このプロジェクトは、スイス国立科学財団（SNF＃310030-127586/1）によってサポートされていました。

1。椎間板の収穫<ol><li>皮膚の存在は、汚染の可能性（図2）を増加させるので、全体の長さのウシの尾は皮なしで可能であれば、地元の食肉処理場から得られる。 </li><li>大きなまな板を準備して、カッティングボード（図2）の上に滅菌ワークステーションと楽器を準備。 </li><li>滅菌ガーゼは、55mmのクエン酸ナトリウムを含む0.9％塩化ナトリウムで湿らせ、6ウェルプレートの各ウェルに入れ、無菌層流フードの下準備。 </li><li>流域を準備し、水道水で1:100 Betadineソリューションを希釈する。 </li><li> 5分間、1％Betadine溶液を含む流域の全体の牛の尾を浸し。 </li><li>簡潔に乾いた滅菌ガーゼで尾と無菌ワークステーション上に置きます。 </li><li>慎重に手術用メスの刃＃22とテール周りの筋肉を取り除く。 </li><li>比較的大きいと非常に小さいが含まれて通常必要とされていないテールの部分、尾の両端を、離れてチョップIVDs（図3）。 </li><li>さらに手術用メスの刃＃10を使用してIVD周囲の筋肉や腱をトリミング。ディスクの外側の環を切らないように注意してください。 </li><li>外科皮膚マーカとIVDの前にマークを付ける（このステップはオプションです、それは私たちのバイオリアクターでの軸回転の中心を決定するのに役立ちます）。 </li><li>静かに尾を移動することによって、IVDと脊椎の接続ポイントを見つけます。手術用メスの刃の鈍い側でIVDと骨の間に境界線を感じる。切断部位は、脊椎骨に向かってIVDの離れて1〜2ミリメートルでなければなりません。場所のカスタムは、切断部位（図4）産業のブレードホルダーを作った。 </li><li>カスタムメイドの工業用ブレードホルダー（図3）の上にハンマーで開裂するIVDと椎。 </li><li> IVD -椎接続の反対側に繰り返します。 </li><li>ラップは、55mmのクエン酸ナトリウムを含む0.9％塩化ナトリウムで湿らせた滅菌ガーゼでIVD分離。 </li><li>希望のサンプル番号（通常は約6缶にIVDsの分離を継続する10〜20ミリメートル）との間の直径で収穫される。 </li><li>滅菌乳酸リンゲル液でジェット洗浄システムを（ジマー、税込）に接続、5L袋は便利なサイズ（代わりに滅菌PBSまたは0.9％生理食塩水）です。 </li><li>鉗子とジンマーPulsavacジェット洗浄システム（図1C）を使用して、エンドプレートの表面のジェット洗浄両面でIVDを保持する。ジェットガンは、30の間の角度で終板の表面に撮影してください - 60 °各側面に30秒程度のために。 （図1及び図7） </li><li>ジェットは別のディスクのサンプルを洗浄しながら、湿らせたガーゼで6ウェルプレートとラップにIVDsを元に戻します。 </li><li> IVDsは、器官培養とダウンストリームのアプリケーション（例えば、細胞の生存率、機械的荷重、器官培養）（図1D）のための準備が整いました。 </li></ol> 2。代表的な結果成功した椎間板の準備を判断するアウトカムの測定値は、小さな分子量の蛍光色素（例えばprocion赤）拡散実験することができますPBS 2の1％溶液として調製される。 IVDは、色素溶液と色素の少なくとも100 mLをして自由膨張の下で24時間のためのIVDに受動的に拡散することを許可されて保持されます。 IVDは、° Cと、最終的に室温に液体窒素で瞬間凍結し、室温に戻し、アセトン転送（最初-80にプリ℃まで冷却し、C、-20 °次にC、4シリーズで脱水です）。ディスクは、ポリメチルアクリレート（PMMA）に埋め込まれ、厚さ100μmのセクションを生成するために鋭利な刃でカットすることができます。これらのセクションは、任意の標準的な明視野顕微鏡ではなく優先的にprocion赤は赤色蛍光（図5-6）を発するので、蛍光顕微鏡の使用によって見ることができます。骨表面のtrabeculiはジェットlavagingのステップ（図7）の後に非常にきれいに表示されます。 成功した器官培養のためのパラメータは、汚染のすべての不在の最初の維持、細胞生存率（図8-9）、ディスクの高さとです。組織切片およびサフラニンO /ファストグリーン染色やマイヤーのヘマトキシリン​​3で測定された全体的な&quot;ディスクの整合性&quot;、。我々は、ライブディスクの組織を染色し、共焦点レーザー走査型顕微鏡とLIVE / DEAD染色キット（Molecular Probes社、Invitrogen社、バーゼル、スイス）を使用して3Dで細胞の生存性をスキャンするために別の場所で4,5公開プロトコルとマクロを開発した。我々は、さらにNIH ImageJのプラットフォーム4-6を用いて三次元組織または3Dのキャリアで、細胞生存率を推定するために計数自動化されたセルを実行するマクロ、ルーチンを開発した。代替プロトコルはプロナーゼとコラゲナーゼの種類を使用して細胞外マトリックスの連続消化を使用して初期消化によってディスクの細胞の細胞生存率を決定するために2とし、細胞懸濁液7を染色するために提案されている。十倍を含むPBSでのインキュベーションステップを関連するプロトコルの以前のバージョンのディスク前に臓器Cuの収穫後の10分のためのより高濃度のペニシリン/ストレプトマイシン濃度lture。このステップは、培養の目的のために有益と思われるので、ジェットの洗浄ステップでは、このステップは不要になります。 <img alt="図1" src="/files/ftp_upload/3490/3490fig1.jpg" /> 図1方法論の手順の概要は、そのままエンドプレートと骨の薄層（1 - 2mm）をウシ尾骨椎間板を準備する。椎間板（IVD）とその栄養経路のA）の模式図。 B）カスタマイズしたブレードホルダーと産業鋭い刃とIVDをカットする手順。 C）ジェット洗浄ステップでは、高い細胞生存率を確保し、エンドプレートと両側に接続されている骨の1〜2 mmを維持する栄養交換を可能にする。 D）最後に、IVDは、バイオリアクターの試料室で培養することができますまたは任意のダウンストリームアプリケーションに使用される。 <img alt="図2" src="/files/ftp_upload/3490/3490fig2.jpg" /> 図2トップ：全長新鮮な牛テール（理想的にobta高い細胞の生存を確保するために事後の2〜3時間以内にined。底部：骨の終板（EP）と椎体（VB）との間で、成長板（GP）、骨化の二次中心の存在を示す6ヶ月歳前後牛テールのX線画像。 <img alt="図3" src="/files/ftp_upload/3490/3490fig3.jpg" /> 図3。）ツールは、無菌条件下でウシの尾を解剖する。椎間板を削減しながらB）カスタマイズされたブレードホルダーは、アクションの牛テールから運動のセグメントを切り出しする。標準的な工業用ブレード（ルッツ、ドイツ）とカスタマイズされたブレードホルダーのC）サイドビュー。 <img alt="図4" src="/files/ftp_upload/3490/3490fig4.jpg" /> 図4。運動セグメントをカットする切断部位を示す周囲の筋肉と靭帯を除去した後、牛テールの描画と画像。部分的な椎骨はwiを&quot;クラック&quot;する必要がある離れて軟骨終板からカスタムメイドのブレードホルダー番理想的に1〜2ミリメートルの完全な骨の表面を確実にする。 <img alt="図5" src="/files/ftp_upload/3490/3490fig5.jpg" /> 図5。の前に（）と（B）の後、ジェット洗浄ステップの効果的な洗浄のデモンストレーション。噴霧手順の後に椎間板セグメントのクリーンな骨の表面に注目してください。 <img alt="図6" src="/files/ftp_upload/3490/3490fig6.jpg" /> 図6。作りたての成長板によって閉塞効果を発揮、1％procion赤色の溶液に24時間放置牛テール椎間板（IVDs）を切り出したの自由膨潤拡散実験。のデジタルX線矢状画面に2つの後も軟骨や骨端板と尾骨ウシ椎間板の外植片を介して成長板と矢状カットの骨蛍光顕微鏡画像の第二層を含むIVD4時間自由拡散。 GP：成長板、2ndry CO：骨化の二次中心。 <img alt="図7" src="/files/ftp_upload/3490/3490fig7.jpg" /> 図7。1％procion赤色の溶液に24時間放置、新たに調製した摘出したウシの尾部椎間板（IVDsが）、ジェット洗浄療法の洗浄効果を示すの自由膨潤実験。治療（右）ジェットlavaged処理なしジェット洗浄療法（左）制御側を受けた〜1.5 mmthick骨端板と摘出したウシIVDsの矢状厚いセクション（〜100mm）に。入口は1％procionの赤のウシ椎間板の外植片のジンマーの傷デブリドマンシステム24時間無料の拡散実験から使用されたスプレーパターンを示しています。 <img alt="図8" src="/files/ftp_upload/3490/3490fig8.jpg" /> 図8。共焦点イメージングスタックの投影のデッド/ライブ画像（250μm）bovinから撮影電子椎間板セグメントは髄核（NP）、内側の線維輪（IA）と外側の線維輪（OA）のためそれぞれ0と14日間無料の膨潤性の下に置かこのジェット洗浄法を用いて調製。緑色のセルは=生きている細胞は、カルセインAM（アセチルメチルエステル）、赤血球エチジウムホモダイマー-1で染色=死んだ細胞の核で染色。 <img alt="図9" src="/files/ftp_upload/3490/3490fig9.jpg" /> 図9。生きている組織や3Dスタックのスキャンの共焦点レーザー走査顕微鏡を用いて椎間板の細胞バイアビリティ。自由膨潤性条件下で21日間の培養上髄核（NP）およびアニュラス線維症（AF）の細胞の生存。 （± SEM、N = 6の平均）統計的な違いは、ノンパラメトリッククラスカルワリスのグループ間での順位和検定を署名した使用してテストされました。有意な差は21日目とNPの他のすべての時間の点の間に発見された。 AFに有意な差で7日目までの間に発見されたが0日目。 （* P &lt;0.05、** P &lt;0.01）。

<ol>
	<li>Lee, C. R., Iatridis, J. C., Poveda, L., Alini, M. <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 (Phila Pa 1976). 31, 515-522 (1976).</li><li>Chan, S. C. W., Gantenbein-Ritter, B., Leung, V. Y., Chan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreserved+intervertebral+disc+with+injected+bone+marrow-derived+stromal+cells:+a+feasibility+study+using+organ+culture.">Cryopreserved intervertebral disc with injected bone marrow-derived stromal cells: a feasibility study using organ culture.</a> Spine. J. 10 (6), 486-496 (2010).</li><li>Chan, S. C., Ferguson, S. J., Wuertz, K., 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=Biological+Response+of+the+Intervertebral+Disc+to+Repetitive+Short+Term+Cyclic+Torsion.">Biological Response of the Intervertebral Disc to Repetitive Short Term Cyclic Torsion.</a> Spine (Phila Pa 1976). , (2011).</li><li>Gantenbein-Ritter, B., Sprecher, C. M., Chan, S., Illien-J&#252;nger, S., Grad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+imaging+protocols+for+live/dead+staining+in+three-dimensional+carriers.">Confocal imaging protocols for live/dead staining in three-dimensional carriers.</a> Methods Mol. Biol. 740, 127-140 (2011).</li><li>Gantenbein-Ritter, B., Potier, E., Zeiter, S., van der Werf, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+three+techniques+to+determine+cell+viability+in+3D+tissues+or+scaffolds.">Accuracy of three techniques to determine cell viability in 3D tissues or scaffolds.</a> Tissue Engineering Part C Methods. 14, 353-358 (2008).</li><li>Rasband, W. 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> ImageJ. , (1997).</li><li>Haschtmann, D., Stoyanov, J. V., Ettinger, L., Nolte, L. P., Ferguson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+novel+intervertebral+disc/endplate+culture+model:+analysis+of+an+ex+vivo+in+vitro+whole-organ+rabbit+culture+system.">Establishment of a novel intervertebral disc/endplate culture model: analysis of an ex vivo in vitro whole-organ rabbit culture system.</a> Spine. 31, 2918-2925 (2006).</li><li>Gantenbein, B., Gr&#252;nhagen, T., Lee, C. R., van Donkelaar, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+organ+culturing+system+for+intervertebral+disc+explants+with+vertebral+endplates:+a+feasibility+study+with+ovine+caudal+discs.">An in vitro organ culturing system for intervertebral disc explants with vertebral endplates: a feasibility study with ovine caudal discs.</a> Spine. 31, 2665-2673 (2006).</li><li>Korecki, C. L., Kuo, C. K., Tuan, R. S., Iatridis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intervertebral+disc+cell+response+to+dynamic+compression+is+age+and+frequency+dependent.">Intervertebral disc cell response to dynamic compression is age and frequency dependent.</a> J. Orthop. Res. 27, 800-806 (2009).</li><li>Korecki, C. L., MacLean, J. J., Iatridis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+compression+effects+on+intervertebral+disc+mechanics+and+biology.">Dynamic compression effects on intervertebral disc mechanics and biology.</a> Spine (Phila Pa 1976). 33, 1403-1409 (1976).</li><li>Jim, B., Steffen, T., Moir, J., Roughley, P., Haglund, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+intact+intervertebral+disc+organ+culture+system+in+which+degeneration+can+be+induced+as+a+prelude+to+studying+repair+potential.">Development of an intact intervertebral disc organ culture system in which degeneration can be induced as a prelude to studying repair potential.</a> European Spine Journal. , 1-11 (2011).</li></ol>

我々は、開示することは何もない。

成功した器官培養のための最初のステップは、外植片が汚染されるべきではないことを確認することです。この手順を始める前に尾にはスキンは適用されるべきである。無菌室に持ち込むあらゆる動物の毛は、汚染の点で問題である可能性があります。牛テールは、理想的に（これは初期の細胞の生存に影響を与える）可能な限り新鮮なはずです。さらに、betadineの洗浄工程は、さらなる汚?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（省略可能） </td></tr><tr><td colspan="4">ローカル虐殺家（理想的に時間の事後内および皮なし）から牛テールから新鮮なウシ椎間板組織、。 </td></tr><tr><td> PulsavacプラスACシステム</td><td>ジンマー株式会社、スイス</td><td> 00-5150-486-01 </td><td>ヒップスプレーヘッド付きやAC電源で最高のパフォーマンス（8つのAA電池パックを持つものがも動作しますが、あまり便利ではありません） </td></tr><tr><td>大容量ファンスプレーのw /スプラッシュシールド、12.7センチメートル長さ</td><td>ジンマー株式会社、スイス</td><td> 00-5150-175-00 </td><td>利用可能ないくつかのスプレーヘッドが存在する、我々が正常にこれをテスト</td></tr><tr><td>メスの刃＃22と＃10 </td><td>スワン-モートイングランド、上</td><td> ＃10：0201 ＃22：0208 </td><td></td></tr><tr><td>手術用メスの刃ホルダー＃3と＃4 </td><td>ハウスマン、ドイツ</td><td> ＃3：06.103.00 ＃4：06.104.00 </td><td></td></tr><tr><td>ルッツ工業用ブレード</td><td>ルッツ、ドイツ</td><td> 1022.0884 </td><td></td></tr><tr><td>リン酸緩衝生理食塩水（PBS） </td><td>インビトロジェン、スイス</td><td> 10010-023 </td><td></td></tr><tr><td>ダルベッコ改変イーグル培地（DMEM） </td><td> Gibco社、スイス</td><td> 11960-044 </td><td></td></tr><tr><td>乳酸リンゲル液（ブドウ糖なし） </td><td>ビクセル、スイス</td><td> 133 0002 </td><td></td></tr><tr><td> 6ウェルマルチウェルプレート</td><td> TPP、スイス</td><td> 92006 </td><td></td></tr><tr><td> Betadineソリューション</td><td>ムンディファーマ、スイス</tD&gt; <td> 10055025 </td><td></td></tr><tr><td>外科皮膚マーカー</td><td> Pore​​x外科、スイス</td><td> 9560 </td><td></td></tr><tr><td>大きなまな板</td><td></td><td></td><td>どんなブランドが可能です。 </td></tr></tbody></table>

preparation of intact bovine tail intervertebral discs for organ culture

椎間板（IVD）は椎骨に椎を接続する脊椎の関節です。それは、背骨の荷重を伝達し、背骨に柔軟性を与えるために機能します。線維輪（AF）で取り囲む最も内側の髄核（NP）、および両側の椎体にNPとAFを接続する2つの軟骨終板：それは3つの区画から構成している。おそらく変性椎間板疾患（DDD）と椎間板ヘルニアが原因で椎間板の痛みは私達の現代社会における主要な問題として同定されている。 IVD変性の機序を調べるために、ライブディスクの細胞を用いたin vitro器官培養系で非常に魅力的です。そのままウシ尾骨IVDsのin vitro培養十分に制御生理学的および機械的な環境におけるメカノ生物学的側面 ​​の研究を可能にする、関連するモデルシステム、に進んでいる。牛テールIVDsは高い数値で、比較的容易に得ることができますdは、細胞密度、細胞集団と寸法に関する人間の腰椎IVDsと非常によく似ています。栄養経路が明らかに凝固した血液によってブロックされたしかし、軟骨終板と骨のエンドプレートを保持し、前のウシ尾IVD収穫テクニックは、文化の1〜2日後に失敗しました。 IVDsが最大の無血管器官である、したがって、NPの細胞への栄養素は、隣接する椎体から毛細管芽を経由して拡散にもっぱら依存しています。エンドプレートの表面上に骨の破片と血餅の存在は、ディスクと妥協の細胞の生存の中心に栄養の拡散を妨げることができる。当社グループは、汚染のリスクが低いとアウトテールからIVDsを&quot;クラック&quot;することは比較的迅速なプロトコルを確立した。我々は、血栓や切削屑を除去し、非常に効率的に栄養の拡散経路を再オープン外科ジェット洗浄システムを使用して、切りたての骨端板の表面を透過することができますIVDの中心に。脊椎骨の両側の成長板の存在は、避けるべきと前の文化に削除する必要があります。このビデオでは、我々は準備中に重要なステップを概説し、無料の腫れ文化で14日間高い細胞生存率を維持し成功した器官培養するキーを示しています。培養時間は、適切な機械的な環境が機械的荷重のバイオリアクターを使用して維持することができるときに拡張することができる。ここで示した手法は、ブタ、ヒツジ及びウサギの尾と腰IVD分離など他の動物種に拡張することができます。

Watch this Scientific Journal Video about Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture at JoVE.com

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

このプロトコルは、のための器官培養のための尾骨ウシ椎間板のための収穫技術を示しています。<em&gt; in vitroで</em&gt;器官培養。

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  ...

preparation-intact-bovine-tail-intervertebral-discs-for-organ

Research

JoVE Journal

Bioengineering

14.4K ビュー.  University of Bern. このプロトコルは、のための器官培養のための尾骨ウシ椎間板のための収穫技術を示しています。 in vitroで器官培養。

ビデオ: Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

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 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

幹細胞培養アプリケーションのための3Dフィブリン足場の調製

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

3次元細胞培養モデルの質量分析イメージング用試料調製の戦略

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

相関顕微鏡を用いて、無傷の細胞における上皮成長因子受容体の化学量論を学びます

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

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 ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

作製と内皮下電気抵抗を直接定量化する統合型電極を用いたオンチップ器官システムの検証

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Fragility Assessment of Bovine Cortical Bone Using Scratch Tests

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the fragility of bone, making it fracture-prone. Owing to the large socio-economic impact of bone fracture in our society, there is a need for novel ways to assess the mechanical performance of each hierarchical level of bone. Although stiffness and strength can be probed at all scales &#8211; nano-, micro-, meso-, and macroscopic &#8211; fracture assessment has so far been confined to macroscopic testing. This limitation restricts our understanding of bone fracture and constrains the scope of laboratory and clinical studies. In this research, we investigate the fracture resistance of bone from the microscopic to the mesoscopic length scales using micro scratch tests combined with nonlinear fracture mechanics. The tests are performed in the short longitudinal orientation on bovine cortical bone specimens. A meticulous experimental protocol is developed and a large number (102) of tests are conducted to assess the fracture toughness of cortical bone specimens while accounting for the heterogeneity associated with bone microstructure.

This study assesses the fracture toughness of bovine cortical bone at the sub-meso levels using microscopic scratch tests. This is an original, objective, rigorous, and reproducible method proposed to probe fracture toughness below the macroscopic scale. Potential applications are studying changes in bone fragility due to diseases like osteoporosis.

スクラッチ テストによるウシ皮質骨の脆弱性評価

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

伸縮性、デュアル チャネル、オルガン マイクロ流体チップのスケーラブルな作製

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...

Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and medical intervention in rural environments and military medicine applications where ophthalmic care is not readily available. Untreated injuries are susceptible to infection after the eye has lost its watertight seal, as well as loss of tissue viability due to intraocular hypotension. Therapeutics to temporarily seal open globe injuries, if properly developed, may be able to restore intraocular pressure and prevent infection until proper ophthalmic care is possible. To facilitate product development, detailed here is the use of an anterior segment organ culture open globe injury platform for tracking therapeutic performance for at least 72 h post-injury. Porcine anterior segment tissue can be maintained in custom-designed organ culture dishes and held at physiological intraocular pressure. Puncture injuries can be created with a pneumatic-powered system capable of generating injury sizes up to 4.5 mm in diameter, similar to military-relevant injury sizes. Loss of intraocular pressure can be observed for 72 h post-injury confirming proper injury induction and loss of the eye&#39;s watertight seal. Therapeutic performance can be tracked by application to the eye after injury induction and then tracking intraocular pressure for multiple days. Further, the anterior segment injury model is applicable to widely used methods for functionally and biologically tracking anterior segment physiology, such as assessing transparency, ocular mechanics, corneal epithelium health, and tissue viability. Overall, the method described here is a necessary next step toward developing biomaterial therapeutics for temporarily sealing open globe injuries when ophthalmic care is not readily available.

Open globe eye injuries may go untreated for multiple days in rural or military-relevant scenarios, resulting in blindness. Therapeutics are needed to minimize loss of vision. Here, we detail an organ culture open globe injury model. With this model, potential therapeutics for stabilizing these injuries can be properly evaluated.

オープングローブ損傷と治療性能を追跡するための前セグメント臓器培養プラットフォーム

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and ...

Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional (3D) structures. One of the main functions of epithelia is the formation of barriers that protect the underlining tissues against physical and chemical insults and infectious agents. In addition, epithelia mediate the transport of nutrients, hormones, and other signaling molecules, often creating biochemical gradients that guide cell positioning and compartmentalization within the organ. Owing to their central role in determining organ-structure and function, epithelia are important therapeutic targets for many human diseases that are not always captured by animal models. Besides the obvious species-to-species differences, conducting research studies on barrier function and transport properties of epithelia in animals is further compounded by the difficulty of accessing these tissues in a living system. While two-dimensional (2D) human cell cultures are useful for answering basic scientific questions, they often yield poor in vivo predictions. To overcome these limitations, in the last decade, a plethora of micro-engineered biomimetic platforms, known as organs-on-a-chip, have emerged as a promising alternative to traditional in vitro and animal testing. Here, we describe an Open-Top Organ-Chip (or Open-Top Chip), a platform designed for modeling organ-specific epithelial tissues, including skin, lungs, and the intestines. This chip offers new opportunities for reconstituting the multicellular architecture and function of epithelial tissues, including the capability to recreate a 3D stromal component by incorporating tissue-specific fibroblasts and endothelial cells within a mechanically active system. This Open-Top Chip provides an unprecedented tool for studying epithelial/mesenchymal and vascular interactions at multiple scales of resolution, from single cells to multi-layer tissue constructs, thus allowing molecular dissection of the intercellular crosstalk of epithelialized organs in health and disease.

The present protocol describes the capabilities and the essential culture modalities of the Open-Top Organ-Chip for the successful establishment and maturation of full-thickness organ-on-chip cultures of primary tissues (skin, alveolus, airway, and intestine), providing the opportunity to investigate different functional aspects of the human epithelial/mesenchymal and vascular niche interface in vitro.

オープントップ臓器チップ上でのヒト上皮組織の細胞構造と機能の再構成

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional ...