この作品は、科学研究 (26861689, 17 K 11813) の科学振興費日本社会によって一部に支えられました。著者は、校正のナサニエル ・ グリーン氏に感謝したいと思います。

このプロトコルは、対応するひと歯肉組織、歯肉創傷モデル、および歯肉炎モデルを作成する設計されています。ひと皮膚表皮角化細胞 (HaCaT) 親切教授ノルベルト ・ e. Fusenig ドイツ Krebsforschungszentrum (ハイデルベルク、ドイツ)7から提供されていました。ひと歯肉線維芽細胞 (HGFs) は、以前に公開されたプロトコル8によると歯肉組織から分離された.あらかじめ、インフォームド コンセントを得たし、研究が、日本歯科大学生命歯学部東京での倫理委員会で設定されたガイドラインに従って承認された (承認番号: NDU T2012 35)。細胞文化フードでプロトコルの手順 1-3 を実行必要があります。
1. 歯肉 (GTE) (図 1 a) の 3 D 人体組織等価の準備
<ol><li>ミックス タイプ - A コラーゲンゲル 10% 濃縮 MEM-α と 10% の再構築バッファー (2.2 g NaHCO3と 4.47 g HEPES 100 mL 0.05 N 水酸化ナトリウム) ピペッティングで 15 mL の生殖不能の管に。氷上を使用するまで混合コラーゲン溶液を維持します。</li>
	<li>24 ウェル プレートに 24 の文化挿入 (細孔サイズ 3.0 μ m) を配置します。</li>
	<li>
0.25% トリプシン-EDTA 溶液を使用して文化面から得られたを削除します。10 mL の培地 A に細胞を中断 (MEM-α + 20 %fbs + 1 %glutamax)。Bürker トゥルク細胞カウンターによってセルをカウントします。
	<ol>
<li>セル (1-5 x105セル/mL)、必要な数量を抽出し、190 x g で 4 分間遠心します。</li>
		<li>コラーゲン混合溶液中の細胞を中断します。次のステップまで氷の混合物をしてください。</li>
	</ol>
</li>
	<li>任意の気泡を生じることがなく文化挿入セル コラーゲン混合物 (0.5 – 2.5 x 105細胞/ウェル) の 0.5 mL を追加します。ジェルの中に混合物を凝固する 37 ° C で 5% CO2加湿雰囲気で 10-30 分のための混合物を孵化させなさい。 注: は、コラーゲン ・ ゲルの不均一の形成を避けるために文化挿入部にコラーゲン混合物を追加します。</li>
	<li>まあ、挿入に 0.5 mL に培養液 1 mL を追加します。24 時間の培養プレートを孵化させなさいまたは 37 ° C で 5% CO2加湿雰囲気の中で一晩</li>
	<li>0.25% トリプシン-EDTA 溶液を使用して文化面から細胞を削除します。10 ml の培地 B の細胞を中断 (DMEM + 10 %fbs + 1 %glutamax) とセルをカウントします。抽出細胞 (1-5 105セル/mL x) 190 × g. で 4 分間遠心分離、ピペットの必要量を中断中 B の細胞</li>
	<li>誤嚥により HGF コラーゲン混合物を含む文化挿入からメディアを削除します。HGF コラーゲン混合物の上に HaCaT 細胞懸濁液 (0.5 – 2.5 x 105細胞/ウェル) の 0.5 mL を加えます。24 時間または一晩文化を孵化させなさい。 注: この時点で文化の媒体も間べきである媒体、文化挿入中は中 b. をする必要があります。</li>
	<li>KSR 媒体 (共培養中) 培養液に置き換える (MEM-α + 15% ノックアウト血清交換 + 1 %glutamax) + 5 %fbs。まあ、文化挿入に 0.5 mL 文化に培養液 1 mL を追加します。24-48 h の文化を孵化させなさい。</li>
	<li>培養液に置き換えて KSR ミディアム + 1 %fbs。よく文化に 1 mL と文化挿入に 0.5 mL を追加します。24 時間または一晩文化を孵化させなさい。</li>
	<li>0.7-1 文化に培養液をよく置き換えます mL KSR 媒体。(文化の表面は、空気にさらされる必要があります) 文化挿入から媒体を完全に取り外します。1-2 週間の文化を孵化させなさい。週二回も文化内のメディアを変更します。 注: (ケラチノ サイトの最上位のレイヤーで構成される) 文化表面上中レベル上昇てはいけないです。常に乾いた状態に保つ培養面。</li>
</ol>2. 負傷者 GTE (図 1 b) の準備
<ol><li>1-3 mm 径組織パンチャーを準備します。それを 20 分 121 ° C のオートクレーブで殺菌します。</li>
	<li>300 μ m、深さについて GTE に垂直に組織のパンチャーをプッシュし、傷をシミュレートするために組織で穴を開けます。</li>
	<li>この手順では、創傷治癒と組織再生を調査するため負傷 GTE を使用します。また、GTE 10% 中性緩衝ホルマリンを使用した組織学的解析の実行を修正します。</li>
</ol>3. 炎症性 GTE (日本支社は 1996) (図 1 b) の準備
<ol><li>以前レポート9によると thp-1 細胞を養います。</li>
	<li>ミックス タイプ - A コラーゲンゲル 10% 濃縮 RPMI 1640 年 10% 再構成バッファー (2.2 g NaHCO3と 4.47 g HEPES 100 mL 0.05 N 水酸化ナトリウム) と 10 ng/mL PMA。使用するまで氷の上混合コラーゲン溶液を維持します。</li>
	<li>カウント 1-2 x 10 の6 THP 1 セルと 190 x g に 4 分間遠心をコラーゲン混合溶液 1 mL のセル中断します。次のステップまで氷の混合物をしてください。</li>
	<li>GTE の負傷者の領域に 10-30 μ L THP 1 コラーゲン混合物を追加します。0.7-1 に培養液を交換 mL KSR 培 PMA の 10 ng/mL。</li>
	<li>72 h の 37 ° C で 5% CO2加湿雰囲気の中で混合物を孵化させなさい。</li>
	<li>歯肉炎や歯周病を調査するためにモデルを使用 (たとえば、日本支社は 1996 サイトカインのリリース10にその効果を見るために潜在的な抗炎症薬を追加します)。また、72 h PMA 処理を省略すると LPS の 2 μ G/ml、文化を追加し、48 h11,12の 37 ° C で 5% CO2加湿雰囲気で組織を孵化させなさい。</li>
</ol>4. 固定および GTE の日本支社は 1996 全体マウント免疫染色文化
<ol><li>
文化の固定。
	<ol>
<li>24 ウェル プレートから培養培地を削除します。リン酸緩衝生理食塩水 (PBS) で 2 回組織を洗浄します。</li>
		<li>文化にも挿入して 1 mL の 10% ホルマリン中性緩衝液の 1 mL を追加します。4 ° C で冷蔵庫で一晩 24 ウェル プレートを残します。</li>
		<li>次の日 10% 中性緩衝ホルマリンを削除します。</li>
		<li>全体のマウント是非、固定後 PBS に組織を 3-4 回の洗浄します。</li>
		<li>ヘマトキシリンとエオシン (H & E) 染色や正規免疫染色、脱水、パラフィン包埋、切片と進みます。 注: 全体のマウントの是非、この手順を省略できます。</li>
	</ol>
</li>
	<li>
全体のマウント免疫染色 注: このプロトコルは、13 の参照の 1 つから変更されました。<ol>
<li>3 組織を洗う x PBS 0.5-1% トリトン、10-30 分たびに。</li>
		<li>ブロック バッファー内で 30 分間 2 回組織を孵化させなさい (PBS 1% トリトン + 10 %bsa)、室温で。</li>
		<li>5-10 分毎回ブロック バッファーで組織 2 × を洗ってください。</li>
		<li>挿入に必要な希釈/濃度に一次抗体溶液 50 μ L を追加します。薄いプラスチックしがみつくフィルムを使用して、組織を乾燥を避けるために挿入をラップします。</li>
		<li>4 ° C で 1 ~ 2 日間組織を孵化させなさい</li>
		<li>組織 3 回洗浄、PBS で 30 分間 1% トリトン + 10 %bsa。洗浄 3 回再び、PBS で 5 分 1% トリトン。</li>
		<li>二次抗体の 50 μ L を加えてブロック バッファー (PBS 1% トリトン + 10 %bsa) と各挿入するヘキスト 33342 ソリューション (NucBlue ライブ細胞染色) の 5 μ L。薄いプラスチックしがみつくフィルムを使用して、組織を乾燥を避けるために挿入をラップします。</li>
		<li>4 ° C で 1 ~ 2 日間インキュベートします。湿らせたペーパー タオルで 24 ウェル プレートをラップし、インキュベーションを通して使用されるプラスチック パックに入れます。</li>
		<li>PBS 1% トリトンの 10 分の 3 回、組織を洗浄します。</li>
		<li>鋭い針を使用して、組織を取得する文化挿入の膜をカットします。スライドに膜を配置します。 注: 組織は、膜にする必要があります。</li>
		<li>蛍光マウント中の 2-3 滴を追加します。解析までカバーガラスと暗闇の中で 4 ° C でストアを持つ組織をカバーしてください。</li>
		<li>共焦点レーザ走査型顕微鏡 (LSM) と組織を表示します。</li>
	</ol>
</li>
</ol>

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このプロトコルは、歯肉組織等価および皮下脂肪組織相当前レポート8,21,22で説明を作成する方法に基づいています。これは、シンプルで簡単な方法は、いくつかの手順特別な注意が必要です。たとえば、コラーゲン混合物は、ソリューションのゲル形成を避けるために使用するまで氷で冷やす必要があります。コラーゲ?...

歯周病は、成人の歯の喪失の主要な原因です。歯肉炎と歯周炎は、最も一般的な歯周疾患です。両方は歯肉でバイオ フィルムを介した急性または慢性炎症性変化を提示します。歯肉炎は、歯周炎は、通常慢性炎症として提示に対し、急性炎症によって特徴付けられます。組織学的レベルでは、微生物の構成要素は、マクロファージ、リンパ球、形質細胞、肥満細胞の1,2などの免疫細胞の活性化をトリガーします。(歯肉上皮細胞、線維芽細胞、血管内皮細胞、骨芽細胞を含む) ローカルの細胞と対話するこれらの免疫細胞、特にマクロファージ炎症歯周組織3,4病変の結果します。ラットなどの動物の様々 な種類の歯周疾患の実験モデルを確立されているハムスター、ウサギ、フェレット、イヌ、霊長類。ただし、モデル動物の生理病理学、細胞および分子メカニズムを分析し、歯周疾患5の新しい薬を評価しにくく、人間のそれとは異なる。歯周病細菌や単分子膜ひと口腔上皮細胞の共同栽培は、歯周感染症6のメカニズムを調査する使用されています。口腔細胞の単層培養がそのまま組織の三次元 (3 D) 細胞アーキテクチャを欠いているにもかかわらず、したがって、培養状況を模倣することはできません。
ここでは、体外で歯周疾患を表す 3 D 炎症人体組織等価な歯肉 (日本支社は 1996) が確立されます。歯周疾患のこの 3 D モデルは、単層細胞培養および動物モデル間の中間位置を占めています。HaCaT ケラチノ サイト、歯肉線維芽細胞、THP 1 マクロファージなどの細胞の 3 種類はコラーゲンのゲルの共同栽培、日本支社は 1996 を構築する炎症性のイニシエーターによって刺激されます。日本支社は 1996 は密接に、細胞分化、細胞間の相互作用、および歯肉組織におけるマクロファージ活性化の生体内での条件をシミュレートします。このモデルは、薬物スクリーニングと歯周疾病の新しい薬理学的アプローチをテストのためだけでなく、創傷治癒、炎症、組織再生で細胞および分子メカニズムを分析するため多くのアプリケーションを持っています。

<table><tbody><tr><td>Collagen type I-A</td><td>Nitta Gelatin Inc</td><td></td><td>For making dermis of GTE</td></tr><tr><td>MEM-alpha</td><td>Thermo Fisher Scientific</td><td>11900073</td><td>Cell culture medium</td></tr><tr><td>Cell Culture Insert (for 24-well plate), pore size 3.0 &amp;mu;m</td><td>Corning, Inc.</td><td>353096</td><td>For tissue culture</td></tr><tr><td>GlutaMAX</td><td>Thermo Fisher Scientific</td><td>35050061</td><td>Cell culture reagent</td></tr><tr><td>DMEM</td><td>Thermo Fisher Scientific</td><td>31600034</td><td>Cell culture medium</td></tr><tr><td>KnockOut Serum Replacement</td><td>Thermo Fisher Scientific</td><td>10828028</td><td>Cell culture reagent</td></tr><tr><td>Tissue puncher</td><td>Shibata system service co., LTD</td><td>SP-703</td><td>For punching holes in GTE</td></tr><tr><td>RPMI 1640</td><td>Thermo Fisher Scientific</td><td>31800022</td><td>Cell culture medium</td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A3294</td><td>For immunostaining</td></tr><tr><td>Hoechst 33342 (NucBlue Live Cell stain)</td><td>Thermo Fisher Scientific</td><td>R37605</td><td>For labeling nuclei</td></tr><tr><td>Fluorescence mount medium</td><td>Dako</td><td></td><td>For mounting samples after immunostaining</td></tr><tr><td>Anti-Cytokeratin 8+18 antibody [5D3]</td><td>abcam</td><td>ab17139</td><td>For identifying epithelium</td></tr><tr><td>Scaning electron microscope</td><td>Hitachi, Ltd.</td><td>HITACHI S-4000</td><td>For observing samples&#039; surface topography and composition</td></tr><tr><td>Confocal laser scanning microscopy</td><td>LSM 700; Carl Zeiss Microscopy Co., Ltd.</td><td>LSM 700</td><td>For observing samples&#039; immunofluorescence staining</td></tr><tr><td>Anti-Cytokeratin 19 antibody</td><td>abcam</td><td>ab52625</td><td>For identifying epithelium</td></tr><tr><td>Anti-vimentin antibody</td><td>abcam</td><td>ab92547</td><td>For identifying fibroblasts and activated macrophages</td></tr><tr><td>Anti-TE-7 antibody</td><td>Millipore</td><td>CBL271</td><td>For identifying fibroblasts in the dermis</td></tr><tr><td>Anti-CD68 antibody</td><td>Sigma-Aldrich</td><td>SAB2700244</td><td>For identifying macrophages</td></tr><tr><td>Human CD14 Antibody</td><td>R&amp;amp;D Systems</td><td>MAB3832-SP</td><td>For identifying macrophages</td></tr><tr><td>Alexa Fluor 594-conjugated secondary goat anti-rabbit antibody</td><td>Thermo Fisher Scientific</td><td>A11012</td><td>For immunofluorescence staining</td></tr><tr><td>Alexa Fluor 488-conjugated secondary goat anti-mouse antibody</td><td>Thermo Fisher Scientific</td><td>A11001</td><td>For immunofluorescence staining</td></tr><tr><td>EVOS FL Cell Imaging System</td><td>Thermo Fisher Scientific</td><td></td><td>For observing the sample&#039;s immunofluorescence staining</td></tr><tr><td>THP-1 cells</td><td>Riken BRC cell bank</td><td>RCB1189</td><td>For making iGTE</td></tr><tr><td>PMA(Phorbol 12-myristate 13-acetate)</td><td>Sigma-Aldrich</td><td>P8139</td><td>For differentiatting THP-1 cells</td></tr></tbody></table>

three-dimensional inflammatory human tissue equivalents of gingiva

(歯肉炎・歯周炎などの歯周病は、成人の歯の喪失の主要な原因です。歯肉の炎症は、歯周疾病の根本的な生理病理学です。歯周疾患の実験モデルを現在は動物の様々 なタイプに設けられています。ただし、モデル動物の生理病理学、細胞および分子メカニズムを分析し、歯周疾患の新薬を評価しにくく、人間のそれとは異なる。ここでは、ヒトの炎症性組織培養歯肉 (日本支社は 1996) 相当を再構築するための詳細なプロトコルを提案する.2 種類のひと歯肉線維芽細胞 (HGF) と三次元条件下でヒトの皮膚表皮角化細胞 (HaCaT) などを含む人間の細胞を用いた人体組織等価な歯肉 (GTE) を構築します。GTE に穴をパンチする組織パンチャーを使用して傷のモデルを作成します。次に、人間の THP 1 球がコラーゲン ・ ゲル混合は、GTE の穴に挿入されます。10 ng/mL ホルボール 12-ミリスタート 13-アセタート (PMA) 72 時間の adimistration、thp-1 細胞では GTE (日本支社は 1996) でフォーム炎症巣にマクロファージ分化 (日本支社は 1996 もすることができます stumilated 2 μ g/ml のリポ多糖 (LPS) の炎症を開始する 48 時間).日本支社は 1996 は、ひと細胞を用いた三次元アーキテクチャ炎症歯肉の最初の in vitroモデルです。日本支社は 1996 は、歯周疾病の主要な病理学的変化 (免疫細胞 activition、fibryoblasts、上皮細胞、単球やマクロファージの細胞相互作用) を反映します。GTE、負傷者 GTE、日本支社は 1996 は、歯周疾病の創傷治癒過程を研究する汎用性の高いツール、組織再生、炎症、細胞間相互作用や画面潜在的な医薬品として使用できます。

細胞には、位相差顕微鏡による観察 (図 2 a) の下で典型的な表皮細胞の形態が表示されます。スキャナー電子顕微鏡 (SEM) 像は、HaCaT 細胞表面が多く微絨毛で覆われていたことを示した。HaCaT 細胞間の細胞間の接続は、膜プロセス (図 2 b) によって仲介されました。細胞は、歯肉上皮マーカー K8/1814HaCaT 細胞...

Watch this Scientific Journal Video about Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva at JoVE.com

Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

プロトコルの目的は、炎症性歯肉モデル体外を構築することです。この組織モデルは共同ひと細胞、HaCaT ケラチノ サイト、歯肉線維芽細胞および三次元条件下での THP 1 マクロファージの 3 種類を栽培します。このモデルは、歯肉炎や歯周炎などの歯周病を調査に適用することができます。

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 ...

three-dimensional-inflammatory-human-tissue-equivalents-of-gingiva

Research

JoVE Journal

Developmental Biology

9.7K ビュー.  The Nippon Dental University. プロトコルの目的は、炎症性歯肉モデル体外を構築することです。この組織モデルは共同ひと細胞、HaCaT ケラチノ サイト、歯肉線維芽細胞および三次元条件下での THP 1 マクロファージの 3 種類を栽培します。このモデルは、歯肉炎や歯周炎などの歯周病を調査に適用することができます。

ビデオ: Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

人間の3次元の組織工学<em&gt; in vitroで</em&gt;腫瘍テストシステム

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

生物工学

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 ...

免疫・感染学

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三次元の全層皮膚等価物と自動傷つけ処理の発生

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

生産、特性評価および3D組織工学ヒト食道粘膜モデルの使用可能性

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

細胞外マトリックス内に埋め込まれたエンジニアリング三次元上皮組織

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

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.

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 ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

スクリーニング用途のための線維芽細胞由来ヒト工学的結合組織

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids direct contact with the measured area, it is suitable for monitoring blood flow changes during wound healing in human patients. Vestibuloplasty is periodontal surgery to the oral vestibule, aiming to restore vestibular depth with simultaneous enlargement of the keratinized gingiva. In this special clinical case, a split thickness flap was elevated at the first upper premolar and a xenogenic collagen matrix was adapted to the resulting recipient bed. LSCI was used to monitor the re- and neovascularization of the graft and the surrounding mucosa for one year. A protocol is introduced for the correct adjustment of microcirculation measurement in the oral mucosa, highlighting difficulties and possible failures.
The clinical case study presented demonstrated that &#8212; following the appropriate protocol &#8212; LSCI is a suitable and reliable method for following up microcirculation in a healing wound in the human oral mucosa and gives useful information on graft integration.

This study introduces a protocol for measuring microcirculation in human oral mucosa by laser speckle contrast imaging. The monitoring of wound healing after vestibuloplasty combined with a xenogenic collagen graft is presented on a clinical case.

ひと歯肉移植血管新生を監視への新しいアプローチ

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids ...

医学

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 ...

新規マウスギャグを用いた歯髄炎症マウスモデルの作成 

Establishment of a Murine Pulp Exposure Model with a Novel Mouth-Gag for Pulpitis Research

Pulpitis, a common cause of natural tooth loss, leads to necrosis and loss of bioactivity in the inflamed dental pulp. Unraveling the mechanisms underlying pulpitis and its efficient treatment is an ongoing focus of endodontic research. Therefore, understanding the inflammatory process within the dental pulp is vital for improving pulp preservation. Compared to other in vitro experiments, a murine pulpitis model offers a more authentic and genetically diverse context to observe the pathological progression of pulpitis. However, using mice, despite their cost-effectiveness and accessibility, poses difficulties due to their small size, poor coordination, and low tolerance, complicating intraoral and dental procedures. This protocol introduces a novel design and application of a mouth-gag to expose mouse pulp, facilitating more efficient intraoral procedures. The mouth-gag, comprised of a dental arch readily available to most dentists and can significantly expedite surgical preparation, even for first-time procedures. Micro-CT, hematoxylin-eosin (HE) staining, and immunofluorescence staining were used to identify changes in morphology and cell expression. The aim of this article is to help researchers establish a more reproducible and less demanding procedure for creating a pulp inflammation model using this novel mouth-gag.

著者スポットライト:改良されたマウスモデルによる歯科歯髄研究の強化

This article presents a streamlined protocol for establishing a pulpitis model in mice using an innovative mouth-gag, followed by subsequent histological analysis.

歯肉の炎症性三次元人体同等

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人間の3次元の組織工学