サラA. Alfaqeehは歯科、高等教育省、サウジアラビア王国の種類サウド大学によって資金を供給される。

1。セットアップする<ol><li> （鉱物油で潤滑研ぎ石を使用して）解剖器具をシャープにし、70％エタノールスプレーや乾熱滅菌を使用して器官培養に使用する前に滅菌する。 12 V電源を使用して、2M水酸化ナトリウムで電気分解を使用して解剖針を研ぐ。 </li><li> 1％のグルタおよび1％ペニシリン - ストレプトマイシンを補充したアドバンスト·ダルベッコ改変イーグル培地、F12（DMEM F12キー）からなる、胚の器官の解剖と培養に用いる培養液を準備します。 </li></ol> 2。胚解剖<ol><li>ホームオフィスやその他の管理機関によって承認されたスケジュール1の方法を使用して、時限交配、妊娠女性を間引く。下腹部の周りの皮膚を離れて解剖し、ハサミを使って腹部を開いてカット。下正中線で一緒に接続する2つの子宮角を見つけます。 </li><li>氷の上の媒体で満たされたチューブ内の子宮と場所を解剖する。 WHEnは氷上で、組織は、数時間放置することができる。 </li><li>子宮を取り出して、顕微鏡下で培地中のペトリ皿に置く。可能な胚の解離は、汚染を最小限に抑えるために層流フード内で実行されるべきある。 </li><li>子宮から胚を解剖し、その胚体外組織からそれらを解放。 </li><li>タングステン針を使用して胚を斬首し、培地の新鮮な皿に頭を移す。 </li><li>口腔内に針を配置することで、ヘッドから下顎を隔離し、頭の後ろ（ 図1Aおよび図1B）に向かってカットスルー。チョッピングを待っている間に単離されたあごを一時的に氷上で媒体に格納することができる。 </li><li>全体下顎のスライスラインステージのためE11.5-E15.5歯胚培養のための理想的です。 E15.5の後に下顎骨の途上骨は、チョッピングのためにあまりにも難しいです。歯のスライスは、まだすることができますが、骨（歯骨や歯槽骨を形成する）は、最初にして除去されなければならない9チョッピングの前に解剖。タングステン針とピンセットを使って慎重に骨を除去。 </li></ol> 3。胚スライス<ol><li>標準テーブル組織チョッパー（ 図1C）を使用して、下顎骨組織切片を準備します。約200顎後に新鮮な刃を使用し、それが低下したときにはディスク装着と同一平面にあることを確認してください。 </li><li>使用前に70％エタノールでディスク装着し、ブレードを清掃してください。マシンを使用している場合、ブレードは非常にシャープでよう注意が必要があります。 </li><li>標本の年齢に応じて200〜400ミクロンの間の切断距離でチョッパーを設定し、全体の歯胚が必要であるかどうかに応じて。最大でブレード力を設定。 </li><li>ピペットまたはピンセットを用いてプラスチック製の取り付けディスクに下顎を転送します。向きがまだ明確で確認して、ディスク上に下顎を配置します。現像舌配向を決定するのに有用なランドマークとして使用することができる。臼歯ためのAFを使用切歯のために、彼らは（ 図2Aにチョッピングの面を参照）成長するにつれて、これらの歯の様々な方向を反映するように、下顎の矢状スライスを使用している間rontal /横方向（ 図1（b）にチョッピングの面を参照）。 </li><li>少し取り付けディスクに下顎を乾燥させるために、ろ紙を用いて組織の周りから過剰な培地を除去。 </li><li>すぐに乾燥させた後、チョッパーの円形のステンレス製のテーブルの上にディスクを置きます。マシンの電源を入れ、[開始]を押します。表は、同時にブレードを担持するチョッピングアームを上昇さ/分、最大200ストロークで滴下しながら左から右に自動的に横断する。 </li><li>組織が完全にスライスされた後、マシンの電源をオフにします。ブレードアームが上昇位置にあることを確認して、ディスクを取り出します。ブレードアームの下に指を入れたりチョッピング時に無人の機械を放置しないでください。 </li><li>ディスク装着を取るとすぐにメディアトンを追加過度の乾燥を防ぐために、ディスク上のスライスO。注意深く培地の小さなペトリ皿（ 図2A）内にタングステン針、次いでピペットを用いて、ディスクの底からスライスされた組織を取り除く。 </li><li>針を使用してスライスを分離した後、実体顕微鏡（ 図1D、1E、および図2B）の下で、目的の組織を含む組織切片を選択します。培地で新鮮な培養皿に選択したスライスを配置します。 </li></ol> 4。スライス文化<ol><li> 、脱水を防ぐために、外側のウェルにオートクレーブ処理水約2mlを添加して皿の中央を横切って金属グリッドを配置することによって、培養の準備が中央ウェル器官培養皿を準備し（ 図3Aおよび3B）。 </li><li>ステンレスメッシュのシートからX 1.5センチメートル約4センチのストリップを切断した金属グリッドを準備します。中央に穴を作るために穴パンチを使用し、曲げ千鳥エンドを作成するための側面。グリッドは、ディッシュ（ 図3Aと図3B）の底に平行に横たわって、中央だけでなく全体に平らに適合する必要があります。使用前に滅菌するグリッドをオートクレーブ。 </li><li>グリッドの穴を​​カバーするのに十分な大き透明なポリエチレンテレフタレート（PET）膜（孔径0.4μm）での片を切り取る。選択されたスライスと皿にカット膜を置きます。 </li><li>膜の上にスライスを配置し、慎重に中央に平らにスライスして、媒体からメンブレンを持ち上げます。 </li><li>中央にある円形の穴の上にグリッド上にフィルターを置き、膜のレベルまで、ウェルに培養液に約1ミリリットルを追加。 </li><li>操作のために（例えば、8）を培地にタンパク質または小分子阻害剤を追加します。 </li><li>文化の日の0時に形態学のレコードのスライスを撮影しています。 </li><li> 5％のCOの加湿雰囲気中で食器を置く<sインキュベーター内で37℃で&gt; 2 UB。 </li><li>平均して7日間、定期的に文化を撮影しています。培養液ごと48〜72時間を変更します。写真撮影のための下（ 図2C-E）からの光源を用いてスライスを表示します。 </li></ol> 5。リネージュトレース系譜トレースが必要な場合は、このようにDiIまたはDiOのような親油性染料は、ステップ4、スライス培養の前に歯胚に注入することができる。 <ol><li>ニードルプラーを用いてガラスキャピラリー針を引き出します。 </li><li>ピンセットを用いて、針の先端を破壊し、ダウン色素溶液にチップを置く。染料は、毛細管現象によってチップを埋めながら、数分間のままにしておきます。 </li><li>ホルダーに充填された注射針を配置し、注射器や口アスピレーターに管を介して取り付けます。 </li><li>ラベルされることになったスライスの領域をタッチし、培地中の針の先端の位置を決めます。針ANから少量の染料を変位させる空気圧を適用する組織上にD。 </li><li>針を取り外し、蛍光下にラベルを確認してください。 </li><li>正常に標識されたスライスは、フィルターに移し、工程4に記載のように培養することができる。 </li></ol>

<ol>
	<li>Rothova, M., Peterkova, R., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+map+of+the+dental+mesenchyme:+dynamic+development+of+the+dental+papilla+and+follicle.">Fate map of the dental mesenchyme: dynamic development of the dental papilla and follicle.</a> Developmental biology. 366, 244-254 (2012).</li><li>Ferguson, C. A., 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=Activin+is+an+essential+early+mesenchymal+signal+in+tooth+development+that+is+required+for+patterning+of+the+murine+dentition.">Activin is an essential early mesenchymal signal in tooth development that is required for patterning of the murine dentition.</a> Genes &amp; development. 12, 2636-2649 (1998).</li><li>Trowell, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+culture+of+mature+organs+in+a+synthetic+medium.">The culture of mature organs in a synthetic medium.</a> Experimental cell research. 16, 118-147 (1959).</li><li>Matalova, E., Antonarakis, G. S., Sharpe, P. T., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+of+primary+and+secondary+enamel+knots.">Cell lineage of primary and secondary enamel knots.</a> Developmental dynamics : an official publication of the American Association of Anatomists. 233, 754-759 (2005).</li><li>Mitsiadis, T. A., Tucker, A. S., De Bari, C., Cobourne, M. T., Rice, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+regulatory+relationship+between+Tbx1+and+FGF+signaling+during+tooth+morphogenesis+and+ameloblast+lineage+determination.">A regulatory relationship between Tbx1 and FGF signaling during tooth morphogenesis and ameloblast lineage determination.</a> Developmental biology. 320, 39-48 (2008).</li><li>Diep, L., Matalova, E., Mitsiadis, T. A., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+tooth+bud+mesenchyme+to+alveolar+bone.">Contribution of the tooth bud mesenchyme to alveolar bone.</a> Journal of experimental zoology. Part B, Molecular. 312B, 510-517 (2009).</li><li>Rothova, M., Feng, J., Sharpe, P. T., Peterkova, R., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+mesoderm+to+the+developing+dental+papilla.">Contribution of mesoderm to the developing dental papilla.</a> The International journal of developmental biology. 55, 59-64 (2011).</li><li>Buchtova, 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=Initiation+and+patterning+of+the+snake+dentition+are+dependent+on+Sonic+hedgehog+signaling.">Initiation and patterning of the snake dentition are dependent on Sonic hedgehog signaling.</a> Developmental biology. 319, 132-145 (2008).</li><li>Buchtova, M., Stembirek, J., Glocova, K., Matalova, E., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+regression+of+the+dental+lamina+underlies+the+development+of+diphyodont+dentitions.">Early regression of the dental lamina underlies the development of diphyodont dentitions.</a> Journal of dental research. 91, 491-498 (2012).</li><li>Cho, S. W., 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=The+primary+enamel+knot+determines+the+position+of+the+first+buccal+cusp+in+developing+mice+molars.">The primary enamel knot determines the position of the first buccal cusp in developing mice molars.</a> Differentiation; research in biological diversity. 75, 441-451 (2007).</li><li>Sakano, 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=Cell+dynamics+in+cervical+loop+epithelium+during+transition+from+crown+to+root:+implications+for+Hertwig's+epithelial+root+sheath+formation.">Cell dynamics in cervical loop epithelium during transition from crown to root: implications for Hertwig's epithelial root sheath formation.</a> Journal of periodontal research. , (2012).</li><li>Fisher, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+development+in+vitro+of+the+whole+and+halved+lower+molar+tooth+germ+of+the+mouse.">Morphological development in vitro of the whole and halved lower molar tooth germ of the mouse.</a> Archives of oral biology. 16, 1481-1496 (1971).</li><li>Coin, R., Schmitt, R., Lesot, H., Vonesch, J. L., Ruch, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+halved+embryonic+lower+first+mouse+molars:+correlation+with+the+distribution+pattern+of+non+dividing+IDE+cells,+the+putative+organizers+of+morphogenetic+units,+the+cusps.">Regeneration of halved embryonic lower first mouse molars: correlation with the distribution pattern of non dividing IDE cells, the putative organizers of morphogenetic units, the cusps.</a> The International journal of developmental biology. 44, 289-295 (2000).</li><li>Kavanagh, K. D., Evans, A. R., Jernvall, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+evolutionary+patterns+of+mammalian+teeth+from+development.">Predicting evolutionary patterns of mammalian teeth from development.</a> Nature. 449, 427-432 (2007).</li><li>Munne, P. M., Tummers, M., Jarvinen, E., Thesleff, I., Jernvall, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tinkering+with+the+inductive+mesenchyme:+Sostdc1+uncovers+the+role+of+dental+mesenchyme+in+limiting+tooth+induction.">Tinkering with the inductive mesenchyme: Sostdc1 uncovers the role of dental mesenchyme in limiting tooth induction.</a> Development. 136, 393-402 (2009).</li></ol>

著者は、彼らが競合する経済的利益を持っていないことを宣言します。

歯の文化のこの方法は、歯胚へのアクセスの利点は、上皮または間葉内のビーズの正確な系譜トレースと配置を可能にする優れています。発展途上歯胚の決定された領域は、したがって、特に標的とすることができる。培養中に歯胚の変化形態を続けることができ、かつ操作の効果はすぐに評価した。 これらは正確に細断することができないような方法は、しかし、そ?...

多くの実験のためには、培養組織ex vivoでの開発に従うことができることが重要である。組織を発症する培養物は、開発の定義された周期でアクセスを提供し、培養培地への因子の添加による遺伝子の操作、または装填ビーズ上、およびトランスフェクションおよびエレクトロポレーション1を用いて可能にする。多くの実験のために、それが成長するにつれて、組織は、形態形成を受けるように系統標識細胞の運命を追跡するために、例えば、組織を可視化できることが重要である。これは、胚からの組織のブロックを培養するときに明らかにされていません胚の中の深い開発組織に特に問題となり得る。彼らは下顎、上顎骨および前頭鼻プロセス内で開発するように歯が、この良い例です。全体顎骨を培養したときに歯の表面的な構造が見ることができますが、形態の変化は、固定された組織の切片化した後に分析することができます<s&gt; 2まで。私たちは、私たちが歯胚の発達に従うことを可能にし、開発中に歯の様々な部分へのアクセスを提供するライブスライス培養技術を適応している。修正されたTrowellの方法3を使用して気液界面での技術培養歯胚スライス、。これらのスライス培養を直接歯の形態形成に続いて、このようなエナメルの結び目と歯乳頭や毛包1,4-7などの個別のコンポーネントの系譜トレースを可能に非常に有用であった。技術は、マウス胚に限定されず、正常ブタの培養生のスライスとヘビ歯組織8,9に用いられてきた。歯の発生を可視化することができるという利点に加えて、スライス方法はまた、組織の薄いスライスをインキュベーターから媒体と空気から栄養分へのアクセスが増加しているという利点を有する。これが改善され、生体内での開発と一致する歯胚の成長、およびshow invasになり乳頭7への内皮細胞のイオン。これとは対照的に、全体の下顎の文化で歯胚は、 生体内でのものよりも遅く発展と文化の中心地は、多くの場合、長期培養中に壊死性である。スライス培養において、歯は、顎のスライス内で発達し、周囲の現像骨および他の組織との相互作用を監視することができる。本手法では、組織は、直線支持媒体10,11に埋め込 ​​む必要はありませんし、チョッピングブロックに組織を取り付けるための任意のシステムを必要とせず、解剖後に切り刻まれている。方法は、多くの下顎骨が1セッションで区分けすることができるように、そのための非侵襲的かつ迅速である。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>101077Y</td>
			<td>100% ethanol was diluted in distilled H2O to 70%.</td>
		</tr>
		<tr>
			<td>DMEM F12</td>
			<td>Gibco</td>
			<td>12634-010</td>
			<td>Advanced Dulbecco&#39;s Modified Eagle Medium F12</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Sigma</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>DiI (Molecular probes)</td>
			<td>Vybrant</td>
			<td>V-22885</td>
			<td>Cell-labeling solution</td>
		</tr>
		<tr>
			<td></td>
			<td>Invitrogen</td>
			<td></td>
			<td>Cell tracker CM-DiI, C-7000</td>
		</tr>
		<tr>
			<td>DiO (Molecular probes)</td>
			<td>Vybrant</td>
			<td>V22886</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Geminator 500</td>
			<td>Thomas</td>
			<td>Thomas No. 3885A20</td>
			<td>Dry-heat sterilization</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>McIlwain tissue chopper</td>
			<td>Ted Pella, Inc.</td>
			<td>10180</td>
			<td>Standard table</td>
		</tr>
		<tr>
			<td>Organ culture dish (Center-Well Organ Culture Dish)</td>
			<td>Falcon</td>
			<td>353037</td>
			<td></td>
		</tr>
		<tr>
			<td>Membranes (Cell Culture Inserts, 0.4 &#956;m pore size)</td>
			<td>BD Falcon</td>
			<td>353090</td>
			<td>PET track-etched membrane, 6-well format</td>
		</tr>
		<tr>
			<td>Metal grids (Stainless Steel - AISI 304 - Mesh)</td>
			<td>Goodfellow</td>
			<td>FE228710</td>
			<td>(Fe/Cr18/Ni10)</td>
		</tr>
		<tr>
			<td>AutoFlow Direct Heat CO2 Incubator</td>
			<td>Nuaire</td>
			<td>NU-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Picospritzer III</td>
			<td>Intracel Ltd</td>
			<td>051-0500-900 0-100 psi</td>
			<td>Single channel picospritzer III</td>
		</tr>
		<tr>
			<td>Glass capillary with filament 1 mm</td>
			<td>WPI</td>
			<td>TW100F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten wire 0.1 mm</td>
			<td>Goodfellow</td>
			<td>W005138</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten wire 0.38 mm</td>
			<td>Goodfellow</td>
			<td>W005155</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirator tubes</td>
			<td>Sigma</td>
			<td>A5177</td>
			<td>Used for mouth aspiration lineage tracers</td>
		</tr>
	</tbody>
</table>

the slice culture method for following development of tooth germs in explant culture

外植片培養物は、特定の時点で現像器官の操作を可能にし、従って、発生生物学者のための重要な方法である。多くの器官のために、ex vivoで培養中に監視できるように、発達中の組織にアクセスすることは困難である。形態形成運動に従うことができ、特定の細胞集団が、系統操作またはトレースの対象とすることができるようにスライス培養法は、組織へのアクセスを可能にする。 本論文では、種の範囲の歯胚の培養に非常に成功しているスライス培養する方法を説明します。この方法は、他の顎の組織に囲まれた、生体内で観察されたものと同様の速度にて開発歯胚への優れたアクセスを提供します。これは、歯と周囲組織との間の組織の相互作用を監視することを可能にする。本論文では、歯胚に集中していますが、同じプロトコルは数値の発展をたどるために適用することができますこのような唾液腺、メッケル軟骨、鼻腺、舌、耳などの他の臓器の。

第一大臼歯歯小嚢の動きに追従するためには、250μmの正面切片を上記の方法を用いて下顎を介して採取した。歯小嚢は、発生中の歯の外側のエナメル上皮（OEE）を取り囲む間葉の層であり、先に6の歯周組織の形成に関与することが示されている。顎はE14.5、歯の発生のキャップ段階で解剖した。スライスにおける歯科上皮の輪郭が明らかになった、と凝縮歯科間充織は、歯上皮（ <str...

Watch this Scientific Journal Video about The Slice Culture Method for Following Development of Tooth Germs In Explant Culture at JoVE.com

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

ここでは、詳細な組織チョッパーを用いて下顎骨のスライスにおける培養歯胚へのメソッド。この方法は、より伝統的な培養法を用いて、利用できない、操作および系統の追跡のための素晴らしい機会を提供し、開発中に歯に固有のアクセスを許可します。

外植片培養での歯胚の開発に続いてのためのスライス培養法

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

the-slice-culture-method-for-following-development-tooth-germs

Research

JoVE Journal

Biology

14.0K ビュー.  King's College London. ここでは、詳細な組織チョッパーを用いて下顎骨のスライスにおける培養歯胚へのメソッド。この方法は、より伝統的な培養法を用いて、利用できない、操作および系統の追跡のための素晴らしい機会を提供し、開発中に歯に固有のアクセスを許可します。 

ビデオ: The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

E18ラット脳の器官型スライス培養

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

生物学

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.  

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

遺伝子銃の箇条書きとスライス培養における神経細胞の微粒子銃トランスフェクションの準備

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

初期のニワトリ胚をex vivo（新文化）の文化のための方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

神経毒性の高いコンテンツ分析のための神経とアストロサイト共培養アッセイ

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

ヒト胚性幹細胞の文化とメンテナンス

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

腱板断型ボトルの培養システムを使用してげっ歯類全胚培養の方法

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food.
Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out.
To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area.
We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.

We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.

人間の腸粘膜の外植片の文化と偏刺激する新規な方法

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

小説<em&gt;エクスビボ</emマウス胎児ハート&gt;文化法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

無血清全胚培養系で、マウス胚発生

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.