Name: an in vitro organ culture model of the murine intervertebral disc
Uploaded: 2017-04-11T14:00:00.000+00:00
Duration: 8 min 3 s
Description: Watch this Scientific Journal Video about An In Vitro Organ Culture Model of the Murine Intervertebral Disc at JoVE.com

この作品は、ワシントン大学の筋骨格系研究センター（NIHのP30のAR057235）、分子イメージングセンター（NIHのP50のCA094056）、メカノトレーニンググラント（NIH 5T32EB018266）、NIH R21AR069804、およびNIH K01AR069116によってサポートされていました。著者は、データ収集の彼の貢献のためのパトリック・ウォン感謝したいと思います。

全ての動物実験は、セントルイス動物実験委員会のワシントン大学を遵守して行われました。 1.動物<ol><li> 2系統のマウスを得る：10週齢のBALB / c（N = 6、BALB-M、BALB / cAnNTac）及び10週齢の核因子カッパBルシフェラーゼレポーター動物（NF-κβ-LUC）の上に飼育BALB / cバックグラウンド（N = 6、BALB / C-TG（RELA-LUC）31Xen）。 </li><li>解剖前に、時間を住居の追加の2分間、続いて5分間2.5-3リットル/分の流量でCO 2過剰投与して動物を安楽死させます。 </li><li>解剖前に2分間70％エタノール浴中に浸すことによって、動物の外側の領域を消毒します。 </li></ol> 2.解剖<ol><li>体腔を露出させるために小さな切開鋏を用いてマウスの背側表面に縦方向の垂直カットを行います。 </li><li>から始まる動物の背骨の両側に二つの長手方向の垂直カットを作ります尾椎骨（C8）への最初の腰椎（L1）。 </li><li>メス、細かい鉗子、細かい解剖ハサミを使用して、慎重に動物の体腔からL1-C8から背骨を取り除きます。 </li><li>こすり又は腹側のIVDを傷つけないように確保しながら慎重に、脊柱を取り囲む過剰軟組織を除去します。 </li><li>さらに、L1 / L2、L3 / L4、L5 / L6、C3 / C4、C5 / C6、およびC7 / C8ディスクにおける椎骨ディスク、脊椎の機能的脊椎ユニット（FSUs）に脊柱を解剖。 </li><li> 1％2分間ペニシリン - ストレプトマイシンを補充したハンクス平衡塩溶液中FSUsをすすぎます。 </li><li>無培養および未処理FSUs（フレッシュ）、培養されたが、未処理FSUs（コントロール）、および培養し、スタブ処理しFSUs（スタブ）：ランダムに三つのグループにFSUsを割り当てます。 </li><li>スナップは、直ちに-20℃の冷凍庫に解剖して格納した後、液体窒素で新鮮FSUサンプルを凍結します。 </li></ol> 3.器官培養条件<ol><li>無菌の培養培地の500ミリリットルを調製1：1ダルベッコ改変イーグル培地：栄養混合F12：20％ウシ胎児血清（FBS）および1％ペニシリン - ストレプトマイシンを補充した（DMEM F12）。 </li><li> 24ウェル培養プレートの各ウェルに、10mLの血清学的ピペット、ピペット培地の2ミリリットルを使用。 </li><li>解剖以下と媒体が充填された24ウェルプレートの個々のウェルに各FSUを配置し、すすぎます。 </li><li> 37°C、5％CO 2、20％O 2及び&gt; 90％の湿度を維持する滅菌インキュベーター中でサンプルをインキュベートします。 </li><li> 図1Aに示すように、24時間の初期培養期間後、機械的に無菌の27ゲージ針を使用して、線維輪の針穿刺を介してスタブ群サンプルを傷つけます。 </li><li>新しい使用滅菌ピンセットに古いプレートから新しい媒体が充填された24ウェル培養プレートを調製することにより、メディアごとに48時間を変更し、転送サンプル。古いメディアとdisposを吸引バイオハザード廃棄物ビンの古い培養プレートの電子。 </li><li>培養の最後の日に、24時間0.75 mg / mlとニトロブルーテトラゾリウムクロリドを含む培地中ですべてのサンプルをインキュベートします。 </li><li>その後、機械的試験または組織学的検査（ 図1B）の準備ができるまで-20℃のフリーザー中の液体窒素とストアとすべてFSUs凍結。 </li></ol> 4.縦測定NF-κB <ol><li> NF-κBの発現を評価するための1、5、13、および19日目NF-κβルシフェラーゼ動物からの画像FSUs。 </li><li>各ウェルに1mg / mlのルシフェリン溶液10μLを添加し、37℃で10分間滅菌したインキュベーター中でインキュベートします。 </li><li>画像1分間の露光時間と2のビンの設定と画像化システムを使用して生物発光用のサンプル。 </li><li>同時に、写真を撮るとIVDに発光の解剖学的位置を決定するために、生物発光画像とをオーバーレイする機械を使用します。 </li></ol><pクラス= &quot;jove_title&quot;&gt; 5。機械的な評価<ol><li>変位制御動的圧縮9を用いてたIVDの機械的挙動を決定します。 </li><li>解剖顕微鏡、メス、ピンセットを使用して、無傷の軟骨終板を維持しながら、各試料からFSUの骨の椎体を除去し、IVDに取り付けられました。 </li><li>シアノアクリレート接着剤を使用して1センチメートルX 1センチメートルX 0.2cmのアルミニウム板に単離されたIVDを取り付けます。 </li><li>レーザーマイクロメータを使用して、各ディスクの縦軸に3回の測定の平均を取ることによって、椎間板の高さと幅を測定します。最大ディスク幅で平均椎間板の高さを分割することにより椎間板の高さの比を計算します。 </li><li>機械的試験に使用される入力歪み値を計算するために測定された椎間板の高さを使用してください。平均的な椎間板の高さは約690ミクロン±39μmであったことに留意されたいです。 </li><li> compressiの下でリン酸緩衝生理食塩浴にサンプルを置き、マシン上と0.02 Nにディスクをプリロード</li><li>周期的に3つの試験それぞれ20 1％の歪みレベルでのサイクルおよび5％の歪みレベルに対して1 Hzでの正弦波形を使用してディスクを圧縮し、IVDの荷重と変位値を記録します。リラックスして、けがを防止するために、ディスクの臨床試験の間の時間を休憩の10分を許可します。 </li><li>最後のサイクルのローディング相から平均剛性を計算し、荷重と変位データとの間の位相角の正接を計算します。 </li></ol> 6.プロテオグリカンとコラーゲンの定量化<ol><li>機械的試験に続いて、プレ風袋遠心管に、単離されたIVDを配置し、化学天秤を用いて重量を測定することにより、ディスク湿重量を測定します。 </li><li>ブロックヒーターを用いて65℃で一晩250μLで単離されたIVDパパイン消化溶液（0.1 M酢酸ナトリウム、0.01 M EDTA、0.005 MシステインHClを、pHが6.4）を消化します。 </li><li> 10分間の遠心分離サンプル2,000×gで、上澄み液を集めます。 </li><li>コンドロイチン硫酸標準とジメチルメチレンブルー（DMMB）アッセイを使用して、IVDのプロテオグリカン含有量を測定します。 </li><li> DMMB溶液（21 mgのDMMB、5mLの無水エタノールを、1 LのddH 2 O中に2gのギ酸ナトリウム）を準備。 </li><li>各ウェル中のDMMB溶液の250μLと一緒に96ウェルプレートに三重で標準および試料のピペット30μL、。 </li><li>分光光度計を用いて525 nmでプレートの吸光度を読みます。 </li><li>製造者の指示に従ってヒドロキシプロリンアッセイキットを使用してコラーゲン含有量を測定します。 </li></ol> 7.組織学<ol><li> 24時間、4％パラホルムアルデヒド中でサンプルのサブセットを修正し、48時間、5％ギ酸中で脱石灰化、エタノールで脱水し、パラフィンに埋め込みます。 </li><li>矢状10μmの厚さでセクション、サンプルをミクロトームを用いて、ガラススライドに切片を適用します。サフラニンO /ファストGrとを使用してセクションを染色EENおよびDAPI。 </li><li>光学顕微鏡を使用して、画像は10倍の倍率でスライドします。 </li></ol> 8.造影microComputed断層撮影（マイクロCT） <ol><li> 7日の時点で縦イオベルソールコントラスト増強10、及び末端リンモリブデン酸（PMA）、コントラスト強調を用いて0、2、5、及び7日の時点でサンプルのサブセットをスキャンします。 </li><li>前マイクロCTスキャン時間〜4時間、約50ミリグラム/ mLのヨウ素含有イオベルソール溶液の最終濃度のために培地に350 mg / mlとヨウ素イオベルソール溶液の300μLを添加し、4 37℃で滅菌インキュベーター中でインキュベート時間。 </li><li>インキュベーション後、1 mlのマイクロ遠心チューブに滅菌ガーゼと場所でサンプルを包みます。 </li><li>マイクロCTシステム内の場所のサンプルと45 keVp、177μA、10.5μmのボクセルサイズ、および300ミリ秒の積分でスキャンします。 </li><li>エクスポートDICOMファイルなどのマイクロCTからのデータやソフトウェアを使用して可視化します（ <em&gt;例えば、OsiriXの）。 </li><li> 7日の時点で、3日間、5％のPMA溶液、続いて24時間、4％パラホルムアルデヒド溶液を用いて試料を固定し、そして工程8.4で使用したものと同じ設定を使用してスキャンします。 </li></ol> 9.三次元有限要素モデリング<ol><li>セグメントマニュアルしきい値（ 例えば 、OsiriXの）でソフトウェアを使用したIVDのDICOMファイル。 </li><li> IVDのNPとAFボリュームはMeshlabを使用して、ボクセルベースの有限要素メッシュに変換します。 </li><li>完全なIVDを形成し、有限要素ソフトウェアで実験的に決定された境界条件を適用するためにNPとAFの微細構造を組み合わせます。 </li></ol>

<ol>
	<li>Dagenais, S., Caro, J., Haldeman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+of+low+back+pain+cost+of+illness+studies+in+the+United+States+and+internationally.">A systematic review of low back pain cost of illness studies in the United States and internationally.</a> Spine J. 8 (1), 8-20 (2008).</li><li>Urban, J., Roberts, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degeneration+of+the+intervertbral+disc.">Degeneration of the intervertbral disc.</a> Arthritis Res Ther. 5 (6), 1-48 (2003).</li><li>Mirza, S. K., White, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+intervertebral+disc+and+pathophysiology+of+herniated+disc+disease.">Anatomy of intervertebral disc and pathophysiology of herniated disc disease.</a> J Clin Laser Med Surg. 13 (3), 131-142 (1995).</li><li>Acaroglu, E. R., 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=Degeneration+and+aging+affect+the+tensile+behavior+of+human+lumbar+anulus+fibrosus.">Degeneration and aging affect the tensile behavior of human lumbar anulus fibrosus.</a> Spine. 20 (24), 2690-2701 (1995).</li><li>Abraham, A. C., Liu, J. W., Tang, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+changes+in+the+structure+and+inflammatory+response+of+the+intervertebral+disc+due+to+stab+injury+in+a+murine+organ+culture+model.">Longitudinal changes in the structure and inflammatory response of the intervertebral disc due to stab injury in a murine organ culture model.</a> J Orthop Res. 34 (8), 1431-1438 (2016).</li><li>Ohtori, S., Inoue, G., Miyagi, M., Takahashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathomechanisms+of+discogenic+low+back+pain+in+humans+and+animal+models.">Pathomechanisms of discogenic low back pain in humans and animal models.</a> Spine J. 15 (6), 1347-1355 (2015).</li><li>Pelle, D. 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=Genetic+and+functional+studies+of+the+intervertebral+disc:+A+novel+murine+intervertebral+disc+model.">Genetic and functional studies of the intervertebral disc: A novel murine intervertebral disc model.</a> PLoS ONE. 9 (12), (2014).</li><li>Zhang, 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=Time+course+investigation+of+intervertebral+disc+degeneration+produced+by+needle-stab+injury+of+the+rat+caudal+spine.">Time course investigation of intervertebral disc degeneration produced by needle-stab injury of the rat caudal spine.</a> J Neurosurg: Spine. 15 (4), 404-413 (2011).</li><li>Liu, J. W., Abraham, A. C., Tang, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+high-throughput+phenotyping+of+the+viscoelastic+behavior+of+whole+mouse+intervertebral+discs+using+a+novel+method+of+dynamic+mechanical+testing.">The high-throughput phenotyping of the viscoelastic behavior of whole mouse intervertebral discs using a novel method of dynamic mechanical testing.</a> J Biomech. 48 (10), 2189-2194 (2015).</li><li>Lin, K. H., Wu, Q., Leib, D. J., Tang, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+for+the+contrast-enhanced+microCT+imaging+of+murine+intervertebral+discs.">A novel technique for the contrast-enhanced microCT imaging of murine intervertebral discs.</a> J Mech Behav Biomed Mater. 63, (2016).</li><li>Pasparakis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+tissue+homeostasis+by+NF-kappaB+signalling:+implications+for+inflammatory+diseases.">Regulation of tissue homeostasis by NF-kappaB signalling: implications for inflammatory diseases.</a> Nat Rev: Immunol. 9 (11), 778-788 (2009).</li><li>O&#8217;Connell, G. D., Vresilovic, E. J., Elliott, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+animals+used+in+disc+research+to+human+lumbar+disc+geometry.">Comparison of animals used in disc research to human lumbar disc geometry.</a> Spine. 32 (3), 328-333 (2007).</li><li>Lee, C. R., 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=In+vitro+organ+culture+of+the+bovine+intervertebral+disc:+effects+of+vertebral+endplate+and+potential+for+mechanobiology+studies.">In vitro organ culture of the bovine intervertebral disc: effects of vertebral endplate and potential for mechanobiology studies.</a> Spine. 31, 515-522 (2006).</li><li>Chan, S. C., Gantenbein-Ritter, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Intact+Bovine+Tail+Intervertebral+Discs+for+Organ+Culture.">Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture.</a> J. Vis. Exp. (60), e3490 (2012).</li><li>Holguin, N., Aguilar, R., Harland, R. A., Bomar, B. A., Silva, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+aging+mouse+partially+models+the+aging+human+spine:+lumbar+and+coccygeal+disc+height,+composition,+mechanical+properties,+and+Wnt+signaling+in+young+and+old+mice.">The aging mouse partially models the aging human spine: lumbar and coccygeal disc height, composition, mechanical properties, and Wnt signaling in young and old mice.</a> J Appl Physiol. 116 (12), (2014).</li></ol>

この原稿の著者は、彼らが競合する金融利害関係を持たないことを宣言します。

このプロトコルは、IVDにおける生物学的変化を監視することに重点を置いて、マウスFSUの器官培養の概要を説明します。これらの培養の成功メンテナンスは慎重な無菌技術を必要とします。具体的には、切開は、2.1から2.6ステップと培養物を3.1から3.6の無菌状態が維持されることを保証するために特別な注意を必要とするステップ、及びこれらの手順は、汚染物質を?...

腰痛（LBP）は、職場での世界的な障害や生産性の損失のための主要な因子であり、そして一人のアメリカ人は、LBP処理1に500億ドルを超えると過ごします。流行が、LBPの病因は複雑かつ多因子のまま。しかし、椎間板（IVD）の変性は、LBP 2のための最も重要な危険因子の一つです。 外側線維輪（AF）、内部の髄核（NP）、及び近位および遠位3全体構造を挟む2つの軟骨終板：IVDは、三の微細構造で形成されています。加齢および変性に、IVD成分は、組成、構造的変化、および機械4。これらの変化は、NPにおけるプロテオグリカン及び水和の損失を含む、椎間板の高さを減少し、機械能力5を悪化。これらの変化はあります多くの場合、炎症反応だけでなく、LBP症状6につながる事象のカスケードで最高潮に達する関節腔への好中球および後根神経節の侵入を促進するサイトカインを伴います。 腰痛の発生前に、変性の原因を特定できないことが多いので、IVD変性のメカニズムを研究することは、ヒトでの挑戦です。このように、IVDの臓器まで実験システムを簡素化するの還元主義的アプローチは因果変数のメカニズムの微調整を可能にし、その下流効果5を調べます。システムは、このようにIVDの変性に対する外部刺激の影響の直接的な解釈を可能にする、唯一のネイティブ細胞集団と細胞外マトリックスを周囲に縮小されます。また、低コスト及びマウスモデルのスケーラビリティ、ならびに遺伝子改変動物7の多数は、Tを可能にします彼IVD変性のメカニズムと潜在的な治療法の迅速な目標スクリーニング。ここでは、IVD細胞および組織安定性たIVD、恒常的、機械的、構造的、および炎症性パターンに与えられる特定の焦点で、21日間にわたって維持されたマウス器官培養系を記載しています。この方法を使用して、我々は、椎間板変性の背後にあるメカニズムを理解するために刺し誘発性損傷モデル8でしたIVD機能の変更を監視します。さらに、我々は造影マイクロCTイメージングおよびIVDの三次元高解像度モデリングを含むさらなる研究を行うために、この器官培養法のいくつかのアプリケーションを記載しています。

<table><tbody><tr><td>96 well plate</td><td>Midwest Scientific</td><td>TP92096</td><td>Used for biochemical assays</td></tr><tr><td>24 well plate</td><td>Midwest Scientific</td><td>TP92024</td><td>Used for organ culture</td></tr><tr><td>25 mL pipettes</td><td>Midwest Scientific</td><td>TP94024</td><td>Used for organ culture</td></tr><tr><td>10 mL pipettes</td><td>Midwest Scientific</td><td>TP94010</td><td>Used for organ culture</td></tr><tr><td>5 mL pipettes</td><td>Midwest Scientific</td><td>TP94005</td><td>Used for organ culture</td></tr><tr><td>500 mL bottle top filters, 22 &amp;micro;m</td><td>Midwest Scientific</td><td>TP99505</td><td>Used for filter media</td></tr><tr><td>10 &amp;micro;L pipette tips</td><td>Midwest Scientific</td><td>NP89140098</td><td>Used for biochemical assays</td></tr><tr><td>200 &amp;micro;L pipette tips</td><td>Midwest Scientific</td><td>NP89140900</td><td>Used for biochemical assays</td></tr><tr><td>1,000 &amp;micro;L pipette tips</td><td>Midwest Scientific</td><td>NP89140920</td><td>Used for biochemical assays</td></tr><tr><td>DMEM/F-12</td><td>Invitrogen</td><td>11330032</td><td>Used for culture media</td></tr><tr><td>Optiray 350</td><td>Guebert</td><td>19133341</td><td>Used for contrast enhanced microCT</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>F2442</td><td>Used for culture media</td></tr><tr><td>Penicillin Streptomycin&amp;nbsp;</td><td>Sigma</td><td>P4333</td><td>Used for culture media</td></tr><tr><td>Tetrazolium Blue Chloride</td><td>Sigma</td><td>T4375</td><td>Used for biochemical assays</td></tr><tr><td>D-Luciferin</td><td>Sigma</td><td>L6152</td><td>Used for bioluminescence imaging</td></tr><tr><td>Chondroitin Sulfate</td><td>Sigma</td><td>C9819</td><td>Used for biochemical assays</td></tr><tr><td>10% Phosphomolybdic Acid Solution</td><td>Sigma</td><td>HT152</td><td>Used for contrast enhanced microCT</td></tr><tr><td>Safranin O</td><td>Sigma</td><td>S8884</td><td>diluted to .1% concentration (in water)</td></tr><tr><td>Fast Green FCF</td><td>Sigma</td><td>F7258</td><td>.001% concentration</td></tr><tr><td>Papain from papaya latex</td><td>Sigma&amp;nbsp;</td><td>P3125</td><td>Used for biochemical assays</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td>Nucleic acid staining</td></tr><tr><td>Cyanoacrylate Glue</td><td>Loctite</td><td>234790</td><td>Adhesive&amp;nbsp;</td></tr><tr><td>1.5 mL Microcentrifuge Tubes</td><td>Fischer Scientific</td><td>S348903</td><td>Used for biochemical assays</td></tr><tr><td>&lt;strong&gt;Big Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BioDent</td><td>ActiveLife</td><td></td><td>For mechanical testing</td></tr><tr><td>Cytation 5</td><td>Biotek</td><td></td><td>Spectrophotometer</td></tr><tr><td>AxioCam503</td><td>Carl Zeiss AG</td><td></td><td>Microscope</td></tr><tr><td>VivaCT40</td><td>Scanco</td><td></td><td>MicroCT</td></tr><tr><td>Analytical balance</td><td>Denver Instrument Company</td><td>A-200DS</td><td>Analytical balance</td></tr><tr><td>Incubator HERAcell 150i</td><td>Thermo Scientific</td><td></td><td>Organ Culture</td></tr><tr><td>Dissection Scope</td><td>VistaVision</td><td></td><td>Used during dissection</td></tr><tr><td>Laser Micrometer</td><td>Keyence</td><td>LK-081</td><td>Measuring disc height</td></tr><tr><td>Microcentrifuge 5810 R</td><td>Eppendorf</td><td></td><td>Used for biochemical assays</td></tr><tr><td>Microtome</td><td>Leica&amp;nbsp;</td><td>RM2255</td><td>Used for histology</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Prism 7</td><td>GraphPad</td><td></td><td>For statistics</td></tr><tr><td>MATLAB R2014a</td><td>Mathworks</td><td></td><td>For modeling</td></tr><tr><td>Osiri-LXIV</td><td>Pixmeo</td><td></td><td>Open Source</td></tr><tr><td>MeshLab v1.3.3</td><td>Visual Computing Lab - ISTI - CNR</td><td></td><td>Open Source</td></tr><tr><td>PreView/FEBio 2.3</td><td>Utah MRL &amp;amp; Columbia MBL</td><td></td><td>Open Source</td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td></td></tr><tr><td>Microsoft Excel</td><td>Windows</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Dissection Tools&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cohan-Vannas Spring Scissors&amp;nbsp;</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>15000-02</td><td>Or any nice pair of spring scissors</td></tr><tr><td>Fine Scissors - Sharp&amp;nbsp; (small)</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>14060-09</td><td></td></tr><tr><td>Fine Scissors - Sharp&amp;nbsp; (larger)</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>14060-11</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>11252-40</td><td>At least 2; can also use #3&amp;nbsp;</td></tr><tr><td>Extra Fine Graefe Forceps, serrated</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>11150-10</td><td>At least 2</td></tr><tr><td>Micro-Adson Forceps, serrated</td><td>World Precision Instruments</td><td>503719-12</td><td></td></tr><tr><td>Micro-Adson Forceps, teeth</td><td>World Precision Instruments</td><td>501244</td><td></td></tr><tr><td>Scalpel Handle - #3</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>10003-12</td><td></td></tr><tr><td>Scalpel Handle - #4</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>10004-13</td><td></td></tr><tr><td>Scalpel Blades - #23</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>10023-00</td><td></td></tr><tr><td>Insect Pins, size 000</td><td>Fine Science Tools</td><td>26000-25</td><td></td></tr><tr><td>27 G Needle</td><td>BD PrecisionGlide Needles</td><td>BD305109</td><td></td></tr></tbody></table>

an in vitro organ culture model of the murine intervertebral disc

椎間板（IVD）変性が腰痛に多大な貢献です。 IVDは、脊椎の荷重を伝達し、減衰させる働きを線維軟骨関節です。 IVDは、プロテオグリカンが豊富な髄核（NP）と軟骨終板に挟まれたコラーゲンが豊富な線維輪（AF）から成ります。一緒に隣接する椎骨と椎骨-IVDの構造は、機能的脊椎ユニット（FSU）を形成します。これらの微細構造はユニークな細胞型だけでなく、ユニークな細胞外基質が含まれています。 FSUの全体の器官培養は、天然の細胞外マトリックス、細胞分化表現型、および細胞 - マトリックス相互作用を保存します。これにより、器官培養技術は、IVDの複雑な生物学的メカニズムを調査するために特に有用です。ここでは、IVDのための病気のメカニズムや治療法を研究するための理想的なプラットフォームを提供し、全体腰椎マウスFSUsを培養するためのハイスループットアプローチを説明します。さらに、我々はSを記述する造影マイクロCTイメージングおよびIVDの三次元高解像度の有限要素モデリングを含む、さらなる研究を行うために、この器官培養法を利用everalアプリケーション。

図2-3は、培養マウスたIVD用プロテオグリカン分布、NF-κBの発現、剛性、粘性、椎間板の高さ、及び湿重量の代表的な結果を示します。適切に培養した場合は、コントロール群のIVDパラメータは、新鮮なグループと有意差があってはなりません。培養物が感染または他の方法で侵害された場合、対照群には、（結果は示さず）、特にNF-κBの発現およびプロテオグリカン分布は、新?...

Watch this Scientific Journal Video about An In Vitro Organ Culture Model of the Murine Intervertebral Disc at JoVE.com

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

椎間板（IVD）の全体の器官培養は、天然の細胞外マトリックス、細胞表現型、および細胞 - マトリックス相互作用を保存します。ここでは、マウスの腰椎とその機能脊髄単位で尾したIVDと、このシステムを利用する複数のアプリケーションを使用してIVD培養系を説明します。

アン<em&gt;インビトロ</emマウス椎間板の&gt;器官培養モデル

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

an-in-vitro-organ-culture-model-of-the-murine-intervertebral-disc

Research

JoVE Journal

Bioengineering

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

9.4K ビュー.  Washington University in St. Louis. 椎間板（IVD）の全体の器官培養は、天然の細胞外マトリックス、細胞表現型、および細胞 - マトリックス相互作用を保存します。ここでは、マウスの腰椎とその機能脊髄単位で尾したIVDと、このシステムを利用する複数のアプリケーションを使用してIVD培養系を説明します。 

ビデオ: An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

卵巣と卵管の三次元器官培養用アルギン酸ゲル

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

生物工学

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.

器官培養のための無傷牛テール椎間板の準備

The intervertebral disc (IVD) is the joint of the spine connecting  vertebra to vertebra. It functions to transmit loading of the spine and give  ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

マウスモデルにおける腸のティッシュエンジニアリング

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

有毒吸入化学試験のためのin vitroでの細胞培養モデルにおける

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

再構成ヒト角膜上皮様（RHCE）組織モデルを使用して眼刺激性化学物質の有害性の同定のための眼刺激性試験（EIT）

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Ovine Lumbar Intervertebral Disc Degeneration Model Utilizing a Lateral Retroperitoneal Drill Bit Injury

Intervertebral disc degeneration is a significant contributor to the development of back pain and the leading cause of disability worldwide. Numerous animal models of intervertebral disc degeneration have been developed. The ideal animal model should closely mimic the human intervertebral disc with regard to morphology, biomechanical properties and the absence of notochordal cells. The sheep lumbar intervertebral disc model fulfils these criteria. We present an ovine model of intervertebral disc degeneration utilizing a drill bit injury through a lateral retroperitoneal approach. The lateral approach significantly reduces the incision and potential morbidity associated with the traditional anterior approach to the ovine spine. Utilization of a drill-bit method of injury affords the ability to produce a consistent and reproducible injury, of precise dimensions, that initiates a consistent degree of intervertebral disc degeneration. The focal nature of the annular and nucleus pulposus defect more closely mimics the clinical condition of focal intervertebral disc herniation. Sheep recover rapidly following this procedure and are typically mobile and eating within the hour. Intervertebral disc degeneration ensues and sheep undergo necropsy and subsequent analysis at periods from eight weeks. We believe that the drill bit injury model of intervertebral disc degeneration offers advantages over more conventional annular injury models.

Intervertebral disc degeneration is a significant contributor to back pain and a leading cause of disability worldwide. Numerous animal models of intervertebral disc degeneration exist. We demonstrate an ovine model of intervertebral disc degeneration, utilizing a drill bit, which achieves a consistent disc injury and reproducible level of disc degeneration.

側方腹腔ドリルビット傷害を利用した羊の腰椎椎間板変性モデル

Intervertebral disc degeneration is a significant contributor to the development of back pain and the leading cause of disability worldwide. Numerous ...

医学

A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.

Symptomatic intervertebral disc (IVD) degeneration (IDD) is a major socioeconomic burden and is characterized by inflammation and tissue degradation. Due to the lack of causative therapies, there is an urgent need for innovative experimental organ culture models to study the mechanisms involved in the progression of the disease, find therapeutic targets, and reduce the need for animal models. We here present a novel, three-dimensional organ culture model protocol mimicking the proinflammatory and catabolic microenvironment, which is present during IDD. 
Initially, bovine caudal IVDs were dissected, cleaned, and cultured in the tissue culture medium. Dynamic physiologic or pathologic loading was applied in a custom-made bioreactor for 2 hours per day. IVDs were assigned to a control group (high glucose medium, physiological loading, phosphate-buffered saline injection) and a pathological group (low glucose medium, pathological loading, tumor necrosis factor-alpha injection) for four days. Gene expression analysis from collected nucleus pulposus cells of the IVDs and enzyme-linked immunosorbent assay of the conditioned organ culture media was performed. 
Our data revealed a higher expression of inflammatory markers and reduced disc heights after loading in the pathological group compared to the control group. This protocol is reliable to simulate IVD inflammation and degeneration and can be further expanded to broaden its application scope.

This protocol presents a novel experimental model of proinflammatory, degenerative bovine organ culture to simulate early-stage intervertebral disc degeneration.

初期段階の椎間板疾患をシミュレートする炎症性の退行性臓器培養モデル。

Symptomatic intervertebral disc (IVD) degeneration (IDD) is a major socioeconomic burden and is characterized by inflammation and tissue degradation. Due ...

Optical Sectioning and Visualization of the Intervertebral Disc from Embryonic Development to Degeneration

Intervertebral disc (IVD) degeneration is a leading cause of low back pain and it entails a high degree of impairment for the affected individuals. To decode disc degeneration and to be able to develop regenerative approaches a thorough understanding of the cellular biology of the IVD is essential. One aspect of this biology that still remains unanswered is the question of how cells are spatially arranged in a physiological state and during degeneration. The biological properties of the IVD and its availability make this tissue difficult to analyze. The present study investigates spatial chondrocyte organization in the anulus fibrosus from early embryonic development to end-stage degeneration. An optical sectioning method (Apotome) is applied to perform high resolution staining analyses using bovine embryonic tissue as an animal model and human disc tissue obtained from patients undergoing spine surgery. From a very high chondrocyte density in the early embryonic bovine disc, the number of cells decreases during gestation, growth, and maturation. In human discs, an increase in cellular density accompanied the progression of tissue degeneration. As had already been demonstrated in articular cartilage, cluster formation represents a characteristic feature of advanced disc degeneration.

We present a method to investigate spatial chondrocyte organization in the anulus fibrosus of the intervertebral disc using an optical sectioning method.

胚発生から変性までの椎間板の光学断面化と可視化

Intervertebral disc (IVD) degeneration is a leading cause of low back pain and it entails a high degree of impairment for the affected individuals. To ...

生物学

Generating a Pro-Inflammatory Organ Culture Model to Simulate an Early Intervertebral Disc Disease

Source: Saravi, B., et al. <a href="https://app.jove.com/v/62100">A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.</a> J. Vis. Exp. (2021).
This video demonstrates a technique for culturing bovine intervertebral discs in bioreactors to develop a three-dimensional organ culture model. Specific nutrient-deprived media and degenerative pressure conditions are applied to the discs, mimicking the pro-inflammatory and degenerative microenvironment observed during early-stage intervertebral disc disease. The disc heights are measured post-treatment to analyze the effects of nutrient deprivation and degenerative loading.

初期の椎間板疾患をシミュレートするための炎症誘発性臓器培養モデルの生成

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Dissection of the bovine ...

JoVE Encyclopedia of Experiments

神経科学

神経細胞培養技術

高度な神経細胞モデル

Generating an Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease

Source: Saravi, B. et al., <a href="https://app.jove.com/v/62100">A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.</a>&#160;J. Vis. Exp. (2021).
This video demonstrates the development of a pro-inflammatory and degenerative organ culture model for early-stage intervertebral disc (IVD) disease using TNF-&#945;. It outlines the procedure for injecting TNF-&#945; into the intervertebral disc and explains the mechanisms through which TNF-&#945; induces inflammation and degradation of the IVD.

Source: Saravi, B. et al., A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.&#160;J. Vis. Exp. ...

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