現在のプロジェクトは、概念資金のカンザス大学証明書によって賄われていた。我々 感謝したい看護スタッフ院長 (カンザスシティ、カンザス) で取得の蝸牛文化を支援するためひと臍帯やデビッド ・ ヨルゲンセンのことで私たちを支援のため。

カンザス大学医療センター (院長) で承認された機関動物ケアおよび使用委員会 (IACUC) のプロトコル (御急ぎでなかったら、#2014-2234) によると、動物の安楽死を含めたすべての処理を行った。
注: HWJCs は、インフォームド コンセントを提供される患者が寄贈したひと臍帯から分離した、標本 (区 IRB #15402) カンザス大学人間科目委員会によって承認されたプロトコルに従って使用されました。
1. 側頭骨収穫と蝸牛の分離
<ol>
	<li>外科はさみの鋭いペアで頭蓋骨と第一頚椎の間に切断によって適切に安楽死させた、15 週齢マウスの首をはねるし、(図 1 a) を消毒する 70% エタノールで頭をスプレーします。</li>
	<li>外科はさみ (図 1 bの-C破線) の同じシャープな対を用いた正中面で頭蓋骨を二等分します。</li>
	<li>鉗子 (図 1の矢印) のペアを使用して頭蓋骨から脳組織を削除します。</li>
	<li>存在の聴覚と前庭神経根 (図 1E-F矢印) 頭蓋骨と (図の矢印 1f) 外から外耳道の奥の頭骨を識別します。頭蓋骨 (図 1) の残りの部分から側頭骨を分離する頭蓋骨を慎重にカットします。 注: いくつかのインスタンス可能性があります可能である繊細な加工を必要とせず頭骨から頭蓋骨の周囲の骨を詮索します。</li>
	<li>ヒントは蝸牛に穴をあけるリスクはないので、外耳道 (図 1 H)、約 5 mm の開口部に微細鉗子を挿入します。優しくこじ開ける胞 (図 1 H、トップの赤斜線領域) の骨骨折 (1I を図、赤い点線線) ので。 注: 必要に応じて、解剖顕微鏡役立つ可能性がありますこのプロセスの正確な配置や楽器の操作を確保するため。</li>
	<li>微細鉗子を使用すると、(図 1 j, 赤いブラケット) 蝸牛の骨迷路の公開側頭骨からブラの残りの部分で詮索離れて。</li>
	<li>シャーレの頭骨を保持するために十分な大きさの場所 (例えば、30 mm 以上)、解離性の下にあるビューのフィールドを対象範囲し、満タンで十分なリン酸緩衝生理食塩水 (PBS) 側頭骨 (例えば1、0.5 mL) をカバーします。</li>
	<li>ブラ直面している蝸牛の楕円形や円形の窓が PBS で削除と側頭骨が水没します。 注: 前庭系を操作して定位 (1I を図と図 2 b) 蝸牛の機能のためのハンドルとしても優れていますと、蝸牛から前庭系の除去する、必要はありません。</li>
	<li>鉗子の超微細のペアを使用して、あぶみ骨を通過するアブミ骨動脈を削除します。</li>
	<li>動脈渡される、アブミ骨のアーチを超微細鉗子の先端を挿入し、繊細な上向きあぶみ骨を持ち上げます。縫合のアブミ骨は卵円窓から持ち上げる必要があります。</li>
	<li>超微細鉗子の先端を使用して、楕円の穴をあけるし、windows のラウンドします。</li>
	<li>28.5 ゲージ注射器とチューブを充填 1 × PBS を使用する (内側直径 0.28 mm、外径 0.61 mm) 管の楕円形の窓を置きます、接続されている (図 2 a -B)。 注: 管の開口部は、正確な配置を確保するため支援超微細鉗子を使用して操作できます。<ol>
		<li>各蝸牛に新鮮な注射器とチューブを使用します。</li>
	</ol>
	</li>
	<li>2 mL の 10% 抗生物質抗真菌薬 (対策) と 10% ペニシリン-ストレプトマイシン (ペン-連鎖球菌) PBS で蝸牛の 5 分以上を外リンパを削除するを灌流します。あまりにも多くの圧力で速すぎる灌蝸牛内微細構造に損傷を与えます。 注: 蝸牛を介して流体を灌流して手で注射器を手動で運転が効果的な灌流ポンプを代替として使用できます。<ol>
		<li>灌手動で場合は、側頭骨の前庭部分をつかんで灌蝸牛 (図 2 a) を安定させるために鉗子を自己終了の罰金ペアを使用します。 注意: 必須ではありませんが、これは両手を自由に楽器を処理を残します1 つの手はチューブを配置できる他、注射器 (図 2 a) を運転できる適切に (図 2 b)。</li>
		<li>このステップから転送、空気に不必要に蝸牛を公開しないように注意してください。流体の循環をブロック流体の空間に空気の泡を導入、蝸牛内の微細損傷さらに乾燥します。</li>
	</ol>
	</li>
	<li>削除し、残った微細鉗子を用いた筋肉組織や骨フラグメントを破棄します。</li>
	<li>必要に応じて、抗生物質溶液ごと 48 時間の定期的な変更で 7 日まで 10% 対策で PBS で 10% ペン連鎖球菌性ソリューション 4 ° C で分離の蝸牛を格納します。</li>
</ol>
2. 蝸牛処理
<ol>
	<li>Decellularization
	<ol>
		<li>各蝸牛の塗りつぶし 1% ナトリウム ドデシルと 20 mL ガラス シンチレーション バイアルの硫酸塩 (SDS) 水溶液のイオン (DI) の水があり、蝸牛を decellularize するために使用。</li>
		<li>開口部は十分な大きさを通過する蝸牛のかみそりの刃を使用してプラスチック製の 7 mL 転送ピペットの先端をカットします。</li>
		<li>転送ピペットを使用して、ゆっくりと引いて、蝸牛ピペットにのでそれは数ミリでカットオフ端を渡します。</li>
		<li>1 %sds ソリューションいっぱいシンチレーション バイアルに蝸牛を追放します。</li>
		<li>転送ピペットを用いたシンチレーション バイアルに蝸牛 (DI) 脱イオン水に 1 %sds の 2 mL を循環します。</li>
		<li>、ローテーターにシンチレーション バイアルを置き、部屋の温度で 72 時間 10 rpm で回転するシンチレーション バイアルを許可します。<ol>
			<li>72 時間の期間にわたって、1 %sds ソリューションすべての 24 h を変更します。決して空気に蝸牛を公開する SDS を変更するとき注意してください。</li>
		</ol>
		</li>
		<li>3 回の DI 水で各 30 分の蝸牛を洗います。</li></ol></li></ol>SDS 料金; 同じシンチレーション バイアルの洗浄を行うこの段階では、蝸牛が脱です。
	
	
	<li>脱灰
	<ol>
		<li>蝸牛を維持するのに十分なだけを残して水没、シンチレーション バイアルから残りの DI 水を削除します。</li>
		<li>脱灰を開始するには、蝸牛を含むシンチレーション バイアルに DI 水で 10% エチレンジアミン四酢酸 (EDTA) (0.02 M) 5 mL を追加します。</li>
		<li>転送ピペットを用いたシンチレーション バイアルに蝸牛を純水で 10 %edta の 2 mL を循環します。</li>
		<li>回転子に戻るシンチレーション バイアルを置き、部屋の温度で 72 時間 10 rpm で回転するシンチレーション バイアルを許可します。72 時間の期間にわたって 10 %edta 溶液のすべての 24 h を変更します。蝸牛が空気にさらされないようにソリューションを変更するときに注意します。</li>
		<li>すすぎ 3 回 1 × PBS です。 で、2 h の蝸牛この段階では、蝸牛が脱灰します。</li>
	</ol>
	</li>
	<li>ストレージ
	<ol>
		<li>72 h までの蝸牛溶液中で保存、10%、反反 10% ペン連鎖球菌 PBS で 4 ° C で 注: ストレージ期間を長く可能性があります抗生物質溶液; の定期的な変更が可能ただし、加工の蝸牛の拡張記憶域は、このプロトコルでは検証されていません。</li>
	</ol>
	</li>

3. 調達と hWJCs の拡大
<ol>
	<li>以前に発行されたプロトコル22によると hWJCs を分離します。簡単に言えば、3 cm のセクションにへその緒を分割します。</li>
	<li>慎重には、メスで浅い切開縦各臍帯のセグメントを広げるし、臍帯ワルトンを公開します。血管を削除するのに鉗子を使用します。</li>
	<li>組織が水没する (例えば、40 mL で 60 mm のペトリ皿) 十分な PBS で 2 回臍帯セグメントを洗います。メスで組織のセグメントを細かくみじん切り。100 mm ペトリ皿消化中の 50 mL の組織を消化 (0.2 %2 型コラゲナーゼ、低グルコース環境における 1% ペニシリン-ストレプトマイシン、ダルベッコ変更されたワシの媒体 (DMEM))。</li>
	<li>5% CO2、37 ° C のインキュベーターで一晩 50 回転軌道シェーカーの中の消化に組織を配置します。次の日、1:16 pbs は、抗生物質抗真菌 2% の割合で消化中を希釈し、室温で 500 × g で遠心分離によって細胞をペレット (~ 27 ° C) 10 分破棄上清の。</li>
	<li>細胞と間葉系幹細胞成長培地 (MSCGM) にペレットを再懸濁します。プレート細胞培養の 7 x 103セル/cm2の密度では、T-75 フラスコを扱われます。文化の MSCGM、HWJCs、通路 5 実験用に展開します。 注: HWJCs がサブ培養される大きい T フラスコに (例えば、T-150、T-300) 継 hWJCs 実験のための収穫を増加するとき。</li>
</ol>
4. 脱蝸牛への hWJCs の注入
<ol>
	<li>脱の蝸牛を準備します。
	<ol>
		<li>無菌技術を使用して、3 回の 30 分の 1 × PBS でストアドの蝸牛を洗浄します。</li>
		<li>24 ウェル プレートの別の井戸に蝸牛を転送し、37 ° C の 1 つの mL を追加 MSCGM で加温します。</li>
		<li>蝸牛 5% CO2セルの注入の前に 1 時間の最小値の 37 ° C 細胞文化インキュベーターで孵化させなさい。</li>
	</ol>
	</li>
	<li>HWJCs を切り離して、再懸濁します
	<ol>
		<li>37 ° C と洗浄 hWJCs 前回 1x PBS を温めた。</li>
		<li>十分な 37 ° C を追加セル容器表面に 0.5 %edta でトリプシンで加温します。</li>
		<li>HWJCs 5% CO2、37 ° C 細胞文化のインキュベーターで最大 5 分間インキュベートします。<ol>
			<li>細胞の 90% が倒立顕微鏡の下のセルを表示することによって表層文化から切り離されることを確認します。 注: 移動文化容器を軽くタップすると、細胞をデタッチします。静止したとき、細胞が分離されていません。</li>
			<li>さらに添付された細胞を取り除くためフラスコの側面が軽きます。</li>
		</ol>
		</li>
		<li>無菌技術を使用して、トリプシンのボリュームに MSCGM の同等のボリュームを含む 50 mL の円錐管に培養器から転送 hWJCs はセルを分離するに使用されます。</li>
		<li>常温 500 × g で遠心分離によって、hWJCs をペレット (~ 27 ° C) 5 分。</li>
		<li>上清を吸引し、500,000 細胞/ml の濃度で MSCGM で hWJCs を再懸濁します。</li>
	</ol>
	</li>
	<li>蝸牛を灌流します。
	<ol>
		<li>転送ピペットを使用して新しい 24 ウェル プレートに蝸牛を転送します。</li>
		<li>任意の蝸牛が乾燥するを防ぐために各の蝸牛に MSCGM のドロップを追加します。</li>
		<li>繊細な楕円形や円形の窓を向いているように超微細鉗子を使用して蝸牛に合わせます。 注: は、際は蝸牛が簡単に破損している各蝸牛を直接取り扱いに細心の注意を取る。</li>
		<li>接続チューブ、滅菌 28.5 ゲージ インスリン注射器の使用を再停止される 0.2 mL hWJCs (100,000 細胞) を描画します。</li>
		<li>滅菌微細鉗子を使用して、楕円形の窓にチューブを置きます。</li>
		<li>繊細かつゆっくり 0.2 ml ポリエチレン チューブに接続されている 28.5 ゲージ インスリン注射器を使用して再懸濁の hWJCs の蝸牛を灌流 (外径: 0.61 mm、内径: 0.28 mm) で約 5 分。</li>
		<li>後、0.8 mL 37 ° C の追加も 1 mL に井戸の総容積をもたらすに蝸牛を含む、MSCGM で加温します。</li>
		<li>各蝸牛、新鮮な注射器を使用して、各時間をチューブの 4.3.3-4.3.7 の手順を繰り返します。</li>
		<li>5% CO2、37 ° C 細胞文化のインキュベーターで灌流の蝸牛を置き、週 3 回のメディアを変更します。</li>
	</ol>
	</li>
</ol>
5. 蝸牛収穫と保存
注: 蝸牛培養があります、任意の時点で収穫まで 30 日後灌流。
<ol>
	<li>蝸牛を保持するには、ロッキング プラットフォーム上の 4 ° C で一晩 1x PBS で 4% パラホルムアルデヒドで修正します。</li>
	<li>固定後、3 回各 5 分の 1 × PBS と蝸牛を洗います。</li>
	<li>徐々 に脱水エタノールと蝸牛とのパラフィン包埋する前に脂肪族炭化水素溶剤でクリアします。</li>
	<li>セクションは、ミクロトームを使用して 10 μ m の厚さにサンプル、ガラス顕微鏡スライドをマウントします。 注: サンプルはする準備が整いました組織学的や免疫組織化学的処理の標準プロトコルを使用して。</li>
</ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+ear+tissues+as+scaffolds+for+stem+cell+differentiation.">Decellularized ear tissues as scaffolds for stem cell differentiation.</a> J Assoc Res Otolaryngol. 14 (1), 3-15 (2013).</li><li>Davies, D., Holley, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+alpha+3+and+alpha+6+integrins+in+the+developing+mouse+inner+ear.">Differential expression of alpha 3 and alpha 6 integrins in the developing mouse inner ear.</a> J Comp Neurol. 445 (2), 122-132 (2002).</li><li>Gerchman, E., Hilfer, S. R., Brown, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+extracellular+matrix+in+the+formation+of+the+inner+ear.">Involvement of extracellular matrix in the formation of the inner ear.</a> Dev Dyn. 202 (4), 421-432 (1995).</li><li>Goodyear, R. J., Richardson, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrices+associated+with+the+apical+surfaces+of+sensory+epithelia+in+the+inner+ear:+molecular+and+structural+diversity.">Extracellular matrices associated with the apical surfaces of sensory epithelia in the inner ear: molecular and structural diversity.</a> J Neurobiol. 53 (2), 212-227 (2002).</li><li>Mellott, A. J., 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=Improving+Viability+and+Transfection+Efficiency+with+Human+Umbilical+Cord+Wharton's+Jelly+Cells+Through+Use+of+a+ROCK+Inhibitor.">Improving Viability and Transfection Efficiency with Human Umbilical Cord Wharton's Jelly Cells Through Use of a ROCK Inhibitor.</a> Cell Reprogram. , (2014).</li><li>Mellott, A. J., Shinogle, H. E., Moore, D. S., Detamore, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Photo-conversion:+A+second+chance+to+label+unique+cells.">Fluorescent Photo-conversion: A second chance to label unique cells.</a> Cell Mol Bioeng. 8 (1), 187-196 (2015).</li><li>Mitchell, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+cells+from+Wharton's+jelly+form+neurons+and+glia.">Matrix cells from Wharton's jelly form neurons and glia.</a> Stem Cells. 21 (1), 50-60 (2003).</li><li>Mellott, A. J., 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=Nonviral+Reprogramming+of+Human+Wharton's+Jelly+Cells+Reveals+Differences+Between+ATOH1+Homologues.">Nonviral Reprogramming of Human Wharton's Jelly Cells Reveals Differences Between ATOH1 Homologues.</a> Tissue Eng Part A. 21 (11-12), 1795-1809 (2015).</li><li>Buytaert, J. A., Johnson, S. B., Dierick, M., Salih, W. H., Santi, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+versus+sTSLIM+3D+imaging+of+the+mouse+cochlea.">MicroCT versus sTSLIM 3D imaging of the mouse cochlea.</a> J Histochem Cytochem. 61 (5), 382-395 (2013).</li><li>Nonoyama, H., 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=Investigation+of+the+ototoxicity+of+gadoteridol+(ProHance)+and+gadodiamide+(Omniscan)+in+mice.">Investigation of the ototoxicity of gadoteridol (ProHance) and gadodiamide (Omniscan) in mice.</a> Acta Otolaryngol. 136 (11), 1091-1096 (2016).</li><li>Dai, C., 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=Rhesus+Cochlear+and+Vestibular+Functions+Are+Preserved+After+Inner+Ear+Injection+of+Saline+Volume+Sufficient+for+Gene+Therapy+Delivery.">Rhesus Cochlear and Vestibular Functions Are Preserved After Inner Ear Injection of Saline Volume Sufficient for Gene Therapy Delivery.</a> J Assoc Res Otolaryngol. , (2017).</li><li>Sutherland, A. J., Converse, G. L., Hopkins, R. A., Detamore, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bioactivity+of+cartilage+extracellular+matrix+in+articular+cartilage+regeneration.">The bioactivity of cartilage extracellular matrix in articular cartilage regeneration.</a> Adv Healthc Mater. 4 (1), 29-39 (2015).</li></ol>

著者が明らかに何もありません。

我々 は正常に蝸牛の複雑な三次元組織足場としての使用を可能にする decellularization プロセスを介して蝸牛からネイティブ蝸牛細胞を削除できることを実証しました。サンティら。15 decellularizing の蝸牛の最初のメソッドを開発し、光シート顕微鏡23の助けを借りてを通じて多くの人工内耳構造のボリュームを正確に推定しています。このような初期?...

蝸牛は、側頭骨は、複雑なスパイラル構造です。外側の骨迷路と、同心の内側の膜迷路1から成り立っています。膜迷路は、流体の 3 つのスペースで構成されています: Scala スカラー座 vestibuli メディア、およびスカラー座索1。Scala メディア住宅感覚の上皮細胞の種類の多数で構成されているが、感覚有毛細胞 (HC)、神経インパルス2音波の機械的エネルギーを変換、特に重要です。音響外傷3,4、5薬6疾患7,8、および高齢化9への暴露は、すべての HC の死を介して聴覚機能障害で起因できます。哺乳類の毛細胞損失は傷害10後再生することができます鳥の HCs とは異なり、永続的なです。
様々 な現代的な研究努力を失われた HCs を復元する特定の実験的アプローチは異なるが求めています。感覚上皮における遺伝子発現の操作と体の外分化した幹細胞の注入、支配的なアプローチにおいて、蝸牛オルガノイドの幹細胞の分化を誘導方法がされているが11,12,13を試みた。それぞれのアプローチは、直接幹細胞や幹細胞の発達の手がかりに依存ただし、2 番目は共有で、潜在的に重要な要素はそれ自体蝸牛の ECM14,15。
ECM だけでなく細胞と細胞の接着、増殖、生存、および移行の面を含んでいますが、また HCs やらせん神経節報15,16 の開発に重要な役割を果たしている組織の物理的なサポートを提供します ,17。自然発生する ECM は、細胞の表現型決定および/または細胞の接着・増殖・生存の18のガイドが誘導信号を提供します。その結果、脱蝸牛培養 hWJCs との組み合わせでの使用は、ECM と HC の再生の役割を探検するユニークな機会を提供しています。HWJCs は、間葉系幹細胞の19のように振る舞うひと臍帯由来容易に利用できる、非論争の携帯タイプです。HWJCs は、麻痺細胞系統20,21を区別する能力を示しています。したがって、現在のプロトコル詳細分離、decellularization、および内耳組織工学用 hWJCs と C57BL マウスの死骸から蝸牛の血流を示します。

<table><tbody><tr><td>Allegra X-14R Centrifuge</td><td>Beckman-Coulter</td><td>B08861</td><td></td></tr><tr><td>Intramedic Semi-Rigid Tubing</td><td>Becton Dickinson</td><td>427401</td><td></td></tr><tr><td>New Brunswick Innova 2000 Orbital Shaler</td><td>Eppendorf</td><td>M1190-0002</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fine Science Tools</td><td>14060-10</td><td></td></tr><tr><td>Fine Forceps</td><td>Fine Science Tools</td><td>11370-40</td><td></td></tr><tr><td>Ultra-Fine Forceps</td><td>Fine Science Tools</td><td>18155-13</td><td></td></tr><tr><td>50-mL Conical Tubes</td><td>Fisher Scientific</td><td>12565271</td><td></td></tr><tr><td>Petri Dish</td><td>Fisher Scientific</td><td>FB087579B</td><td></td></tr><tr><td>U-100 Insulin Syringe</td><td>Fisher Scientific</td><td>14-829-1B</td><td></td></tr><tr><td>Scintillation Vial</td><td>Fisher Scientific</td><td>03-341-73</td><td></td></tr><tr><td>Rotator</td><td>Fisher Scientific</td><td>88-861-049</td><td></td></tr><tr><td>Transfer Pipette</td><td>Fisher Scientific</td><td>22-170-404</td><td></td></tr><tr><td>Razor Blade</td><td>Fisher Scientific</td><td>12-640</td><td></td></tr><tr><td>Antibiotic-Antimycotic</td><td>Fisher Scientific</td><td>15-240-062</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Fisher Scientific</td><td>15-140-122</td><td></td></tr><tr><td>24-Well Plate</td><td>Fisher Scientific</td><td>07-200-84</td><td></td></tr><tr><td>SuperFrost PLUS Glass Microscope Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Transfer Pipette</td><td>Fisher Scientific</td><td>22-170-404</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant with DAPI</td><td>Fisher Scientific</td><td>P36935</td><td></td></tr><tr><td>Clear-Rite 3</td><td>Fisher Scientific</td><td>22-046341</td><td></td></tr><tr><td>Thermo Scientific Forma Series II 3110 Water-Jacekted CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Fisher Scientific</td><td>13-998-078</td><td></td></tr><tr><td>Mesenchymal Stem Cell Growth Medium</td><td>Lonza</td><td>PT-3001</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Lonza</td><td>CC-3232</td><td></td></tr><tr><td>TPP T-75 Culture Flask</td><td>MidSci</td><td>TP90076</td><td></td></tr><tr><td>TPP T-150 Culture Flask</td><td>MidSci</td><td>TP90151</td><td></td></tr><tr><td>TPP T-300 Culture Flask</td><td>MidSci</td><td>TP90301</td><td></td></tr><tr><td>Dissection Microscope</td><td>Nikon Instruments</td><td>SMZ800</td><td></td></tr><tr><td>Nikon Eclipse Ts2R-FL Inverted Microscope</td><td>Nikon Instruments</td><td>MFA51010</td><td></td></tr><tr><td>NuAire Class II, Type A2 Biosafety Cabinet</td><td>NuAire</td><td>NU-425-600</td><td></td></tr><tr><td>1X PBS</td><td>Sigma-Aldrich</td><td>P5368-10PAK</td><td></td></tr><tr><td>1% SDS Solution</td><td>Sigma-Aldrich</td><td>436143-100G</td><td></td></tr><tr><td>10% EDTA</td><td>Sigma-Aldrich</td><td>E9884-100G</td><td></td></tr></tbody></table>

a protocol for decellularizing mouse cochleae for inner ear tissue engineering

哺乳類のヒアリング不足を再生する能力を促進する機械刺激受容の有毛細胞では、難聴の治療法を限られています。現在の再生医療戦略は、移植幹細胞や内耳難聴を修正する損傷した幹細胞の交換を奨励するためにサポート細胞を周囲の遺伝子操作に焦点を当てています。しかし、細胞外マトリックス (ECM) は誘導と有毛細胞の機能を維持する重要な役割を果たすこと、十分に調査されていません。人工内耳を使用して成体幹細胞を成長する足場として ECM 方法、組成と細胞外の環境のアーキテクチャにエイズ聴覚機能の維持の細胞に独特な洞察力があります。分離し、成体幹細胞灌流を受け入れる足場として使用するマウスからの蝸牛を decellularizing する方法をご紹介します。現在のプロトコルの蝸牛は安楽死させたマウス、脱と脱灰から分離されます。その後、臍帯から隔離された人間の Wharton のゼリー細胞 (hWJCs) は、各蝸牛に慎重に灌。バイオリアクター, として、蝸牛を用い、解析の処理を受ける前に 30 日間培養します。脱の蝸牛は個人の細胞外構造を保持が、細胞の存在や DNA の顕著なフラグメントを明らかにしなかった。蝸牛に灌流セルは蝸牛の内外面のほとんどを侵略し、30 日間の期間にわたって問題なく育った。したがって、どのように人工内耳の ECM の影響細胞発達や行動を研究する現在のメソッドを使用することができます。

ここに提示されたメソッドを使用すると、蝸牛の成功した decellularization は、4' を介して DNA の存在有無を調べることで評価された 6-diamidino-2-phenylindole (DAPI) 染色します。Decellularized 蝸牛内 DNA が識別されなかった場合、蝸牛を完全に decellularized と考えられていた。Decellularization または脱灰を受けていない以前の実験からネイティブ蝸牛は、構造と伝統的 C57BL マウス蝸?...

Watch this Scientific Journal Video about A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering at JoVE.com

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

内耳組織工学用マウス蝸牛を Decellularizing するためのプロトコル

このプロトコルの目的は、decellularize し、めしマウス蝸牛組織工学用の足場として利用する効果的な方法を示すことです。

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

a-protocol-for-decellularizing-mouse-cochleae-for-inner-ear-tissue

Research

JoVE Journal

Bioengineering

10.7K ビュー.  University of Kansas Medical Center. このプロトコルの目的は、decellularize し、めしマウス蝸牛組織工学用の足場として利用する効果的な方法を示すことです。

ビデオ: A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

エレクトロポレーションによりマウス胎児蝸牛外植の文化と遺伝子導入

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

発生生物学

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

マウス蝸牛外植片の長期的タイムラプスイメージング

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

神経科学

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

蝸牛細胞の変性を誘導するための迅速かつ信頼性の高いテクニック：耳毒性剤送達のためのマウスラウンドウィンドウアプローチ

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

医学

Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest signaling pathways shown to be associated with ciliary function was the non-canonical Wnt signaling pathway, also referred to as planar cell polarity (PCP) signaling. One of the best places in which to study the effects of planar cell polarity (PCP) signaling during vertebrate development is the mammalian cochlea. PCP signaling disruption in the mouse cochlea disrupts cochlear outgrowth, cellular patterning and hair cell orientation, all of which are affected by cilia dysfunction. The goal of this protocol is to describe the analysis of PCP signaling in the developing mammalian cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy. Defects in convergence and extension are manifested as a shortening of the cochlear duct and/or changes in cellular patterning, which can be quantified following dissection from developing mouse mutants. Changes in stereociliary bundle orientation and kinocilia length or positioning can be observed and quantitated using either immunofluorescence or scanning electron microscopy (SEM). A deeper insight into the role of ciliary proteins in cellular signaling pathways and other biological phenomena is crucial for our understanding of cellular and developmental biology, as well as for the development of targeted treatment strategies.

Primary cilia influence various signaling pathways. The mammalian cochlea is ideal for examining planar cell polarity (PCP) signaling. Cilia dysfunction affects cochlear outgrowth, cellular patterning and hair cell orientation, readouts of PCP. Our goal is to analyze PCP signaling in mouse cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy.

繊毛病マウス変異蝸牛における平面セル・-極性表現型の評価

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest ...

Cryosectioning and Immunostaining Mouse Inner Ear Tissue: From Embryonic to Adult Stages

This protocol details general histology methods for preparing inner ear samples from embryonic, neonatal, and adult mice. The purpose of this protocol is to provide a straightforward and standardized method for inner ear tissue processing for researchers, possibly new to the field, interested in working with the mouse cochlea. Included here are protocols for dissection, fixation, embedding, cryosectioning, and immunostaining. Section immunostaining is one of the best methods for examining individual cells within the context of the entire cochlea.
Key steps include dissecting the inner ear away from the skull, fixing the tissue using 4% paraformaldehyde, embedding with Optimal Cutting Temperature compound, cryosectioning, and immunostaining with antibodies targeting specific proteins expressed by the cochlea.
Included in our methods are special considerations made for the cochlea given its 
shape and structure.&#160;Reasonably good focus and dissection skills are needed, as tissue damage can affect the quality of immunostaining and consistency among samples. However, with the procedures we outline, most users can be trained in a few weeks to make beautiful preparations. Overall, this protocol offers a valuable way to promote research to understand auditory development, function, and disease in mouse models.

This protocol, intended for novice users of mouse inner ear tissue, comprehensively details steps for processing mouse inner ears at different developmental stages for section immunostaining.

マウス内耳組織の凍結切片および免疫染色:胚期から成虫期まで

This protocol details general histology methods for preparing inner ear samples from embryonic, neonatal, and adult mice. The purpose of this protocol is ...

Dextran Labeling and Uptake in Live and Functional Murine Cochlear Hair Cells

The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET remain unknown. Electrophysiological studies of hair cells revealed that the MET channel has a large conductance and is permeable to relatively large fluorescent cationic molecules, including some styryl dyes and Texas Red-labeled aminoglycoside antibiotics. In this protocol, we describe a method to visualize and evaluate the uptake of fluorescent dextrans in hair cells of the organ of Corti explants that can be used to assay for functional MET channels. We found that 3 kDa Texas Red-labeled dextran specifically labels functional auditory hair cells after 1&#8211;2 h incubation. In particular, 3 kDa dextran labels the two shorter stereocilia rows and accumulates in the cell body in a diffuse pattern when functional MET channels are present. An additional vesicle-like pattern of labeling was observed in the cell body of hair cells and surrounding supporting cells. Our data suggest that 3 kDa Texas-Red dextran can be used to visualize and study two pathways for cellular dye uptake; a hair cell-specific entry route through functional MET channels and endocytosis, a pattern also available to larger dextran.

Here, we present a method for visualizing the uptake of 3 kDa Texas Red-labeled dextran in auditory hair cells with functional mechanotransduction channels. In addition, dextrans of 3&#8211;10 kDa can be used to study endocytosis in hair and supporting cells of the organ of Corti.

生きた機能性マウス人工内毛細胞におけるデキストランの標識と取り込み

The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET ...

生物工学

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

大人のマウスにおける人工内耳表面製剤

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

Whole Neonatal Cochlear Explants as an In vitro Model

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is characterized by the cumulative and irreversible loss of sensory hair cells and auditory nerves in the cochlea. Entire and vital cochlear explants are one of the fundamental tools in hearing research to detect hair cell loss and to characterize the molecular mechanisms of the inner ear cells. Many years ago, a protocol for neonatal cochlear isolation was developed, and although it has been modified over time, it still holds potential for improvement. 
This paper presents an optimized protocol for isolating and culturing whole neonatal cochlear explants in multi-well culture chambers that enables the study of hair cells and spiral ganglion neuron cells along the entire length of the cochlea. The protocol was tested using cochlear explants from mice and rats. Healthy cochlear explants were obtained to study the interaction between hair cells, spiral ganglion neuron cells, and the surrounding supporting cells. 
One of the main advantages of this method is that it simplifies the organ culture steps without compromising the quality of the explants. All three turns of the organ of Corti are attached to the bottom of the chamber, which facilitates in vitro experiments and the comprehensive analysis of the explants. We provide some examples of cochlear images from different experiments with live and fixed explants, demonstrating that the explants retain their structure despite exposure to ototoxic drugs. This optimized protocol can be widely used for the integrative analysis of the mammalian cochlea.

Whole Neonatal Cochlear Explants as an In vitro Model

The current protocol updates previous protocols and incorporates relatively straightforward approaches for culturing high-quality cochlear explants. This provides reliable data acquisition and high-resolution imaging in live and fixed cells. This protocol supports the ongoing trend of studying inner ear cells.

In vitroモデルとしての新生児蝸牛外植片全体

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is ...

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. The first procedure consists of perforation of the tympanic bulla, termed bullostomy; the second one is a transtympanic injection. Both emulate human clinical intratympanic procedures.
Chitosan-glycerophosphate (CGP) and Ringer&#180;s Lactate buffer (RL) were used as biocompatible vehicles for local drug delivery. CGP is a nontoxic biodegradable polymer widely used in pharmaceutical applications. It is a viscous liquid at RT&#160;but it congeals to a semi solid phase at body temperature. RL is an isotonic solution used for intravenous administrations in humans. A small volume of this vehicle is precisely placed on the Round Window (RW) niche by means of a bullostomy. A transtympanic injection fills the middle ear and allows less control but broader access to the inner ear.
The safety profiles of both techniques were studied and compared by using functional and morphological tests. Hearing was evaluated by registering the Auditory Brainstem Response (ABR) before and several times after microsurgery. The cytoarchitecture and preservation level of cochlear structures were studied by conventional histological techniques in paraformaldehyde-fixed and decalcified cochlear samples. In parallel, unfixed cochlear samples were taken and immediately frozen to analyze gene expression profiles of inflammatory markers by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR).
Both procedures are suitable as drug delivery methods into the mouse middle ear, although transtympanic injection proved to be less invasive compared to bullostomy.

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

Two microsurgery approaches for local drug delivery to the inner ear are described here and compared in terms of impact on hearing parameters, cochlear cytoarchitecture and expression of inflammatory markers.

マウス内耳に標的薬の配布方法の比較研究：Bullostomy<em&gt;対</em&gt; Transtympanicインジェクション

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. ...

生物学

Extracting the Cochlea from a Human Temporal Bone: A Cadaveric Protocol

The extraction of the cochlea from a cadaver human temporal bone may be required for different studies of the inner ear. For histological evaluations, the inner ear must be extracted from the temporal bone to facilitate histologic processing; likewise, some micro-computed tomography devices are too small to accommodate the complete temporal bones; additionally, the image quality can be enhanced when the cochlea is isolated.
The inner ear is located within the petrous part of the temporal bone. The inner ear can be divided into the osseous labyrinth or otic capsule and the membranous labyrinth inside the otic capsule. Furthermore, the inner ear can be divided into the vestibular system (the semicircular canals and the vestibule) and the cochlea. The appreciation of the location and orientation of the cochlea within the temporal bone is difficult, as it is embedded within bony structures and thus cannot be directly visualized. Nevertheless, there are distinct anatomical structures that can help guide the process to allow a reliable drill-out of the cochlea. The landmarks in the posterior parts of the cochlea are the facial nerve, semicircular canals, and the vestibule. In the middle, the inferior borders of the cochlea are identified by the round window and the basal turn of the cochlea. In the anterior border, one encounters the carotid artery; the landmark for the superior border is the genicular ganglion (GG) of the facial nerve. The medial structures are determined by the locations of the internal auditory canal, the superior semicircular canal, and the canal of the internal carotid artery.
In this article, we present a method for extracting the cochlea reliably out of the temporal bone by drill-out while following several anatomical landmarks.

<meta charset="utf8"></head><body>ヒトの側頭骨からの蝸牛の抽出:死体プロトコル

This article presents a reliable method for extracting the human cochlea from the cadaver temporal bone by drill-out while following distinct anatomical landmarks.

ヒトの側頭骨からの蝸牛の抽出:死体プロトコル

The extraction of the cochlea from a cadaver human temporal bone may be required for different studies of the inner ear. For histological evaluations, the ...