この作品は、臨床およびトランスレーショナル科学のための卵巣癌研究基金[助成金LT/UIC/01.2011]、UICセンター、UICがんセンター、および保健助成金C06RR15482の国立研究所によってサポートされていました。 動物実験は、UICで動物実験委員会が承認した動物のケアのプロトコルの下でAAALACによって定められたガイドラインおよび規制に準拠して行った。このプロジェクトのための動物は、シカゴのイリノイ大学で生物資源研究所（BRL）でバリア室に収容されています。 BRLは、少なくとも1日2回、動物を監視し、動物のケアに関するアドバイスを提供する完全に獣医スタッフがいます。

1。卵巣解剖と組織の分離<ol><li>アルギン酸ナトリウムの0.5％（w / v）の溶液を調製、プロトコールの修正版から準備滅菌PBS、37への熱℃を使用して、これまで5に記載反転またはロッキングによるミックスが、ボルテックスしないでください。 25 Gの針を1mlのシリンジにアルギン酸を描画し、37℃に維持</li><li>架橋組織のカプセル化時にアルギン酸および37に暖かい℃で10mMの無菌CaCl 2溶液を準備</li><li>各実験のための24ウェルプレートにウェル当たりの培地1mLの（αMEM+ 5単位ペニシリン、5μg/ mlのストレプトマイシン）を準備する。種々の薬物、ペプチド、または培養液中のウイルスが含まれています。前の臓器のカプセル化にトランスフェクションのために、37℃で4時間のリポフェクトアミン2000とプラスミドDNAと組織をインキュベート</li><li>動物から臓器を解剖し、任意の脂肪や残存組織を除去する、軽く卵巣周囲の嚢の膜をやってのける、とオルガンを二分することによって4つの部分に卵巣や卵管をカット。動物を頸部脱臼に続いてCO 2窒息により安楽死させた。卵管の場合は、軽く結ぶ膜を離れて引っ張り、切断する前にピンセットを使用して反対方向にチューブの端を拡張することによって組織を解く。 </li><li>滅菌メッシュ繊維の上に単一のアルギン酸の液滴（〜1月3日μL）を置きます。 </li><li>滅菌ピンセットを使用して、アルギン酸の液滴と中央に鉗子を用いて液滴または無菌の注射針を臓器の一部を移動する。 </li><li>滅菌ピンセットでメッシュの繊維を把握し、重力のハンギングドロップにアルギン酸を強制的に許可するように上下逆さまにそれを回して、臓器の一部（オルガノイド）を含む液滴を反転。 </li><li>ドロップは、溶液中に入るように10mMのCaCl 2溶液を含む10cmのペトリ皿の端に鉗子をタップします。 </li><li> 2分間インキュベートし、培地中に余分な10mMのCaCl 2で 、転送を解放するために毛穴を含有する滅菌スプーンで取り除く。最大4つのアルギン酸ゲルへの培地を含む24ウェルプレートの1ウェルに移すことができます。定義された期間のための文化。 </li></ol> 2。細胞のアルギン酸とコレクションの劣化<ol><li>アルギン酸ゲルの可溶化のために、リーボビッツのL - 15 37メディア℃で30分間またはゲルが完全に溶解するまで、Cに溶解したアルギン酸リアーゼの文化（3.4 mg / mL）をインキュベートする。 </li><li>アルギン酸リアーゼから組織を削除します。 </li><li> OSEのコレクションの場合、1時間8リーボビッツのL - 15メディアにおけるコラゲナーゼの500台とオルガノイドをインキュベートする。 1分1秒パルス（1秒間ボルテックスし、1秒休息）に続いて1分間ミディアムパワーで2秒のパルス（2秒ボルテックスし、2秒休息）、の渦。臓器片はマイクロ遠心チューブと新しい滅菌マイクロ遠心チューブに上清をピペットの底に落としてください。ペレットの卵巣の表面上皮に3000 rpmで3分間遠心操作する。 </li><li>卵管上皮のコレクションの場合、5単位のペニシリン、5μg/ mlのストレプトマイシン、4％（v / v）のFBSを含むDMEM中で組織培養プラスチック上縦チューブをカットし、場所。 3-5日にわたり、細胞は卵管間質から&quot;クロール&quot;し、組織培養plastic9に成長する。 </li></ol> 3。パラフィン切片用アルギン酸カプセル化された器官培養の固定<ol><li>選択した時点で培養液からビーズをカプセル化アルギン酸を収集する。 </li><li> 2分間滅菌した10mMのCaCl 2溶液中に文化をドロップすることによってゲルを堅くするアルギン酸をRecrosslink。 </li><li>ゲルは固体のまま、完全に固定の間組織をカプセル化することを確実に50 mMスクロース、10mMのCaCl 2及び50mMのカコジル酸ナトリウムを含有する2％パラホルムアルデヒドでアルギン酸カプセル化された臓器を修正。 </li><li>組織の処理に続いて、5μmのセクションを生成するためにミクロトームでパラフィンやセクションに全体のアルギン酸でカプセル化された器官培養片を埋め込みます。 </li><li>アルギン酸塩を溶解し、ベースの抗原の検索を加熱するため、免疫組織化学を実行する場合は、スライドから削除されます。アルギン酸塩が残っていると立ってヘマトキシリン​​とエオシン染色でエオジンのために染色される。 </li></ol> 4。代表的な結果： 卵巣と卵管の三次元器官培養の例を図1に示されています。卵巣は、異なる大きさの片に切断し、OSEは増殖し、メスで切開から負傷した表面を修復するために移行することができます。このプロセス中に、基盤となる間質に対する相対OSEの正しい向きは、卵巣をカプセル化しているOSEで、維持されます。図2に示すように、四半期またはeighthsにカット卵巣に比べて、半分にカット卵巣のOSEは、より完全に卵巣をカプセル化します。卵巣の傷の修復表面は全体の卵巣の文脈で検討することができ、排卵と表面修復が卵巣の表面の変形を誘導する方法に光を当てることができる。 実験のエンドポイントが必要とされる情報によって異なります。組織構造、細胞形態、および特定の細胞の成長率は、簡単にパラフィン切片を使用して監視されます。 7日間、5μg/ mlのインスリンの添加が、卵巣の表面上皮との複数の層の形成の過剰増殖を誘導しながら例えば、卵巣の表面上皮の単層は、7日間（図3A）の無血清αMEMで培養後に観察され細胞（図3B）。固定前に10μMの24時間の最終濃度で培養物にブロモデオキシウリジンを追加することによって、細胞の増殖は（挿入図3Aおよび3B）を測定することができます。顆粒膜細胞の割合が大きく、増殖しながら、無血清αMEMで維持培養のため、OSEの約3〜5％が、24時間の標識期間（挿入図3A）の間に増殖を受ける。培地にインスリンを添加すると、合計OSE（挿入図3B）の約30％にOSEを増殖の割合が高くなります。緑色蛍光タンパク質（GFP）の免疫蛍光は、cDNAをトランスフェクション（図3C）は、次の標準的な顕微鏡とアルギン酸ゲルを介して可視化することができます。アルギン酸の器官培養では卵巣の表面上皮と一次卵胞の成長と分析のために提供していますが、それは成熟した、二次卵胞は、この培養系（図3D）で生き残るために失敗することに留意すべきである。アルギン酸ハイドロゲルマトリックス内に満期までの個々の卵胞の培養は、他の場所で10記載されている。 タンパク質やRNAの変化は、全体の臓器または酵素的分解（図4）を使用して特定の細胞型から分析することができます。一部の間質細胞はまた、（図4D）絶縁されているものの、コラゲナーゼと培養卵巣オルガノイドの治療は、ペレットは、以下の処置を得られた細胞のOSEの濃縮を可能にしながら（図4C）無傷の基本的な間質を残します。 <img alt="図1" src="/files/ftp_upload/2804/2804fig1.jpg" /> 負傷後のOSEを伝播するために使用される図1。マウスの卵巣三次元培養システム。 A）顕微鏡下では、嚢と周囲の脂肪パッドは削除されました。 B）はそのまま卵管チューブと卵巣（O）（T）と脂肪パッド（F）。 C）ブルサと脂肪パッドで卵巣を除去（左）と巻かれていない卵（右）。 D）卵巣や卵管オルガノイドは、アルギン酸の液滴に入れた。 E）アルギン酸包まオルガノイドは、CaCl 2溶液に架橋ゲルを形成するために、アルギン酸塩に滴下した。 F）アルギン酸カプセル化された卵巣オルガノイドは。 G）アルギン酸は、卵管オルガノイドをカプセル化。 H）アルギン酸カプセルオルガノイドは、培養培地中でインキュベートした。 <img alt="図2" src="/files/ftp_upload/2804/2804fig2.jpg" /> 図2表面領域は、サイトケラチン8によって測定された卵巣の表面が再生成されます。 eighths、四半期、半分にカットされた後、OSEによる卵巣オルガノイドのカプセル化は、BSA培地で培養し、サイトケラチン8（CK8）抗体を用いた免疫組織化学分析により測定。 <img alt="図3" src="/files/ftp_upload/2804/2804fig3.jpg" /> 図3。アルギン酸の培養系は、成長因子の通過とOSEへのcDNAのトランスフェクションすることができますが、卵胞の急速な成長と成熟を ​​サポートしていません。 A）卵巣オルガノイドは、OSEをマークCK8表現、について分析した基礎培地で7日間培養。はめ込み、シリアルセクションでは、BrdUの取り込みのために染色。 B）培地にインスリンの添加は、OSEの過形成を誘導する。はめ込み、シリアルセクションでは、BrdUの取り込みのために染色。 C）卵巣オルガノイドは、GFPを発現するようにするcDNAでトランスフェクション。 D）アルギン酸の培養系は、原始卵胞（赤矢印）の成長をサポートしますが、成熟していない二次卵胞（黒矢印）。 <img alt="図4" src="/files/ftp_upload/2804/2804fig4.jpg" /> 図4。OSEは、培養後分析のために単離することができる。 A）オルガノイドは最大2週間のための培地で培養する。 B）カプセル化オルガノイドは、オルガノイドをそのままにしておくアルギン酸リアーゼと扱われます。卵巣は、OSEをマークするCK8で染色される。 C）オルガノイドは、基礎となる間質からOSEを分離するコラゲナーゼで処理される。残りの組織片は、卵巣表面の除去を強調するためにCK8で染色した。 CK8（赤）を染色としてD）処理後に回収した細胞は、OSEのために濃縮されます。細胞は核をマークするためにDAPIで対比されています。

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	<li>Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2009.">Cancer statistics, 2009.</a> CA Cancer J Clin. 59, 225-249 (2009).</li><li>Auersperg, N., Woo, M. M., Gilks, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+ovarian+carcinomas:+a+developmental+view.">The origin of ovarian carcinomas: a developmental view.</a> Gynecol Oncol. 110, 452-454 (2008).</li><li>Crum, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distal+fallopian+tube:+a+new+model+for+pelvic+serous+carcinogenesis.">The distal fallopian tube: a new model for pelvic serous carcinogenesis.</a> Curr Opin Obstet Gynecol. 19, 3-9 (2007).</li><li>Jackson, K. S., Inoue, K., Davis, D. A., Hilliard, T. S., Burdette, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+ovarian+organ+culture+as+a+tool+to+study+normal+ovarian+surface+epithelial+wound+repair.">Three-dimensional ovarian organ culture as a tool to study normal ovarian surface epithelial wound repair.</a> Endocrinology. 150, 3921-3926 (2009).</li><li>Rowley, J. A., Madlambayan, G., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate+hydrogels+as+synthetic+extracellular+matrix+materials.">Alginate hydrogels as synthetic extracellular matrix materials.</a> Biomaterials. 20, 45-53 (1999).</li><li>Augst, A. D., Kong, H. J., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate+hydrogels+as+biomaterials.">Alginate hydrogels as biomaterials.</a> Macromol Biosci. 6, 623-633 (2006).</li><li>Amsden, B., Turner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+characteristics+of+calcium+alginate+gels.">Diffusion characteristics of calcium alginate gels.</a> Biotechnol Bioeng. 65, 605-610 (1999).</li><li>Symonds, D. A., Miller, K. P., Tomic, D., Flaws, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+methoxychlor+and+estradiol+on+cytochrome+p450+enzymes+in+the+mouse+ovarian+surface+epithelium.">Effect of methoxychlor and estradiol on cytochrome p450 enzymes in the mouse ovarian surface epithelium.</a> Toxicol Sci. 89, 510-514 (2006).</li><li>Umezu, T., Hanazono, M., Aizawa, S., Tomooka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+newly+established+clonal+oviductal+cell+lines+and+differential+hormonal+regulation+of+gene+expression.">Characterization of newly established clonal oviductal cell lines and differential hormonal regulation of gene expression.</a> In Vitro Cell Dev Biol Anim. 39, 146-156 (2003).</li><li>Xu, M., Kreeger, P. K., Shea, L. D., Woodruff, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+follicles+produce+live,+fertile+offspring.">Tissue-engineered follicles produce live, fertile offspring.</a> Tissue Eng. 12, 2739-2746 (2006).</li><li>Wilson, R. F., Wiencek, R. G., Balog, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+and+preventing+infection+after+abdominal+vascular+injuries.">Predicting and preventing infection after abdominal vascular injuries.</a> J Trauma. 29, 1371-1375 (1989).</li><li>Xu, 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+Oocyte+Maturation+and+Preantral+Follicle+Culture+from+the+Luteal+Phase+Baboon+Ovary+Produce+Mature+Oocytes.">In Vitro Oocyte Maturation and Preantral Follicle Culture from the Luteal Phase Baboon Ovary Produce Mature Oocytes.</a> Biol Reprod. , .</li><li>Xu, 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+grown+human+ovarian+follicles+from+cancer+patients+support+oocyte+growth.">In vitro grown human ovarian follicles from cancer patients support oocyte growth.</a> Hum Reprod. 24, 2531-2540 (2009).</li><li>Jen, A. C., Wake, M. C., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Hydrogels+for+cell+immobilization.">Review: Hydrogels for cell immobilization.</a> Biotechnol Bioeng. 50, 357-364 (1996).</li><li>Iwamoto, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+bifunctional+alginate+lyase+from+Alteromonas+sp.+strain+no.+272+and+its+action+on+saturated+oligomeric+substrates.">Purification and characterization of bifunctional alginate lyase from Alteromonas sp. strain no. 272 and its action on saturated oligomeric substrates.</a> Biosci Biotechnol Biochem. 65, 133-142 (2001).</li><li>Auersperg, N., Maines-Bandiera, S. L., Dyck, H. G., Kruk, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cultured+human+ovarian+surface+epithelial+cells:+phenotypic+plasticity+and+premalignant+changes.">Characterization of cultured human ovarian surface epithelial cells: phenotypic plasticity and premalignant changes.</a> Lab Invest. 71, 510-518 (1994).</li><li>Auersperg, N., Ota, T., Mitchell, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+events+in+ovarian+epithelial+carcinogenesis:+progress+and+problems+in+experimental+approaches.">Early events in ovarian epithelial carcinogenesis: progress and problems in experimental approaches.</a> Int J Gynecol Cancer. 12, 691-703 (2002).</li><li>Kruk, P. A., Uitto, V. J., Firth, J. D., Dedhar, S., Auersperg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+interactions+between+human+ovarian+surface+epithelial+cells+and+adjacent+extracellular+matrix.">Reciprocal interactions between human ovarian surface epithelial cells and adjacent extracellular matrix.</a> Exp Cell Res. 215, 97-108 (1994).</li><li>West, E. R., Xu, M., Woodruff, T. K., Shea, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+properties+of+alginate+hydrogels+and+their+effects+on+in+vitro+follicle+development.">Physical properties of alginate hydrogels and their effects on in vitro follicle development.</a> Biomaterials. 28, 4439-4448 (2007).</li><li>Auersperg, N., Wong, A. S., Choi, K. C., Kang, S. K., Leung, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+surface+epithelium:+biology,+endocrinology,+and+pathology.">Ovarian surface epithelium: biology, endocrinology, and pathology.</a> Endocr Rev. 22, 255-288 (2001).</li><li>Choi, J. H., Wong, A. S., Huang, H. F., Leung, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonadotropins+and+ovarian+cancer.">Gonadotropins and ovarian cancer.</a> Endocr Rev. 28, 440-461 (2007).</li><li>Fathalla, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incessant+ovulation--a+factor+in+ovarian+neoplasia.">Incessant ovulation--a factor in ovarian neoplasia.</a> Lancet. 2, 163-163 (1971).</li><li>Espey, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+the+hypothesis+that+mammalian+ovulation+is+comparable+to+an+inflammatory+reaction.">Current status of the hypothesis that mammalian ovulation is comparable to an inflammatory reaction.</a> Biol Reprod. 50, 233-238 (1994).</li></ol>

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アルギン酸ハイドロゲルを利用した三次元器官培養系の開発は、生物の多種多様な多くの異なる組織型を分析するための汎用的な方法を表します。それは細胞が11を成長することのできる足場を提供するので、3次元培養の使用は、組織工学と再生医療の分野に拡張することができます。現在、私たちの研究室ではヒト卵巣と卵管組織を用いた初期の研究が行われているが、ヒトおよ?...

<table border="1"><tbody><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（省略可能） </th></tr><tr><td>リーボビッツL - 15培地</td><td>ギブコ</td><td> 11415 </td><td></td></tr><tr><td> αMEM培地</td><td>ギブコ</td><td> 12000-022 </td><td></td></tr><tr><td>アルギン酸ナトリウム</td><td> Novamatrix </td><td></td><td></td></tr><tr><td>カコジル酸ナトリウム</td><td>シグマアルドリッチ</td><td> C0250 </td><td></td></tr><tr><td>ポリプロピレンメッシュ繊維</td><td>マクマスターカー</td><td></td><td> 45&quot;ワイドロール、0.0041&quot;オープニング</td></tr><tr><td>リポフェクタミン2000 </td><td>インビトロジェン</td><td> 11668 </td><td></td></tr><tr><td>ペニシリン/ストレプトマイシン</td><td>ギブコ</td><td> 15070-063 </td><td></td></tr><tr><td>アルギン酸リアーゼ</td><td>シグマアルドリッチ</td><td> A1603 </td><td></td></tr><tr><td> A1603 </td><td>シグマアルドリッチ</td><td> C9697 </td><td> クロストリジウムヒストリから</td></tr><tr><td>ウシ血清アルブミン</td><td> MPのバイオメディカル</td><td> 103700 </td><td></td></tr><tr><td>ウシ胎仔血清</td><td>ギブコ</td><td>ギブコ</td><td></td></tr><tr><td> ITSソリューション</td><td>ロッシュ</td><td> 11074547001 </td><td>インスリン/トランスフェリン/亜セレン酸ナトリウム</td></tr><tr><td>サイトケラチン8抗体</td><td>発達研究ハイブリドーマバンク</td><td> TROMA - 1 </td><td> 1:200で使用</td></tr><tr><td> DAPI </td><td>ベクトル研究所</td><td> H1200 </td><td></td></tr><tr><td>ブロモデオキシウリジン</td><td>シグマ</td><td> B50002 - 1G </td><td>最終濃度10μM </td></tr></tbody></table>

alginate hydrogels for three-dimensional organ culture of ovaries and oviducts

卵巣がんは女性のがん死亡の第五の主要な原因であり、アメリカ1位63％の死亡率を持っています。卵巣癌の起源の細胞型は、質問のままですし、どちらか卵巣表層上皮（OSE）または卵管の線毛2,3の先端部上皮かもしれません。 in vitroでの初代培養のような正常細胞の培養は、科学者はそれによって決定的に起源の細胞型を決定する、明確な上皮で卵巣癌につながる可能性のある特定の変更をモデル化することが可能になります。これはこの病気を対象としたより正確なバイオマーカー、組織特異的遺伝子の変化と動物モデル、そしてより良い予防戦略の開発が可能になります。 アルギン酸ヒドロゲルの正常な細胞を維持することは、その三次元のコンテキスト内で細胞の体外培養において短期を促進し、プラスミドDNA、siRNAを、そして小分子の導入を可能にする。単一の臓器からメス、複数の文化を使って戦略的なカットから派生している部分での培養臓器によっては、単一の動物からの実験の数を増やし、生成することができます。これらは、卵巣癌の形成に関連付けられているOSEの増殖につながる排卵のモデルの側面をカットします。このようなプラスチックの表面にも成長していないOSEなどの細胞型は、この方法を用いて培養し、正常な細胞プロセスまたは癌の形成4の初期のイベントに調査を容易にすることができます。 アルギン酸ヒドロゲルは、組織の5の多くの種類の成長をサポートするために使用することができます。アルギン酸は、損傷組織6,7をしない穏やかなゲル化作用で、その結果、β- D -マンヌロン酸とカルシウムイオンで ​​架橋することができるα- L -グルロン酸の繰り返し単位から成る線状多糖類である。このようなマトリゲルなどの他の三次元細胞培養マトリックスのように、アルギン酸は、組織のための機械的なサポートを提供しますが、タンパク質はアルギン酸マトリックスと反応しないため、セルが5をシグナリング開始しない合成細胞外マトリックスとしてアルギン酸機能。アルギン酸ハイドロゲルは、標準的な細胞培養培地中に浮遊してin vitroでの組織の成長のアーキテクチャをサポートしています。 方法は準備、分離、および最大2週間の培養で維持することができるアルギン酸ヒドロゲル、に卵巣と卵管臓器片の埋め込みのために提示されます。器官培養からタンパク質とRNAサンプルの分析のための細胞の酵素放出についても説明します。最後に、主要な細胞型の成長は、マウスからの遺伝的不死化することなく可能であり、ノックアウトやトランスジェニックマウスを使用するために捜査官を許可します。

Watch this Scientific Journal Video about Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts at JoVE.com

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

それらの三次元コンテキストで正常な細胞の培養は、細胞の形質転換および腫瘍形成に必要な初期の事象を研究するための代替メソッドを表します。このメソッドは、卵巣癌の形成の初期イベントを研究するために、通常の卵巣と卵管の細胞を成長させるために使用されます。

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 ...

alginate-hydrogels-for-three-dimensional-organ-culture-ovaries

Research

JoVE Journal

Bioengineering

21.8K ビュー.  University of Illinois at Chicago. それらの三次元コンテキストで正常な細胞の培養は、細胞の形質転換および腫瘍形成に必要な初期の事象を研究するための代替メソッドを表します。このメソッドは、卵巣癌の形成の初期イベントを研究するために、通常の卵巣と卵管の細胞を成長させるために使用されます。

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

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

導電性インクの平面および三次元印刷

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

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  ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

腫瘍細胞の浸潤を間質液流れに及ぼす影響を測定するための三次元細胞培養モデル

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

三次元コラーゲンマトリックス内の細胞移動の解析

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三次元の全層皮膚等価物と自動傷つけ処理の発生

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

三次元バイオミメティック・テクノロジー：小説Biorubberはコラーゲンハイドロゲルで定義されたマイクロ・マクロスケールのアーキテクチャを作成します。

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

肝組織工学のための三次元細胞培養プラットフォーム：逆コロイド結晶ポリ（エチレングリコール）足場の作製

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

階層化アルギン酸構築：異種細胞集団の共培養のためのプラットフォーム

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

細胞培養アプリケーションのためのヒドロゲルの光媒介形成とパターニング

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

3D細胞培養のための肺細胞外マトリックスから調整可能なヒドロゲル

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...