我々は、滑液の貯蔵及び遠心分離のヘルプを提供するためのロビンナイ（タフツ医療センター）、智也内村とダナケアンズ（タフツ大学）に感謝いたします。この作品は、LZのためのNIH（1R01AR059106 - 01A1）によって賄われていた

滑液の培養ヒト関節軟骨細胞用3Dシステム本研究では、我々は、修正された製造元で推奨されるカプセル化プロトコル（ロンザ、および31）を用いてアルギン酸ビーズで人間の関節軟骨細胞をカプセル化。これらの3次元構造体を使用して、我々は人間の滑液の様々な割合を含有する培養培地で細胞を培養するためのシステムを開発し、軟骨の遺伝子発現のためのこれらの3次元構造を評価している。 1。三次元（3D）カプセル化のためのヒト関節軟骨細胞（HAC）を準備<ol><li> 37 1分間° Cの水浴中で人間の関節軟骨細胞（HAC）（ロンザ）（パッセージ2）のバイアル（1ml）を解凍。 </li><li>軟骨細胞増殖培地（ロンザ）を1mlと混合し、細胞を収集するために3〜5分間3000rpmで遠心する。上清を捨てる。 </li><li>軟骨細胞増殖培地に再懸濁し、細胞ペレット（ロンザ）。 </li><li> 10cmの組織で細胞をプレート培養プレート（BD Biosciences社）、および軟骨細胞増殖培地で培養（ロンザ）細胞がコンフルエントになるまで。日立空調は3または4（P3またはP4）よりも高い継代数で使用してください。 </li><li>アルギン酸ビーズのカプセル化のために細胞を収集するtrypsinzation続いて、15​​5ミリNaClの代わりに、標準PBSでプレート上に日立空調を洗浄してください。一度細胞が切り離されて、5分間3000rpmの時に細胞をスピンダウン。細胞は、今の3Dカプセル化するための準備が整いました。 </li></ol> 2。 3Dビーズに日立空調をカプセル化 8 × 10 5細胞/ mlの密度で1.2％のアルギン酸塩溶液（シグマ）で日立空調を再懸濁します。細胞数は、スタンダードセルのカウンタを使用して、前のカプセル化に決定した。それは、ビーズの細胞の均等な分配を確保するためによく混合することは非常に重要です。 <ol><li>ピペット22ゲージ針（タイコヘルスケア、（株））に接続された12mlのシリンジにHAC /アルギン酸塩溶液の混合物。 </li><li>一方、102メートルと50 mLのビーカーを用意するボリュームでM CaCl 2を 5回HAC / aginateソリューションのこと。ビーカー内の攪拌棒を配置して（約150rpmで）徐々に102 mMのCaCl 2溶液をかき混ぜる。 </li><li> CaCl 2溶液の表面に上記の6インチ約シリンジの先端を保持し、CaCl 2溶液の滴下にHAC /アルギン酸塩混合物を追加。一般的に、結果として得られるアルギン酸ビーズは、約10 4細胞/ビーズのカプセル化の密度と直径で2mmです。注：CaCl 2溶液上にシリンジの高さが重要である。短い距離は、不均一な機械的完全性と細胞のカプセル化を引き起こし、球状ビーズの代わりに、形のティアドロップになります。 </li><li>ビーズは、20〜25分のCaCl 2溶液に撹拌してみましょう。他のアルギン酸カプセル化プロトコルは、10分の非常に短いインキュベーション時間を示す一方で、我々は、CaCl 2溶液と長いインキュベーション時間がなるの整合性を高めることを発見広告。 </li><li> CaCl 2溶液を除去する、軟骨細胞分化培地（ロンザ）で一度して2-3倍のNaClの2-3量のビーズを洗浄し、。他のアルギン酸ビーズのカプセル化プロトコルは、上記の洗浄手順については、フィルタを使用して関与しながら、我々は、アルギン酸ビーズはしばしばこのようにカプセル化の効率を減少させる、フィルターとドライアウトに閉じ込められることがわかった。我々はそれが最も効率的なビーズが廃棄するまでビーカーの底に解決を解決できるようにすることがわかった。 </li><li>培養皿にビーズを転送する標準的なヘラを使用してください。通常、我々の文化軟骨細胞は、それらが滑液と軟骨細胞分化培地（ロンザ）に切り替える前に増殖させた培地でカプセル化のプロセスに適応できるように、軟骨細胞増殖培地で2日間ウェル3 cmあたり12のビーズを。 </li></ol> 3。滑液の培養液で培養軟骨細胞<ol><li>新鮮な滑液は、oから入手できます。utpatientクリニック（我々は、タフツ医療センターから滑液が得られる）。滑液は、実験室に15ミリリットルファルコンチューブに移し、そして直ちに細胞破片を除去するために15分間3000rpmで遠心分離することができます。一定分量の無細胞滑液1.5 mLのマイクロ遠心チューブに、凍結融解を避けるために。上清を-80℃で使用時まで保存することができます。 </li><li>培養前にCaCl 2をこの微量がアルギン酸の整合性を確保するためである、100mMのアスコルビン酸の一定濃度で、体積比率を変化させることで、軟骨細胞分化培地（CDM、ロンザ）で滑液を混ぜると9ミリのCaCl 2（ビーズ）。 </li><li> 37℃インキュベーターで振盪プラットフォーム上でプレートを保持（ロッキング周波数：約75回/分）アルギン酸ビーズ内に栄養分布を手助けするために、ビーズの凝集を軽減する。これは長期間（最長4週間）のために我々は文化これらの軟骨細胞をとして特に重要です。 （注）：軟骨細胞の増殖と分化のメディア（ロンザ）は2つの一般的に使用される抗生物質ゲンタマイシン、アムホテリシンを含んでいますが、我々は軟骨細胞を培養するための滑液中の別の割合を使用していた抗生物質を補足していない。の汚染はこれまで観察されなかった。 </li><li>すべての2〜3日メディアの混合物を変更する。滑液の粘度は、長期培養中にアルギン酸ビーズ内にカプセル化された培養軟骨細胞における大きなハードルとなっています。我々はまた、アルギン酸ビーズの整合性を強化付き102mmのCaCl 2（50％V / V）、と滑液添加培地を希釈することが最も効果的な発見。このように、培地を徐々にビーズを傷つけることなく、ピペットで除去することができます。 </li></ol> 4。遺伝子発現解析のためのアルギン酸ビーズの収穫の日立空調遺伝子発現解析のためのアルギン酸ビーズから日立空調を収穫するときに特別な注意が必要です。 <ol><li>アルギン酸ビーズ3 Tiを洗う約5分間ずつで102 mMのCaCl 2でのMES。 </li><li> 4-5回そのビーズの体積で55 mMのNaCitrateを追加することにより、軟骨細胞を取得する。アルギン酸ビーズが完全にNaCitrate溶液に浸漬されていることが重要です。振ったり、20〜30分の岩。 </li><li>細胞をスピンダウンし、上清を捨てる。 </li><li> RT - PCR解析のために、細胞溶解緩衝液（キアゲンRNA単離キット）で細胞ペレットを再懸濁し、そしてRNAの精製に進みます。 </li></ol> 5。組織学的分析のためにアルギン酸ビーズの日立空調を修正特別なケアは、組織学的分析のための3Dビーズから日立空調を収穫するために取られる必要があります。 <ol><li>アルギン酸ビーズを約5分間ずつで102 mMのCaCl 2で 3回洗浄する。完全に粘性滑液を洗い流すために、我々は、37℃インキュベーター（ロッキング周波数で揺動、一晩軟骨細胞分化培地（ロンザ）で洗浄することが最も効果的な発見：約75時間秒/分）。軟骨細胞増殖培地は、この長期にわたる洗浄の軟骨細胞の生存を確保し、固定剤の最大の浸透を可能にするために使用されていました。 </li><li>一晩70％エタノールでビーズを固定し、組織学的分析を進める。 DAPI染色を実行するには、穏やかな振盪下1時間のためにDAPI溶液（500ng/ml）をアルギン酸ビーズをインキュベートし、15分間付き102mm CaCl 2を各時間で3回洗浄する。 DAPI画像はその後、蛍光顕微鏡で観察することができます。ビーズはまた、アルシアンブルーとH＆E染色用切片にすることができます。 </li></ol> 6。代表的な結果滑液の高い割合で、人間の軟骨細胞のための私達の3次元培養法は、図1に示す模式図に描かれています。人間の軟骨細胞をアルギン酸ビーズ中にカプセル化された後、彼らは滑液の比率を変えて添加した培地で増殖させた。ために滑液の粘度が、それは不可欠です軟骨の構造の凝集を防ぎ、栄養素の均一な分布を確保するために一定の揺動条件下での培養軟骨細胞。それは、固定または溶解バッファーは、（図1）ビーズを貫通できるように、軟骨細胞を取得する前に広範囲にアルギン酸ビーズを洗浄するためにも不可欠です。アルギン酸ビーズ中の軟骨細胞の明視野（BF）画像をFig.2に示します。 DAPI染色は、ビーズ（図2）内のセルのusniform分布を確認するために実施した。 21日間の培養期間の終了時に、遺伝子発現解析は、定量RT - PCRによって行った。リファレンス遺伝子GAPDHは、それが軟骨細胞32上のqPCR解析のための最も信頼できるリファレンス遺伝子の一つであると決定されたとして、すべてのPCR反応のために正規化のために使用されていました。例を図に示します。我々は変形性関節症患者6例からプールされた滑液で培養したヒト関節軟骨細胞からの結果を分析した3。事実と一致することから軟骨細胞ロンザは必然的に脱分化33につながる2次元の文化、で展開されている、我々は、0日目軟骨細胞は軟骨基質遺伝子の最小限のレベル（図3）を発現しています。滑液を補充した軟骨細胞分化培地（ロンザ）または培地で軟骨細胞の3次元培養では有意に21日目（図3）34での再分化軟骨細胞を示すコラーゲン、アグリカンとMMP13、の軟骨の遺伝子発現を増加させた。メディアにおける滑液の割合を増やすと、軟骨基質のマーカーコラーゲンIIとアグリカンのmRNA発現（Fig.3Aおよび3B）の同等のレベルになりました。培地のみ（図3c）の培養と比較してさらに、100％滑液で培養した軟骨細胞は、さらに軟骨分解酵素MMP13のmRNA発現の減少を示した。興味深いことに、細胞死インジケータカスパーゼ3の発現レベルは徐々に示唆し、滑液の増加比で減少しているのynovial液体培養では減少アポトーシスレベル（図3D）につながっている。したがって、我々の結果は、3D設定の滑液中に高レベルの培養ヒト関節軟骨細胞は、実現可能な技術であることを示している。 テーブルと図<img alt="図1。" src="/files/ftp_upload/3587/3587fig1.jpg" /> 図1 3Dアルギン酸ビーズの滑液の割合が高いの文化、ヒト関節軟骨細胞への法の模式図。最初に、軟骨細胞およびアルギン酸塩溶液を混合されています。 CaCl 2溶液に滴下適用された場合、軟骨細胞は、架橋のCa -アルギン酸ハイドロゲルビーズ内に固定されています。これらの3次元構造は、ヒト滑液の比率を変化させた軟骨細胞分化培地（ロンザ）で培養されています。ロッキング条件下で培養21日後、細胞を含むアルギン酸ビーズは細胞が遺伝子のために取得された後に洗浄されています発現解析。 <img alt="図2。" src="/files/ftp_upload/3587/3587fig2.jpg" /> 図2。ブライトフィールド（BF）およびヒト関節軟骨細胞のDAPI画像0％、30％、50％、70％と100％滑液とカプセル化された文化。軟骨細胞は、すべての培養条件で形状が球形であった。明視野（BF）およびDAPIの画像は、軟骨細胞の位置と分布を確認するために重ねていた。インセット、拡大画像。矢尻、BFとDAPI染色像における軟骨細胞の局在。 <img alt="図3。" src="/files/ftp_upload/3587/3587fig3.jpg" /> 図3。一日でカプセル化されたヒト関節軟骨細胞の定量RT - PCR解析0（D0）滑液（SF）（0％、30％、50％、70の様々な比率で添加した培地で培養する（D21）の21日後に％と100％）。つの独立したサンプルの結果がここに表示されます。 GAPDHは、すべてのPCRのための内部基準として使用されていました。 <strオング&gt; A。コラーゲンIIのmRNA発現。B。アグリカンのmRNA発現。C。 MMP13のmRNAの発現。D。カスパーゼ3 mRNAの発現。統計的有意性は、INSTATのソフトウェアを使用して、0日の試料と21日目0％のサンプルと100％関節液（SF）培養に評価した。 *はP &lt;0.05。

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synovial+fluid+and+peripheral+blood+immune+complexes+of+patients+with+rheumatoid+arthritis+induce+apoptosis+in+cytokine-activated+chondrocytes.">Synovial fluid and peripheral blood immune complexes of patients with rheumatoid arthritis induce apoptosis in cytokine-activated chondrocytes.</a> Rheumatol. Int. 27, 901-909 (2007).</li><li>Hegewald, A. A., Ringe, J., Bartel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronic+acid+and+autologous+synovial+fluid+induce+chondrogenic+differentiation+of+equine+mesenchymal+stem+cells:+a+preliminary+study.">Hyaluronic acid and autologous synovial fluid induce chondrogenic differentiation of equine mesenchymal stem cells: a preliminary study.</a> Tissue Cell. 36, 431-438 (2004).</li><li>Xu, Q. R., Dong, Y. H., Chen, S. L., Bao, C. D., Du, H. <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+normal+articular+cartilage+induced+by+late+phase+osteoarthritic+synovial+fluid+in+beagle+dogs.">Degeneration of normal articular cartilage induced by late phase osteoarthritic synovial fluid in beagle dogs.</a> Tissue Cell. 41, 13-22 (2009).</li><li>Kruger, J. P., Endres, M., Neumann, K., Haupl, T., Erggelet, C., Kaps, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrogenic+differentiation+of+human+subchondral+progenitor+cells+is+impaired+by+rheumatoid+arthritis+synovial+fluid.">Chondrogenic differentiation of human subchondral progenitor cells is impaired by rheumatoid arthritis synovial fluid.</a> J. Orthop. Res. 28, 819-827 (2010).</li><li>Steinhagen, J., Bruns, J., Niggemeyer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+culture+system:+Synovial+fibroblasts+modulate+articular+chondrocyte+matrix+synthesis+in+vitro.">Perfusion culture system: Synovial fibroblasts modulate articular chondrocyte matrix synthesis in vitro.</a> Tissue Cell. 42, 151-157 (2010).</li><li>Yang, K. G., Saris, D. B., Verbout, A. J., Creemers, L. B., Dhert, W. 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+effect+of+synovial+fluid+from+injured+knee+joints+on+in+vitro+chondrogenesis.">The effect of synovial fluid from injured knee joints on in vitro chondrogenesis.</a> Tissue Eng. 12, 2957-2964 (2006).</li><li>Skoog, V., Widenfalk, B., Ohlsen, L., Wasteson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+growth+factors+and+synovial+fluid+on+chondrogenesis+in+perichondrium.">The effect of growth factors and synovial fluid on chondrogenesis in perichondrium.</a> Scand. J. Plast. Reconstr. Surg. Hand. Surg.. 24, 89-95 (1990).</li><li>Nuver-Zwart, I., Schalkwijk, J., Joosten, L. A., van den Berg, W. B., van de Putte, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+synovial+fluid+and+synovial+fluid+cells+on+chondrocyte+metabolism+in+short+term+tissue+culture.">Effects of synovial fluid and synovial fluid cells on chondrocyte metabolism in short term tissue culture.</a> J. Rheumatol. 15, 210-216 (1988).</li><li>Beekhuizen, M., Bastiaansen-Jenniskens, Y. M., Koevoet, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritic+synovial+tissue+inhibits+proteoglycan+production+in+human+osteoarthritic+cartilage;+Establishment+and+characterisation+of+a+long-term+coculture.">Osteoarthritic synovial tissue inhibits proteoglycan production in human osteoarthritic cartilage; Establishment and characterisation of a long-term coculture.</a> Arthritis Rheum. , (2011).</li><li>Rodrigo, J. J., Steadman, J. R., Syftestad, G., Benton, H., Silliman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+human+knee+synovial+fluid+on+chondrogenesis+in+vitro.">Effects of human knee synovial fluid on chondrogenesis in vitro.</a> Am. J. Knee. Surg. 8, 124-129 (1995).</li><li>van den Hoogen, B. M., van de Lest, C. H., van Weeren, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loading-induced+changes+in+synovial+fluid+affect+cartilage+metabolism.">Loading-induced changes in synovial fluid affect cartilage metabolism.</a> Br. J. Rheumatol. 37, 671-676 (1998).</li><li>Webb, G. R., Westacott, C. I., Elson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritic+synovial+fluid+and+synovium+supernatants+up-regulate+tumor+necrosis+factor+receptors+on+human+articular+chondrocytes.">Osteoarthritic synovial fluid and synovium supernatants up-regulate tumor necrosis factor receptors on human articular chondrocytes.</a> Osteoarthritis Cartilage. 6, 167-176 (1998).</li><li>Kook, S. H., Son, Y. O., Lee, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+affects+positively+the+proliferation+of+bovine+satellite+cells+and+their+myogenic+differentiation+through+up-regulation+of+MyoD.">Hypoxia affects positively the proliferation of bovine satellite cells and their myogenic differentiation through up-regulation of MyoD.</a> Cell. Biol. Int. 32, 871-878 (2008).</li><li>Knobloch, T. J., Madhavan, S., Nam, J., Agarwal, S., Agarwal, S. <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+chondrocytic+gene+expression+by+biomechanical+signals.">Regulation of chondrocytic gene expression by biomechanical signals.</a> Crit. Rev. Eukaryot. Gene. Expr. 18, 139-150 (2008).</li><li>Guo, J. F., Jourdian, G. W., MacCallum, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+growth+characteristics+of+chondrocytes+encapsulated+in+alginate+beads.">Culture and growth characteristics of chondrocytes encapsulated in alginate beads.</a> Connect. Tissue. Res. 19, 277-297 (1989).</li><li>Toegel, S., Huang, W., Piana, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+reliable+reference+genes+for+qPCR+studies+on+chondroprotective+action.">Selection of reliable reference genes for qPCR studies on chondroprotective action.</a> BMC. Mol. Biol. 8, 13-13 (2007).</li><li>Lin, Z., Fitzgerald, J. B., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+of+human+chondrocytes+during+passaged+monolayer+cultivation.">Gene expression profiles of human chondrocytes during passaged monolayer cultivation.</a> J. Orthop. Res. 26, 1230-1237 (2008).</li><li>Goessler, U. R., Bieback, K., Bugert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+chondrocytes+differentially+express+matrix+modulators+during+in+vitro+expansion+for+tissue+engineering.">Human chondrocytes differentially express matrix modulators during in vitro expansion for tissue engineering.</a> Int. J. Mol. Med. 16, 509-515 (2005).</li><li>Anat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 121, 107-118 (1976).</li></ol>

我々は、開示することは何もない。

このレポートでは、我々は人間の滑液の高濃度を含む培地で、3D環境でのヒト関節軟骨細胞の培養を可能にする方法を開発した。滑液は関節軟骨細胞が存在する関節腔、自然環境を構成する主要コンポーネントの一つです。しかし、滑液の粘度は、軟骨細胞の三次元の長期培養するための大きな課題となっています。粘性環境での3D構造でさえ栄養分布を維持するための課題を克服し、凝集を...

特定の試薬と機器の表： <table border="1"><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td>アルギン酸（アルギン酸ナトリウム塩） </td><td>シグマ</td><td> A2158 - 250G </td><td> 2.4％のソリューションは、40 ° Cで保存</td></tr><tr><td>粒状塩化カルシウム二水和物、 </td><td> JTベーカー</td><td> A19339 </td><td></td></tr><tr><td width="239" rowspan="2" >軟骨増殖培地</td><td width="254" rowspan="2" >ロンザ</td><td width="182" > CC - 3156（ベースのメ​​ディア） </td><td width="141" rowspan="2" ></td></tr><tr><td width="182" > CC - 4409（サプリメント） </td></tr><tr><td width="239" rowspan="2" >軟骨細胞分化のメディア</td><td width="254" rowspan="2" > Lonza </td><td width="182" > CC - 3226（ベースのメ​​ディア） </td><td width="141" rowspan="2" ></td></tr><tr><td width="182" > CC - 4408（サプリメント） </td></tr><tr><td>ヒト関節軟骨細胞</td><td>ロンザ</td><td > CC - 2550 </td><td></td></tr><tr><td> DAPI（4&#39;,6 -ジアミジノ-2 - フェニル二塩酸塩） </td><td>シグマアルドリッチ</td><td > D9542 </td><td></td></tr><tr><td> RNeasyミニキット（RNA抽出用） </td><td>キアゲン</td><td > 74104 </td><td></td></tr><tr><td> PCR試薬：SYBR -緑色</td><td>クアンタ</td><td > 95053-500 </td><td></td></tr><tr><td> 12mlの注射器</td><td>タイコ-ケンダルMonoject </td><td > 512852 </td><td></td></tr><tr><td> 22 - Gague注射針</td><td>タイコ-ケンダルL - Monoject </td><td > 8881 </td><td></td></tr><tr><td>顕微鏡</td><td>オリンポス</td><td > IX71 </td><td></td></tr><tr><td>プラットフォームのロッカー</td><td> Thermoscientific thermolyne </td><td >バリミックス</td><td></td></tr><tr><td></td><td></td><td ></td><td></td></tr><tr><td> プライマーの配列 </td><td></td><td ></td><td></td></tr><tr><td>コラーゲンIIaのフォワード</td><td> 5&#39; - TTC ATC CCA CCC TCT CAC AGT - 3&#39; </td><td ></td><td></td></tr><tr><td>コラーゲンIIA -逆</td><td> 5&#39; - CCTCTGCCTTGACCCGAA - 3&#39; </td><td ></td><td></td></tr><tr><td> MMP13フォワード</td><td> 5&#39; - TGT GCC CTT CTT CAC ACA GAC ACT - 3&#39; </td><td ></td><td></td></tr><tr><td> MMP13 -逆</td><td> 5&#39; - GAG AGC AGA CTT TGA GTC ATT GCC - 3&#39; </td><td ></td><td></td></tr><tr><td>カスパーゼ3フォワード</td><td> 5&#39; - TCA TTA TTC AGG CCT GCC GTG GTA - 3&#39; </td><td ></td><td></td></tr><tr><td>カスパーゼ3の逆</td><td> 5&#39; - TGG ATG AACのCAG GAG CCA TCC TTTの-3&#39; </td><td ></td><td></td></tr></table>

a 3d system for culturing human articular chondrocytes in synovial fluid

軟骨破壊は、変形性関節症の中心的な病理学的特徴、米国における障害の主要な原因です。成人の軟骨は、 生体内で非常に効率的に再生成されません、そしてその結果として、変形性関節症は軟骨の損失を不可逆的につながると慢性的な痛みと不動の1,2が付属しています。軟骨組織のエンジニアリングは、組織機能を再生成し、復元するために有望な可能性を提供しています。この技術は、通常、結果として得られる3Dは、エンジニアリングの目標生体 3-6 に欠損部位に移植することができる生化学的および生体力学的に成熟した組織で、ある期間にわたってバランスのとれた培地で構築する天然または合成の足場と培養に播種した軟骨細胞を含む。軟骨細胞の成長とマトリックスの沈着のための最適な条件を達成することが軟骨の組織工学の成功に不可欠です。 北極でのネイティブ関節腔、軟骨の骨のウラル表面は滑液に包まれている。この透明で粘性のある流体には、無血管性の関節軟骨に栄養を提供し、軟骨細胞の代謝7,8にとって重要な成長因子、サイトカインや酵素が含まれています。さらに、滑液は主に分泌する2つの主要なコンポーネント、ヒアルロン酸とlubricin 9 10を通じて、軟骨表面の間に低摩擦の移動が容易になります。対照的に、軟骨培養組織は、最も頻繁に人​​工培地で培養される。これらのメディアは、おそらく軟骨細胞代謝を研究するための複数の定義された条件を提供することができますが、関節液は、最も正確に関節軟骨細胞が入って置かれての自然環境を反映している確かに、滑液は、取得および保存するために容易であるという利点があり、多くの場合、定期的に体内で補充することができます。いくつかのグループが増殖するヒト、ウシ、ウサギおよびイヌcで滑液で培養液を補充しているhondrocytes、そのほとんどは滑液（20％以下）11月25日の唯一の低レベルを使用していました。鶏、馬と人間の軟骨細胞が滑液のより高い割合と培地で培養されているが、これらの培養システムは、2次元の26から28でした。ここでは、21日の期間にわたって滑液の割合が高く（最大100％）で、3Dシステムで培養ヒト関節軟骨細胞の我々の手法を提案する。そうすることで、我々は滑液の高粘度によって提示される主要なハードルを克服した。このシステムは、さらなるための自然環境を模倣するために軟骨のための自然環境を構成する2つの他の重要な要因（酸素張力および機械的荷重）29,30と組み合わせることができる3D設定で滑液中に人間の軟骨細胞を研究の可能性を、提供します。軟骨の成長。さらに、このシステムはまた、軟骨細胞の滑液の活性を測定するために使用し、開発するためのプラットフォームを提供することができます軟骨再生技術や関節炎の治療オプション。

Watch this Scientific Journal Video about A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid at JoVE.com

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

滑液の高濃度の培養ヒト関節軟骨細胞の3次元システムについて説明する。滑液は関節軟骨のための最も自然な微小環境を反映し、容易に入手し、保存することができます。このシステムは、このように軟骨再生の研究や、関節炎を治療するための治療薬をスクリーニングするために使用することができます。

滑液における培養ヒト関節軟骨細胞用の3Dシステム

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

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Research

JoVE Journal

Biology

20.7K ビュー.  Tufts University School of Medicine. 滑液の高濃度の培養ヒト関節軟骨細胞の3次元システムについて説明する。滑液は関節軟骨のための最も自然な微小環境を反映し、容易に入手し、保存することができます。このシステムは、このように軟骨再生の研究や、関節炎を治療するための治療薬をスクリーニングするために使用することができます。

ビデオ: A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

イモリのレンズ再生を研究するために培養虹彩色素上皮細胞のためのシステム

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

生物学

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

小分子の誘導と多能性ヒト胚性幹細胞からヒト心筋前駆体と心筋細胞の効率的な導出

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

のためのシステム<em&gt; ex vivoで</em胚膵臓&gt;培養

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

Internal organs such as the heart, brain, and gut develop left-right (LR) asymmetries that are critical for their normal functions1. Motile cilia are involved in establishing LR asymmetry in vertebrate embryos, including mouse, frog, and zebrafish2-6. These 'LR cilia' generate asymmetric fluid flow that is necessary to trigger a conserved asymmetric Nodal (TGF-&beta; superfamily) signaling cascade in the left lateral plate mesoderm, which is thought to provide LR patterning information for developing organs7. Thus, to understand mechanisms underlying LR patterning, it is essential to identify genes that regulate the organization of LR ciliated cells, the motility and length of LR cilia and their ability to generate robust asymmetric flow.
In the zebrafish embryo, LR cilia are located in Kupffer's vesicle (KV)2,4,5. KV is comprised of a single layer of monociliated epithelial cells that enclose a fluid-filled lumen. Fate mapping has shown that KV is derived from a group of ~20-30 cells known as dorsal forerunner cells (DFCs) that migrate at the dorsal blastoderm margin during epiboly stages8,9. During early somite stages, DFCs cluster and differentiate into ciliated epithelial cells to form KV in the tailbud of the embryo10,11. The ability to identify and track DFCs&mdash;in combination with optical transparency and rapid development of the zebrafish embryo&mdash;make zebrafish KV an excellent model system to study LR ciliated cells.
Interestingly, progenitors of the DFC/KV cell lineage retain cytoplasmic bridges between the yolk cell up to 4 hr post-fertilization (hpf), whereas cytoplasmic bridges between the yolk cell and other embryonic cells close after 2 hpf8. Taking advantage of these cytoplasmic bridges, we developed a stage-specific injection strategy to deliver morpholino oligonucleotides (MO) exclusively to DFCs and knockdown the function of a targeted gene in these cells12. This technique creates chimeric embryos in which gene function is knocked down in the DFC/KV lineage developing in the context of a wild-type embryo. To analyze asymmetric fluid flow in KV, we inject fluorescent microbeads into the KV lumen and record bead movement using videomicroscopy2. Fluid flow is easily visualized and can be quantified by tracking bead displacement over time.
Here, using the stage-specific DFC-targeted gene knockdown technique and injection of fluorescent microbeads into KV to visualize flow, we present a protocol that provides an effective approach to characterize the role of a particular gene during KV development and function.

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer&#039;s Vesicle

Cilia-generated fluid flow in Kupffer&#x2019;s Vesicle (KV) controls left-right patterning of the zebrafish embryo. Here, we describe a technique to modulate gene function specifically in KV cells. In addition, we show how to deliver fluorescent beads into KV to visualize fluid flow.

クッパー胞における遺伝子機能と繊毛発生する流体の流れの可視化の分析

Internal organs such as the  heart, brain, and gut develop left-right (LR) asymmetries that are critical for  their normal functions1. Motile cilia are ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

エア·液界面での人間の鼻上皮細胞の培養

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

ギャップ結合細胞間コミュニケーション：毒物および毒素の有害効果を評価するための機能バイオマーカー、および天然物の健康上の利点

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

「せん断リング」の組み立てと応用：小説内皮軌道、単方向および定期的な流動のためのモデルとせん断応力

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

磁気活性化細胞を分離し、滑膜液由来間葉系幹細胞が家兎モデルからの浄化戦略を並べ替え

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is compartmentalized into crypts and villi: the stem and proliferating cells are confined to the crypts, whereas the villi epithelium contains only differentiated cells. Hence, the normal intestinal crypts, but not the villi, can give rise to organoids in 3D cultures. The procedure described here is applicable only to villus epithelium undergoing dedifferentiation leading to stemness. The method described uses the Smad4-loss-of-function:&#946;-catenin gain-of-function (Smad4KO:&#946;-cateninGOF) conditional mutant mouse. The mutation causes the intestinal villi to dedifferentiate and generate stem cells in the villi. Intestinal villi undergoing dedifferentiation are scraped off the intestine using glass slides, placed in a 70 &#181;m strainer and washed several times to filter out any loose cells or crypts prior to plating in BME-R1 matrix to determine their organoid-forming potential. Two main criteria were used to ensure that the resulting organoids were developed from the dedifferentiating villus compartment and not from the crypts: 1) microscopically evaluating&#160;the isolated villi to ensure absence of any tethered crypts, both before and after plating in the 3D matrix, and 2) monitoring the time course of organoid development from the villi. Organoid initiation from the villi occurs only two to five days after plating and appears irregularly shaped, whereas the crypt-derived organoids from the same intestinal epithelium are apparent within sixteen hours of plating and appear spherical. The limitation of the method, however, is that the number of organoids formed, and the time required for organoid initiation from the villi vary depending on the degree of dedifferentiation. Hence, depending upon the specificity of the mutation or the insult causing the dedifferentiation, the optimal stage at which villi can be harvested to assay their organoid forming potential, must be determined empirically.

The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

腸内ビリ上皮によるオルガノイドの3D培養

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...