使用して、ストレーナに対して組織をマッシュ、冷たいDNアーゼメディア（30〜35 ml）でフィルタリングします。 </li><li>冷たいDNアーゼメディアを持つセルストレーナーを洗浄します。 </li><li> 4°C、6分間、314×gで遠心分離します。 </li><li>上清を捨て、完全培地1ml中に再懸濁します。 </li><li>自動細胞計数器を用いて、（予想量が&gt; 80％の生存率で1.2×10 6細胞に8×10 5の範囲とすべきである）細胞を数えます。 </li></ol>歯肉細胞の3刺激およびFACS染色<ol><li>ブレフェルジンAの1μL/ mlの存在下でPMA（50ng / ml）およびイオノマイシン（2.5μgの/ ml）で単一細胞懸濁液を刺激</li><li> 37℃で3.5時間、5％CO 2インキュベートします。 </li><li>スピンダウンし、4℃でPBSと遠心機で洗います。 6分間314×gで。 </li><li>製造業者の説明書に従って生存能力染色を用いて細胞を染色します。 </li><li>細胞外マーカー（CD45、TCRγδ、TCRβ、CD4、CD8、TCRγδまたはアリ用ステイン製造者の指示に従って、選択したibodies）。 </li><li> 4°C、5分間、314×gでの冷FACS緩衝液および遠心分離で細胞を洗浄。 </li><li>静かに細胞ペレットを破壊するためにボルテックス。 </li><li>室温で20分間、2％パラホルムアルデヒドで細胞を固定してください。 </li><li>冷たいFACS緩衝液、4°C、細胞内サイトカインのための5分および染色のための314×gで遠心分離で細胞を洗浄。 <ol><li> 4°C、5分間、314×gでFACS緩衝液および遠心機で希釈し、冷0.5％サポニンの溶液でFACSチューブを埋めます。 </li><li> FACSチューブに0.5％サポニン溶液の300μl加え、4℃で暗所で15分間インキュベートし、その後、4℃、5分間、314×gで遠心します。 </li><li>製造者の指示に従って、細胞内のマーカー（IL-17AおよびIFNγまたは選択のサイトカイン）のための細胞を染色。 4°C、5分間、314×gで0.5％サポニン溶液と遠心で細胞を洗浄。 </li><li> FACSで再び洗浄バッファと4℃、5分間、314×gで遠心。 </li><li> FACS緩衝液300μlの中で細胞を再懸濁します。 </li></ol></li><li>フローサイトメーター上の結果を分析します。 </li></ol> 4.フローサイトメトリー分析<ol><li>細胞を可視化するデブリを排除するために、前方（FSC）および側方散乱（SCC）及びゲートに分析されます。 </li><li>高さ（FSC-H）パラメータ対前方散乱面積（FSC-A）を持つ単一のセルを選択します。 </li><li>さらなる分析（ 図2A および 2C）は造血マーカーCD45の+ライブ（生存能力染色によって示されるように）正あるゲート細胞。 </li><li> 図 2と図 3に示すように、特定のマーカーの解析を続けます。 </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

現在の技術では、成功した表現型の特徴付けのためだけでなく、ex vivoでの機能研究のためだけではなく、適切な（シングルマウスからの）免疫細胞の多数をもたらします。別のグループは、以前に分離し、マウスの歯肉の免疫細胞を特徴付けるためのプロトコルを公開し、動物モデル9における歯周炎の研究で、フローサイトメトリー多色を使用しての値を導入していました。...

歯肉組織は、ヒトおよびマウスの歯列を囲み、常に歯 1の複雑なバイオフィルムにさらされています。歯肉障壁をポリシング免疫細胞ネットワークは、組織の完全性を維持するローカル共生微生物に恒常性を確保し、同時に、病原性のチャレンジ2に対して有効な免疫を提供するために不可欠です。恒常性を達成するために、免疫系を注意深くまだ少し詳細は歯肉の免疫細胞集団および組織免疫2の維持における役割の知られている高度に特化した免疫細胞ネットワークを作成歯肉環境に合わせて調整されます。 免疫恒常性が歯肉に破壊される場合には、いずれかの増加ホスト感受性および/ ​​またはdysbiotic微生物群集、炎症状態の存在を通して、歯周炎は、3〜4を発生します。歯周炎は、歯の喪失につながる、一般的な炎症性疾患でありますupporting構造。その重篤な形態では、一般集団5の約10％に見られます。歯周炎感受性および進行に関与する重要な要因を解剖すると6困難であることが判明しました。しかし、動物モデルは、歯周炎の開始および進行7のメカニズムを理解する上で極めて有用でした。モデルは、免疫ホメオスタシスおよび歯周炎の駆動開発を​​維持するために不可欠な重要な細胞集団および分子メディエーターを定義するために使用することができます。このような洞察は、免疫恒常性の歯肉固有の制御の我々の理解を変換し、疾患の病因の我々の現在の理解を促進します。

<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.

プロトコルの適用を説明するために、我々は（ - / - 、 図2A - C LFA対WT）とし、歯周炎のないマウスの歯肉における免疫細胞のネットワークを調べる代表的な結果を示しています。代表的FACSプロットは、歯肉（ 図2A、2C）でのライブCD45 +造血細胞を示します。このプロトコルで免疫細胞の単離および処?...

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

我々は、分離、表現型の特徴およびマウス歯肉からの免疫細胞の機能解析のための手法を確立しました。

単離、特徴付けおよび歯肉免疫細胞ネットワークの機能検討

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

Research

JoVE Journal

Immunology and Infection

10.8K ビュー.  NIH. 我々は、分離、表現型の特徴およびマウス歯肉からの免疫細胞の機能解析のための手法を確立しました。 

ビデオ: 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.

マウスのパイエル板のリンパ球や樹状細胞を単離し、免疫染色

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

免疫・感染学

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.

マウス歯肉細胞の単離、処理と解析

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.

歯肉の炎症性三次元人体同等

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

発生生物学

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.

局所および全身所における病原体により誘導される慢性炎症のためのマウスモデル

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.

16S rRNAのをターゲットジゴキシン標識DNAプローブを用いたパラフィン包埋組織内細菌のその場検出で

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.

宿主細胞と嫌気性菌の相互作用を評価するためのモデル生物としてポルフィロモナス・ジンジバリス

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.

マウスとヒトの歯からの単一細胞の迅速な分離

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.

Y-27632を用いた初原ヒト歯肉上皮細胞の単離と培養

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

生物工学

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.

ヒト粘膜リンパ組織におけるEx Vivo自然免疫応答を研究する扁桃単核細胞の単核細胞の単離

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.

In Vitroでの宿主-病原体相互作用の研究のための器官型組織モデルシステム

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