The authors do not have any acknowledgements.

記載されている全ての手順は、ローカル機関動物実験委員会の事前承認を含む、脊椎動物の使用のためのガイドラインと規則に従って実行されなければなりません。 解剖インスツルメンツおよび培養培地の調製<ol><li>予備洗浄全ての機器は、3％過酸化水素で20分間121℃でオートクレーブに切開に使用します。フード内の真空吸引と0.22μmの細孔サイズのフィルターを使用して、抗生物質や抗真菌溶液でBGJbメディアをフィルタリングします。 </li></ol>文化機器の調製<ol><li> 70％のアルコールでスプレー、小さな三角形で口蓋器官をサポートするポリカーボネートメンブレンフィルターをカットし、それらを乾燥させます。器官培養プレート上で、清潔で滅菌正三角形状のワイヤグリッド（20ミリメートル）を配置することにより、センターウェル器官培養皿（60x15 mm）を用意します。そして、Bの1ミリリットルとよくを埋めますGJBメディア培養培地。注：メディアは舌を沈めることなく、グリッドのレベルに達するのに十分であるべきです。メンブレンフィルターを配置し、光沢のある側を下に、グリッドの上に（グリッドあたり3-4膜）実験に.Depending、治療（タンパク質、抗体または阻害剤）を培地に添加することができます</li></ol> 3.マウスの胚収集と文化のための準備<ol><li>野生型マウスの胚を得るために、使用は13.5妊娠CD-1マウスの遺伝的背景の歪みをタイムアウトしました。 70％エタノールで解剖エリアを清掃してください。 注：マウスの​​他の系統を使用することができます。私たちが原因大きなごみ番号のCD-1マウスを好みます。 </li><li>承認されたプロトコルを使用して死を保証するために、頸椎脱臼に続いて5％イソフルランとE13.5 DPC（日性交後）で妊娠マウスを安楽死させます。 </li><li>商品に腹部ヘアスティックを回避するために腹側腹部表面に70％エタノールをスプレーします。その後、腹側腹腔を開き子宮を見つけるために鋭い鈍い動作はさみとマイクロ解剖ピンセットを使用して。鉗子を使用して、子宮全体を持ち上げ、子宮体部で切断することにより、ライト動作ハサミで子宮角の先端に体から分離。 </li><li> （ケアおよび実験動物の使用のためのプロトコルガイドによる）を氷の上に清潔なペーパータオル上で子宮を置きます。汚染を防止するための別の清潔なペーパータオルで子宮をカバー。さらに使用する前に、70％エタノールのスプレーを使用して器具を滅菌します。子宮胚を氷上で実験室に輸送することができます。 </li><li>オープン層流フードでは、慎重に鋭い鈍い動作はさみとマイクロ解剖ピンセットを用いて卵黄嚢の小さな開口部を作ります。開口部が広いことを確認した後、静かに卵黄嚢から胚を体外に出すためにそれを押してください。 </li><li>冷リン酸緩衝生理食塩水1X（PBS）pH7.4でペトリ皿にすぐに胚を移します。適切な段階を保証するために、元ボディ、頭の大きさ及び四肢の形態のための解剖顕微鏡下で胚アミン。 注：開発の権利の段階ではない胚を実験に使用されていません。 </li></ol><img alt="図2" src="/files/ftp_upload/53063/53063fig2.jpg" /> 図2.肢芽形態。前部と後肢が数字の間ウェビングインデント度がチェックされます。 E13.5では、すべての桁の縮合は非常に明確な登場数字の間のウェビングと遠位くぼみで、顕著です。後肢の桁が半日によって背後にある12上段：前肢、下段：後肢。 4.口蓋解剖<ol><li>解剖顕微鏡下でのPBSでペトリ皿の中で一度に一つの胚を置きます。小さな解剖ハサミとピンセットを使用すると、胚の本体からヘッドを分離します。 </li><li>慎重にピンセットで目の高さ以上のマウスの頭蓋骨を保持する（回避上顎プロセス）は、リップラインの両側にある2つの切開を行います。そして、下顎と舌を削除します。目の高さにカットすることによって上顎処理領域を分離し、脳、目、鼻中隔と隣接する組織を除去します。 </li></ol><img alt="図3" src="/files/ftp_upload/53063/53063fig3.jpg" /> 図 3. 口蓋組織が ​​口蓋棚が上昇した13.5胚から採取したが、接触が行われていない。口蓋棚は静的モデルで72時間空気メディアインタフェースでのフィルタに器官培養に入れた。（A）レベル培養皿のE13.5胚の解剖。台形フィルター膜上に配置された解剖口蓋棚の（B）ブルー星解剖時に鉗子で組織を保持するための領域の境界を定める。（C）の表示。（D）は側面図の配置後味覚及び培地の添加を解剖しました。 <ol start="3"><li>後部に前方から東洋それを助けるために取り付けられた第一の味覚を保ち、口蓋口側を置き、鼻中隔とその周囲の組織を除去します。スプーンヘラを使用し、非常に慎重に培養皿にグリッド上に準備されたフィルターに（鼻側を下）口蓋棚を転送します。注：適切な空気メディア・インターフェースを可能にするために、メディアの音量を調整します。 </li><li>実体顕微鏡下では、一次口蓋を分析し、互いに近接口蓋棚を配置するためにピンセットを使用しています。彼らが接触するように配置されていない場合、口蓋棚が融合されません。口蓋の前方部分が配置されている三角形の膜フィル​​ターをカットします。台形形状は、口蓋の前方部分を局所化に役立ちます。 </li><li>培養口蓋棚単独で又は二種BGJb培地を使用して、同じ器官培養皿三有します。 </li></ol> 5.培養マウス口蓋棚<ol><li> TISSUをインキュベート72時間、加湿気体混合物（5％CO 2及び95％空気）中で37℃ES、及び培地を24時間毎に変更します。 </li></ol>組織学的分析のために6口蓋処理<ol><li> 4℃で4％ホルムアルデヒド/リン酸緩衝生理食塩水のO / Nと文化の中で72時間後に舌を修正&#160; C.次に、静かに転送ピペットを用いてPBSで培養皿に組織を洗います。カセットを埋め込むには、実験室のワイプと場所の一部で舌を包みます。 注：実験用ワイプで口蓋をラップは、脱水工程の間に彼らの損失を防ぎます。 </li><li> 70％、80％以下の、通常のパラフィン包埋手順に続いて1時間100％エタノール洗浄、まで組織を脱水。埋め込むために、トレイに直面して口蓋の前方部分を配置します。 </li><li>後部に前方から冠状方向で連続6μmのセクションをカット。完全なセクションの口蓋は、350〜450 sectiが得られますアドオン。ヘマトキシリンおよびエオシン（H＆E）。10得点、以前に説明した平均融 ​​合スコアを使用して、すべての20 番目のセクション （MFS）スケール（表1）を持つセクションを染色。8 </li></ol>

<ol><li>Nawshad, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Palatal+seam+disintegration+to+die+or+not+to+die+that+is+no+longer+the+question%5BTitle%5D)+AND+%22Developmental+dynamics+an+official+publication+of+the+American+Association+of+Anatomists%22%5BJournal%5D)">Palatal seam disintegration to die or not to die that is no longer the question.</a> Developmental dynamics an official publication of the American Association of Anatomists. 237, 2643-2656 (2008).</li>
<li>Strong, E. B., Buckmiller, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Management+of+the+cleft+palate%5BTitle%5D)+AND+%22Facial+plastic+surgery+clinics+of+North+America%22%5BJournal%5D)">Management of the cleft palate.</a> Facial plastic surgery clinics of North America. 9, 15-25 (2001).</li>
<li>Witt, P. D., Marsh, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+in+assessing+outcome+of+surgical+repair+of+cleft+lip+and+cleft+palate%5BTitle%5D)+AND+%22Plastic+and+reconstructive+surgery%22%5BJournal%5D)">Advances in assessing outcome of surgical repair of cleft lip and cleft palate.</a> Plastic and reconstructive surgery. 100, 1907-1917 (1997).</li>
<li>4miloro, principles of oral and maxillofafcial surgery. 209, 231-249 (1984).</li>
<li>Mima, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+the+epithelial+adhesion+molecule+CEACAM1+is+important+for+palate+formation%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Regulation of the epithelial adhesion molecule CEACAM1 is important for palate formation.</a> PloS one. 8, e61653(2013).</li>
<li>Carette, M. J., Ferguson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+fate+of+medial+edge+epithelial+cells+during+palatal+fusion+in+vitro+an+analysis+by+DiI+labelling+and+confocal+microscopy%5BTitle%5D)+AND+%22Development+%28Cambridge%2C+England%29%22%5BJournal%5D)">The fate of medial edge epithelial cells during palatal fusion in vitro an analysis by DiI labelling and confocal microscopy.</a> Development (Cambridge, England). 114, 379-388 (1992).</li>
<li>Ferguson, M. W., Honig, L. S., Slavkin, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiation+of+cultured+palatal+shelves+from+alligator+chick+and+mouse+embryos%5BTitle%5D)+AND+%22The+Anatomical+record%22%5BJournal%5D)">Differentiation of cultured palatal shelves from alligator chick and mouse embryos.</a> The Anatomical record. 209, 231-249 (1984).</li>
<li>San Miguel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ephrin+reverse+signaling+controls+palate+fusion+via+a+PI3+kinase+dependent+mechanism%5BTitle%5D)+AND+%22Developmental+dynamics+an+official+publication+of+the+American+Association+of+Anatomists%22%5BJournal%5D)">Ephrin reverse signaling controls palate fusion via a PI3 kinase dependent mechanism.</a> Developmental dynamics an official publication of the American Association of Anatomists. 240, 357-364 (2011).</li>
<li>Kang, P., Svoboda, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PI+3+kinase+activity+is+required+for+epithelial+mesenchymal+transformation+during+palate+fusion%5BTitle%5D)+AND+%22Developmental+dynamics+an+official+publication+of+the+American+Association+of+Anatomists%22%5BJournal%5D)">PI 3 kinase activity is required for epithelial mesenchymal transformation during palate fusion.</a> Developmental dynamics an official publication of the American Association of Anatomists. 225, 316-322 (2002).</li>
<li>RD, L. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aRD%2C+L+%22Histopathologic+Technic+and+Practical+Histochemistry%22">Histopathologic Technic and Practical Histochemistry</a>. , 3rd edn, McGraw Hill Book Co. New York. (1965).</li>
<li>Kaufman, M. H. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKaufman%2C+MH+%22The+Atlas+of+Mouse+Development%22">The Atlas of Mouse Development</a>. , Elsevier. (1992).</li>
<li>Taher, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+gene+expression+analysis+of+murine+limb+development%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Global gene expression analysis of murine limb development.</a> PloS one. 6, e28358(2011).</li>
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The authors do not have any competing financial interests.

この記事のプロトコルは、胎生13.5で胚から口蓋棚を解剖する方法を提供します。マウス胚から口蓋棚を、37℃で95％O 2、5％CO 2雰囲気中で無血清培地中で培養しました。成功口蓋解剖を安楽死させたマウスの胚時から培養終了までの手順の間、各段階で複数の要因に決定的に依存しています。口蓋融合に影響を与える最も重要な要因の一つは、器官培養を開始するのに要する時間です?...

口腔顔面裂が最も優勢な頭蓋顔面の先天性欠損症です。また、考慮して可能なすべての頭蓋顔面の欠陥を取って、これらは、新生児2における第二の最も一般的な先天異常があります。裂口蓋は毎年約1米国ではすべての700の出産（米国）で発生し、口蓋裂の発生率は、裂け目月額味覚または3日目あたり裂と15人の子供を持って生まれた475人の子供に等しいです。毎年の展示世界中で生まれた子供の約1％頭蓋顔面​​異形学のいくつかのフォーム。 口蓋や唇の裂け目は、この異常を有する患者のための生涯の影響で非常に高価で複雑な手続きが必要です。経口裂と各患者の推定コストはおよそ$ 100,000個の4です。口唇口蓋裂を有する患者の治療は、頭蓋顔面外科医、耳鼻咽喉科医、遺伝学者、麻酔科医を含む医師のチームを必要とします音声言語病理学者、栄養士、歯科矯正医、補綴、心理学者、神経外科医、および眼科医。 palatogenesisでは、二次口蓋は最初垂直方向に成長し、舌の背の上口蓋棚の上昇を受けて対になった派生物として発生します。標高に続いて、対になった口蓋棚は（ヒトではマウスではE14.5 -E15で週9）正中線に向かって成長します。棚の先端をカバー内側縁上皮（MEE）は正中上皮縫い目を形成する接着します。 これは間葉合流を可能にするために間葉移行、および/またはアポトーシスの上皮が続きます。 MEEに反対の接着は、その変更口蓋裂の原因となる不可欠なイベントです。しかし、研究はわずか口蓋棚5の初期接着性を検討しました。骨芽細胞への間葉細胞の分化により硬口蓋フォーム。音部記号を生成することができます口蓋の発達異常tはまたは唇の関与なしに味覚。 口蓋器官培養技術は、過去30年間6,7に比べて多くのラボのために広く使用されていました。 このプロトコルでは、詳細に口蓋解剖し、静的な器官培養する方法を説明します。静止器官培養の利点は、口蓋棚の融合を可能にすることです。この技術は、多くの融合とシグナル伝達の実験8,9のために私たちの研究室で首尾よく使用されていました。しかし、技術の範囲は膨大であり、静的な器官培養系は、外因性の化学薬剤に対する応答の評価、異なる経路および特定のタンパク質における調節増殖因子の効果を含めて、必要なときにいつでも使用することができます。 <img alt="図1" src="/files/ftp_upload/53063/53063fig1.jpg" /> 図1.マウスPalatogenesis。マウス口蓋の発達段階。（BF）走査 ELE代表的な発達時期に二次口蓋のCTRON顕微鏡写真（SEM）。赤矢印：口蓋接着および融合の最初の部分を示しています。黄色の矢印：ポイント（PLOS 1から許可を得てカウフマン11より転載）融合後に消えますプライマリとセカンダリの口蓋との間の空間に。

<table><tbody><tr><td>BGJ&lt;sub&gt;b&lt;/sub&gt;</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Lonza Inc.</td><td>17-602E</td><td>100X</td></tr><tr><td>Petri dishes</td><td>VWR</td><td>25384-088</td><td>100 x15 mm</td></tr><tr><td>Center-well organ culture dish</td><td>Falcon&amp;nbsp;</td><td>#353037</td><td>60 x15 mm</td></tr><tr><td>Wire triangular grid</td><td>Custom made with stainless steel wire mesh to fit the organ culture dish well.&amp;nbsp;&amp;nbsp;</td><td></td><td></td></tr><tr><td>Polycarbonate filter</td><td>GE&amp;amp; Water and Process Technologies</td><td>K04BP04700</td><td>Black 0.4 micron, 47 mm</td></tr><tr><td>Stereo microscope</td><td>ZEISS&amp;nbsp; Stemi SR</td><td></td><td></td></tr><tr><td>Culture hood</td><td>NUAIRE Laminar Flow</td><td></td><td></td></tr><tr><td>Microdissecting forceps</td><td>Dumont Medical&amp;nbsp;</td><td></td><td>#5</td></tr><tr><td>Microdissecting scissors</td><td>Kent Scientific Corporation</td><td>INS14003-G</td><td>Vannas Scissors, straight, 8 cm long, 0.1mm tips, 5 mm blades, German made</td></tr><tr><td>Microdissecting scissors</td><td>Kent Scientific Corporation</td><td>INS500086</td><td>Vannas Scissors, straight, 8.5 cm long, 0.025 mm x 0.015 mm tips, 7 mm blades</td></tr><tr><td>Microdissecting scissors</td><td>Kent Scientific Corporation</td><td>INS14127-G</td><td>Spring scissors curved curved, 10.5 cm long, 8 mm blades, German made</td></tr><tr><td>Surgical currete (spoon spatula)</td><td>Hu-friedy</td><td></td><td>CM 2/4</td></tr><tr><td>Protector laboratory hood</td><td>Labconco</td><td></td><td></td></tr><tr><td>Incubator</td><td>Thermo Scientific</td><td></td><td>Farma</td></tr><tr><td>Series11 water Jacket</td><td></td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td></td><td></td><td></td></tr><tr><td>PBS&amp;nbsp;</td><td>GIBCO</td><td>10010-023&amp;nbsp;</td><td>1X</td></tr><tr><td>Fiber optic Light source</td><td>Fiber-light&amp;nbsp; Dolan-Jenner</td><td>PL-750</td><td></td></tr><tr><td>Embedding cassette&amp;nbsp;</td><td>Statlab</td><td>EC301</td><td></td></tr><tr><td>Kim Wipes</td><td>VWR</td><td>470173-504</td><td></td></tr></tbody></table>

method of studying palatal fusion using static organ culture

口唇口蓋裂は、すべての先天性欠損症の最も一般的なの一つです。正中上皮縫い目を形成するために付着上皮で覆われた間葉系の棚からの二次口蓋の形（MES）。理論は、MESの細胞が融合した口蓋1を作り、間葉移行（EMT）、アポトーシスおよび移行に対する上皮に従うことを示唆しています。 MESの完全な崩壊は、間葉系細胞とその周辺口蓋合流の最終不可欠相です。私たちは、口蓋の器官培養のための方法を提供します。 試験管プロトコルで開発された溶融中に生物学的および分子プロセスの研究を可能にします。この技術の用途は、外因性の化学薬剤に対する応答を評価するなど、規制および増殖因子と特定のタンパク質の効果数多くあります。口蓋器官培養は 、インビボ研究で使用したことはできない開発の様々な段階での操作を含む、多くの利点を有します。

技術は研究が口蓋融合のin vitroでのニコチンの催奇形効果をテストするために設計された以下のexperiments.Theの異なる種類に適用することができます。 2系統のマウスは、この実験のために利用された、CD1とC57.Palatal組織は、ニコチンヘミ硫酸塩の0.06と6 mMの濃度で処理しました。これは、口蓋融合パターンとタイミングは、2つの異なる株で異なっていたことが観察されました。 CD1マウス...

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Method of Studying Palatal Fusion using Static Organ Culture

口蓋開発の研究は、口蓋裂、驚異的な健康の負担を課し、外観を損なうを持続残すことができ先天異常の発生率が動機とされています。ここでは口蓋の開発および融合に関与する種々のシグナル伝達経路を研究するために使用することができる培養口蓋棚の技術を実証します。

静的器官培養を使用して口蓋の融合を研究の方法

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that ...

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8.7K ビュー.  Texas A&M University Baylor College of Dentistry. 口蓋開発の研究は、口蓋裂、驚異的な健康の負担を課し、外観を損なうを持続残すことができ先天異常の発生率が動機とされています。ここでは口蓋の開発および融合に関与する種々のシグナル伝達経路を研究するために使用することができる培養口蓋棚の技術を実証します。 

ビデオ: Method of Studying Palatal Fusion using Static Organ Culture

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

咽頭アーチのex vivo培養は、ハートと筋前駆細胞とそのニッチを研究します

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

発生生物学

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

蛍光in situハイブリダイゼーションおよび共焦点レーザー走査顕微鏡による口蓋パンダの口腔バイオフィルムの解析

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

医学

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

人間の茸状の味覚乳頭細胞の単離および培養

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

神経科学

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

マウス二次口蓋融合のライブイメージング

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

初代培養およびコルチのマウス臓器のプラスミドエレクトロ。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

生物学

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

We present a protocol for isolation and culture of primary mouse embryonic palatal mesenchymal cells for time-lapse imaging of two-dimensional (2D) growth and wound-repair assays. We also provide the methodology for analysis of the time-lapse imaging data to determine cell-stream formation and directional motility.

集団運動属性を解析するためのマウス胚性口蓋前葉細胞の単離とタイムラプスイメージング

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and ...

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The continuously growing mouse incisor provides an excellent model to study tissue-specific stem cells. The epithelial tissue-specific stem cells of the incisor are located at the proximal end of the tooth in a niche called the cervical loop. They provide a continuous influx of cells to counterbalance the constant abrasion of the self-sharpening tip of the tooth. Presented here is a detailed protocol for the isolation and culture of the proximal end of the mouse incisor that houses stem cells and their niche. This is a modified Trowell-type organ culture protocol that enables in vitro culture of tissue pieces (explants), as well as the thick tissue slices at the liquid/air interface on a filter supported by a metal grid. The organ culture protocol described here enables tissue manipulations not feasible in vivo, and when combined with the use of a fluorescent reporter(s), it provides a platform for the identification and tracking of discrete cell populations in live tissues over time, including stem cells. Various regulatory molecules and pharmacological compounds can be tested in this system for their effect on stem cells and their niches. This ultimately provides a valuable tool to study stem cell regulation and maintenance.

The Trowell-type organ culture method has been used to unravel complex signaling networks that govern tooth development and, more recently, for studying regulation involved in stem cells of the continuously growing mouse incisor. Fluorescent-reporter animal models and live-imaging methods facilitate in-depth analyses of dental stem cells and their specific niche microenvironment.

歯科幹細胞の制御を研究するためのトロウェル型臓器培養の使用

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The ...

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

菌体、円周体、口蓋味覚芽の全山染色、可視化、分析

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

ヒト鼻上皮オルガノイドの培養とイメージング

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

成人マウス口蓋からの一次口腔角化細胞の分離と培養

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...