我々はbioprinterのヘルプはデボラステューダーに感謝します。 作業は、付与契約にN下、欧州連合第七フレームワークプログラム（FP7/2007-2013）°NMP4-SL-2009から229292によって資金を供給された。

1。ポロキサマー407溶液の調製可能であれば、寒い部屋（4℃）でポロキサマー溶液の調製を行います。利用できない場合には、氷のように冷たい水で満たされたビーカーにガラスボトルを置きます。より高い温度では、ポロキサマーは、ゲル化点の上になり、適切に溶解しない。 <ol><li>ガラス瓶に氷冷PBS溶液を60mlを加え、マグネチックスターラーを用いて積極的にかき混ぜる。 </li><li>ポロキサマーの24.5グラムを秤量し、冷PBSに少量でそれを追加。ポロキサマーは、部分的に、よりを追加する前に溶解するまで待ちます。 </li><li>すべてのポロキサマーが溶解するまでのソリューションをかき混ぜる。 </li><li> 100mlと最終体積になるまで冷PBSを加える。最終濃度は24.5％W / Vになります</li><li>溶液を攪拌停止し、溶液中の気泡や泡が消失するまで4℃で残り、それを聞かせて。ゲル内に捕捉された気泡は、pに転送されるrinterカートリッジとプリント犠牲型の欠陥につながる。 </li><li>フィルター（0.22μmのフィルター）を直接針を詰まらせる可能性のある不要な粒子を除去するために、印刷カートリッジにソリューション。フィルタリングステップは、フィルタ内のポロキサマーのゲル化を回避するために、低温室（またはいない場合は冷却のヒント、フィルタなどで使用可能）で実行する必要があります。 °Cまで30分実験前4にロードされたカートリッジを保管してください。 </li></ol> 2。 3Dプリンタの準備この研究で使用される3DプリンタはregenHUから &quot;BioFactory&quot;だった。システムの押し出し部分はいくつかの部分から構成されています。上部の圧力下カートリッジはルアーロックアダプタを介してコネクタに取り付けられている。コネクタは、カートリッジの出口と電磁弁の入口との間のスペースを埋める。電磁弁の出口で、異なる直径を有する針を使用することができる。材料はサブに押し出され真空により移動ステージに保持されている戦略。システムの主要な部分は、図1に示されている。他の押し出しベースのシステムは、印刷処理に使用され、最適化プロセスは、システムごとに行う必要があることができる。 <ol><li>超純水で満たされた別の1.5ml試験管に電磁弁（ノズル径0.3ミリメートル）と針（内径0.15ミリメートル）を配置し、30分間洗浄する温水超音波浴中に置いてください。エタノールで洗浄バルブを洗浄し、窒素銃でそれらを乾燥させます。 </li><li>バルブとニードルプリンタと同様、空、きれいなカートリッジを取り付けます。 </li><li>システムに3バールの圧力を適用し、圧縮空気でインス​​トールバルブとニードルから任意の残留液体を吹き消す。小さな針径のために、針を詰まらせる可能性が小さい粒子の侵入を避けるために、圧縮空気の出口に設置されたフィルタ（共通シリンジフィルター、0.45μmの孔径）を有することが推奨される。 </lI&gt; <li>圧力をオフにして、ポロキサマーでロードカートリッジを取り付けます。ポロキサマーは、室温とゲルに達することができるので、カートリッジがカートリッジを取り付ける前に、約30分冷蔵庫から取り出しておく必要があります。 </li><li>システムに3バールの圧力を印加して、針の先端に到達し、連続ストランドに押出されるまで、ポロキサマーを分配する。 </li></ol> 3。印刷パラメータの最適化正確な3次元構造を作成するには、印刷プロセスは、選択された材料および濃度を最適化しなければならない。粘度および3D印刷システムに応じて、各材料は、パラメータの固定されたセットのための特定の小出し体積と線の太さが得られます。 <ol><li>適切なCADソフト（図面からISOファイルを作成することができる）を使用すると、印刷しようとしている構造と同じ長さについて、単一の線を引く。 </li><li>顕微鏡のスライドガラス25ミリメートル×75ミリメートル×1を置きミリメートルまたはプリンタ内の他の基板と真空をオンにすることによって固定します。 </li><li>プリンタソフトウェアでは、50ヘルツの高周波に電磁弁を設定し、3バールの高圧に設定してください。 </li><li> 300mm /分のステージ速度を持つ単一の行の一つの層を印刷します。 </li><li>所望の線幅に到達するまで圧力を減らす。また、バルブの開放時間を経由して押出されるボリュームを制御することができます。 </li><li>連続的なラインはもう印刷できなくなるまでバルブの頻度を減らす。この値以上の周波数を選択してください。 </li></ol>注：所望の線幅と実線が達成されると、ある印刷層の後に最適なステージ速度とニードルのリフトすなわち、層の厚さを決定する。 <ol start="7"><li>互いの上にいくつかの層を印刷して、針がいくつかの印刷層の後に前の層の上の正しい位置にあるかどうか。層の厚さ（ニードルリフトを調整する）が各層は次のもの（ 図3）の上に印刷されている。 </li><li> 300mm /分からのステージのステージ速度を下げ押出層は以前のもの（ 図4）と同じ位置で開始および終了するように段階的に。高すぎるステージ速度は、押出材は、前の層に触れた前にステージが動いているように原因となる。 </li><li> 。柱構造は3.1.-3.8の手順に従って印刷するためではなく、単一の行を描画する単一のポイントを描画します。柱を印刷する際に集中するパラメータは、柱が堆積されるべき位置に印字ヘッドの圧力（層厚およびポロキサマーの柱の直径を調節）、弁の開時間（押出量）と滞留時間である。 </li><li>パラメータが最適化されるときに、ラインのいくつかの層を印刷すると、固体壁に、またはポイント、ピラーの場合にもたらすべきである。後で使用するためのパラメータを保存します。 </li></ol>この点についての最適化手順の間に発見されたパラメタを使用します。 <ol><li>ガラス顕微鏡スライド上の内部構造（ここでは、それは柱の配列です）を印刷して、それが一晩乾燥させます。この）は、構造体のサイズおよび厚さを減少させ、b）に回避することができるリフトオフ埋め戻し時のように、構造体と基板との間のより良好な接着を提供する。 </li><li> CADソフトウェアでは、あなたが離れて溶出し、記入したつもりな構造を囲む外壁で構成されて構造を描画します。ポロキサマーを持つ構造体を印刷します。壁の印刷は6分かかります。 </li></ol>注意：壁があるため、針の大きさの内部構造から少なくとも3.5 mmの距離まで印刷する必要があります。そうしないと外壁の印刷は内部構造を破壊する<ol start="3"><li>あなたSACRバックフィルしたい溶液を調製とificial型（ここでは1％の脱イオン水でアガロース）。アガロース溶液は、35℃と45℃の間の温度を持っている必要がありこの温度の下には、アガロースは早すぎる固化う。ポロキサマー構造が柔らかくなりますので、この温度を超える、それが印刷された柱を破壊する可能性があります。 </li><li>ゆっくりとピペットを用いて埋め戻し液で犠牲金型を埋める。これは、壁の内部構造の破壊を避けるためにゆっくりと行われるべきである。 </li><li>埋め戻しソリューションゲルを聞かせたり、それが使用されるポリマーに応じて架橋する。アガロースの場合、凝固は、10分間、4℃で行われた。 </li><li>ポロキサマー構造を溶出するために10分間、氷浴中で埋め戻し犠牲型を置きます。 </li><li>ティッシュペーパーで埋め戻し構造をブロットおよび新しいガラス顕微鏡スライド上に置きます。との間に空隙から第三ヒドロゲルの漏れを回避するために、ガラス顕微鏡スライド上に注意深く構造を押す埋め戻し構造とガラス顕微鏡スライド。 </li></ol> 5。ボイドの充填<ol><li>溶出ポロキサマーが残した空隙を埋めるために、30 G針を搭載したシリンジに、意図したポリマー溶液を埋める。この例では、0.05％w / vのリチウムフェニル-2,4,6 - trimethylbenzoylphosphinate（LAP）およびアレクサ488共役フィブリノゲンの2.5％v / vの添加で0.15MのNaCl溶液中1％アルギン酸メタクリレートを用いる。アレクサ-488共役フィブリノゲンは、可視化の目的のために追加されました。 </li><li> 5分間画像に対して高輝度UVランプを有するポリマーを光重合（100ワット、365nmで、基板からの距離が3.5 cmであった）は、エピ蛍光または共焦点顕微鏡を使用して構築する。 </li></ol>

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V., Skardal, A., Atala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+hydrogels+for+bio-printing+applications.">Evaluation of hydrogels for bio-printing applications.</a> Journal of biomedical materials research. Part A. 101, 272-284 (2013).</li><li>He, J., Li, D., Liu, Y., Gong, H., Lu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indirect+fabrication+of+microstructured+chitosan-gelatin+scaffolds+using+rapid+prototyping.">Indirect fabrication of microstructured chitosan-gelatin scaffolds using rapid prototyping.</a> Virtual and Physical Prototyping. 3, 159-166 (2008).</li><li>Sachlos, E., Reis, N., Ainsley, C., Derby, B., Czernuszka, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+collagen+scaffolds+with+predefined+internal+morphology+made+by+solid+freeform+fabrication.">Novel collagen scaffolds with predefined internal morphology made by solid freeform fabrication.</a> Biomaterials. 24, 1487-1497 (2003).</li><li>Lee, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-demand+three-dimensional+freeform+fabrication+of+multi-layered+hydrogel+scaffold+with+fluidic+channels.">On-demand three-dimensional freeform fabrication of multi-layered hydrogel scaffold with fluidic channels.</a> Biotechnology and bioengineering. 105, 1178-1186 (2010).</li><li>Turturro, M., Christenson, M., Larson, J., Papavasiliou, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinase+(MMP)+sensitive+PEG+diacrylate+(PEGDA)+hydrogels+with+spatial+variations+in+matrix+properties+direct+vascular+cell+invasion.">Matrix metalloproteinase (MMP) sensitive PEG diacrylate (PEGDA) hydrogels with spatial variations in matrix properties direct vascular cell invasion.</a> J. Tissue. 6, 302-302 (2012).</li><li>Butterworth, A., Garcia, M. D. L., Beebe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photopolymerized+poly(ethylene)+glycol+diacrylate+(PEGDA)+microfluidic+devices.">Photopolymerized poly(ethylene) glycol diacrylate (PEGDA) microfluidic devices.</a> Roy. Soc. Ch. , 4-6 (2005).</li><li>Shachar, M., Tsur-Gang, O., Dvir, T., Leor, J., Cohen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+immobilized+RGD+peptide+in+alginate+scaffolds+on+cardiac+tissue+engineering.">The effect of immobilized RGD peptide in alginate scaffolds on cardiac tissue engineering.</a> Acta biomaterialia. 7, 152-162 (2011).</li><li>Jeon, O., Bouhadir, K. H., Mansour, J. M., Alsberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photocrosslinked+alginate+hydrogels+with+tunable+biodegradation+rates+and+mechanical+properties.">Photocrosslinked alginate hydrogels with tunable biodegradation rates and mechanical properties.</a> Biomaterials. 30, 2724-2734 (2009).</li><li>Mauck, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+tissue+engineering+of+articular+cartilage+through+dynamic+loading+of+chondrocyte-seeded+agarose+gels.">Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels.</a> J. Biomech. Eng-T Asme. 122, 252-260 (2000).</li><li>D'Arrigo, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronic+acid+methacrylate+derivatives+and+calcium+alginate+interpenetrated+hydrogel+networks+for+biomedical+applications:+physico-chemical+characterization+and+protein+release.">Hyaluronic acid methacrylate derivatives and calcium alginate interpenetrated hydrogel networks for biomedical applications: physico-chemical characterization and protein release.</a> Colloid Polym. Sci. 290, 1575-1582 (2012).</li><li>Pescosolido, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronic+Acid+and+Dextran-Based+Semi-IPN+Hydrogels+as+Biomaterials+for+Bioprinting.">Hyaluronic Acid and Dextran-Based Semi-IPN Hydrogels as Biomaterials for Bioprinting.</a> Biomacromolecules. 12, 1831-1838 (2011).</li><li>Guo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+of+collagen/chondroitin+sulfate/hyaluronan+interpenetrating+polymer+network+for+cartilage+tissue+engineering.">Hydrogels of collagen/chondroitin sulfate/hyaluronan interpenetrating polymer network for cartilage tissue engineering.</a> J. Mater. Sci-Mater. M. 23, 2267-2279 (2012).</li></ol>

著者は宣言することは何もない。

ここでは、初めて、すぐに〜のポロキサマーのゲル - ゾル転移点20℃のために冷たい水に溶出させることができる犠牲型のため熱応答性ポリマーの使用を提示プロセス全体の速度は十分な解像度で印刷することができません生体高分子構造を迅速に作成するためのポロキサマー興味深い。以前に他の材料35について報告されているとしてここに記載された技術は、他のヒドロゲル内ま?...

組織工学アプローチは、ヒト組織および器官の再生19,20に対して過去数年にわたって多くの進歩を遂げている。しかし、今まで、組織工学の焦点は、しばしば簡単な構造、又は膀胱21,22または皮膚23-25 ​​のような小さな寸法を有する組織に限定されている。人間の体は、しかしながら、細胞と細胞外マトリックスは、空間的に定義された方法で配置されている多くの複雑な三次元組織を含んでいる。これらの組織を製造するために、技術が所定位置に3次元構造物内の細胞と細胞外マトリックスの足場を配置することが要求される。 Bioprintingは、製造の複雑な三次元組織のビジョンは10,11,26-28を実現できるような技術になる可能性を持っています。 Bioprintingをパターニングするための物質移動プロセスの使用 &quot;として定義され、相対生物学的に組み立てされているエバント材料-分子、細胞、組織、および生物分解性の生体材料-所定の組織との一つ以上の生物学的機能を達成するために&quot;4これは、2つのサブミクロンの分解能に至るまで、異なる解像度および長さスケールで動作するいくつかの異なる技法を包含する押出印刷1,12,30用420ミクロン〜150ミクロンの解像度に光子重合29。ない単一の材料または材料の組み合わせは、それぞれの方法31の要件を満たします。押し出し印刷の場合、重要なパラメータは、粘度とゲル化時間です高粘度と迅速なゲル化が望まれている32。 3Dプリントは、複雑な形状30,33,34を作成するための犠牲型を簡単に作成可能にする技術です。このプロセスは、押出bioprinterなどのラピッドプロトタイピング技術を用いて金型の構造に基づいている。作成された犠牲鋳型が使用されそれらの低粘度およびゲル化遅い時間に起因する印刷することが困難である材料から複雑な構造を形成する。ここに示された方法は、低温で迅速に溶解かつ正確に押し出すことができる材料からなる犠牲鋳型の生成を伴う。ブロック共重合体、ポリ（エチレングリコール）99 -ポリ（プロピレングリコール）67 -ポリ（エチレングリコール）99（また、プルロニックF127又はポロクサマー407とも呼ばれる）これらの要件を満たす。それは、すでに我々の知る限り、液体環境でその不安定性のためにその変更されていないバージョンで印刷するために使用されていない、押出印刷1で修正されたバージョンで使用されますが、されています。ポロクサマー407はまた、逆の熱応答挙動18 すなわち冷却時にゾルにゲルから、それが変化を示している。最も重要なのは、それは非常に高い忠実度で複雑な任意に湾曲構造に印刷することができます。これはから構造化ヒドロゲルの作成を可能に低粘度材料、この場合遅いゲル化アガロースで、印刷された犠牲鋳型にソリ​​ューションをピペッティングにより。鋳物構造化されたハイドロゲルからの高忠実度とその迅速な溶出犠牲型の印刷の組み合わせは、そのマスクまたはそれはしばしばリソグラフィー法で必要とされるようにタイムスタンプを使用することなく、異なる形状を持つ金型を作成するために、迅速かつ柔軟な方法になります。鋳造構造化されたヒドロゲルは、さらに、その低粘度のため、押出印刷に適していない別の材料を充填することができる。これは、我々の場合には低粘度のアルギン酸メタクリルソリューションです。ここでは、ピラーの配列の一例を用いてヒドロゲルをパターニングするための逆温度応答性犠牲鋳型の方法を提案する。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>Poloxamer (Pluronic F127)</td>
 <td>Sigma</td>
 <td>P2443</td>
 <td ></td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Invitrogen</td>
 <td><a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=10010015" target="_blank">10010-015</a></td>
 <td ></td>
 </tr>
 <tr>
 <td>CAD software</td>
 <td>regenHU</td>
 <td>BioCAD</td>
 <td ></td>
 </tr>
 <tr>
 <td>Alginate methacrylate</td>
 <td>Innovent e.V Technologieentwicklung Jena</td>
 <td></td>
 <td >Synthesized by Innovent for the FP7 Project Nr NMP4-SL-2009-229292</td>
 </tr>
 <tr>
 <td>Fibrinogen From Human Plasma, Alexa Fluor 488 Conjugate</td>
 <td>Invitrogen</td>
 <td>F13191</td>
 <td ></td>
 </tr>
 <tr>
 <td>Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)</td>
 <td>Innovent e.V Technologieentwicklung Jena</td>
 <td></td>
 <td >Synthesized by Innovent for the FP7 Project Nr NMP4-SL-2009-229292</td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Lonza</td>
 <td>50004</td>
 <td ></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Bioprinter</td>
 <td>regenHU</td>
 <td>Biofactory</td>
 <td ></td>
 </tr>
 <tr>
 <td>Valve</td>
 <td>regenHU</td>
 <td>300 &mu;m Nozzel Diameter</td>
 <td ></td>
 </tr>
 <tr>
 <td>Needle</td>
 <td>regenHU</td>
 <td>150 &mu;m Inner Diameter</td>
 <td ></td>
 </tr>
 <tr>
 <td>Zeiss Axioobserver with ApoTome</td>
 <td>Zeiss</td>
 <td></td>
 <td ></td>
 </tr>
 <tr>
 <td>UV Light Source</td>
 <td>UVP</td>
 <td>Blak-Ray B-100AP High Intensity UV Lamp</td>
 <td>100 W</td>
 </tr>
 </tbody>
</table>

printing thermoresponsive reverse molds for the creation of patterned two-component hydrogels for 3d cell culture

Bioprintingは、ラピッドプロトタイピング業界にその起源を持っている新興の技術です。異なる印刷プロセスは、コンタクトbioprinting 1-4（押し出し、ディップペンとソフトリソグラフィ）、非接触bioprinting 5-7（レーザフォワード転写、インクジェット法）、例えば2光子光重合8、レーザベースの技術に分けることができる。そのような組織工学9-13、バイオセンサー微細加工14-16として、例えば、異なる細胞型17の共培養の影響などの基本的な生物学的質問に答えるためのツールとして多くの用途に使用することができる。一般的なフォトリソグラフィーまたはソフトリソグラフィー法とは異なり、押出bioprintingは、別個のマスクやスタンプを必要としないという利点を有する。 CADソフトウェアを使用して、構造の設計を迅速に​​、オペレータの要求に応じて変更して調整することができる。これは、リソグラフィベースより柔軟bioprintingなりアプローチ。 ここでは一例としてヒドロゲル内にピラーのアレイを用いて多重材料の3D構造を作成するために、犠牲モールドの印刷を示す。これらの柱は神経ガイド導管内血管網やチューブのための中空構造を表すことができます。犠牲型のために選択された材料は、ポロキサマー407、4で24.5％w / vのソリューション18℃で、そのゲル化温度〜20℃上記の固体、液体である優れた印刷特性を持つ熱応答性ポリマーであった。このプロパティは、ポロキサマーベースの犠牲型はオンデマンドで溶出することができ、特に狭いジオメトリのための固体材料の溶解が遅い上の利点があります。ポロキサマーは、犠牲モールドを作成するために、顕微鏡ガラススライド上に印刷した。アガロースを金型にピペットでゲル化するまで冷却した。冷たい水の中ポロキサマーの溶出した後、アガロース型内ボイドがアルギン酸メタクリルSPに満ちていたFITCラベルされたフィブリノゲンとiked。いっぱいボイドは、その後UVを架橋された及び構築物は落射蛍光顕微鏡で画像化した。

代表的な結果は、逆型技術は、（ 図2に示す）は、第2の材料で充填することができる構造化されたゲルを作成することを示している。すべての印刷処理の最初に印刷パラメータが最初に最適化される。パラメータの段階的な調整は、 図3および単一の行が印刷され、図4に示されている印刷された多層構造体になります。層の厚さは（一方の後に印刷層ニ?...

Watch this Scientific Journal Video about Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture at JoVE.com

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

bioprinter、犠牲型に基づいてパターン化されたハイドロゲルを作成するために使用された。ポロキサマー型は第二のヒドロゲルで埋め戻した後、溶出し、第三のハイドロゲルを充填した空隙を残していた。この方法では、生体高分子から複雑なアーキテクチャを生成する高速溶出及びポロキサマーの良い印刷適性を使用しています。

模様の作成3D細胞培養のための2つの成分​​のために温度応答性ハイドロゲルリバース金型の印刷

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

printing-thermoresponsive-reverse-molds-for-creation-patterned-two

Research

JoVE Journal

Immunology and Infection

15.0K ビュー.  Cartilage Engineering & Regeneration. bioprinter、犠牲型に基づいてパターン化されたハイドロゲルを作成するために使用された。ポロキサマー型は第二のヒドロゲルで埋め戻した後、溶出し、第三のハイドロゲルを充填した空隙を残していた。この方法では、生体高分子から複雑なアーキテクチャを生成する高速溶出及びポロキサマーの良い印刷適性を使用しています。

ビデオ: Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Vero細胞培養アッセイによるフィールドコレクト蚊から感染性ウイルスの検出

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

免疫・感染学

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

単純ヘルペスウイルスの待ち時間と再活性化の研究の主ニューロン培養システム

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

使用して樹状細胞の移行の監視<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁気共鳴イメージング

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

表現型分析のためのヒト皮膚からリンパ球単離と<em&gt; ex vivoで</em&gt;細胞培養

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

感染症のためのモデルとしてのオルガノイド：人間の文化とマウス胃オルガノイドとピロリ菌のマイクロインジェクション

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

NK細胞の機能を監視するためのサイトメトリーに基づくアッセイを流します

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

再生の研究のための主な胃上皮単層培養のマウスおよびひと由来

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for understanding the function of bacterial cells in the natural environment. Because of technological advances, various bacterial cell functions can be revealed at the single-cell level inside a confined space. GVs are spherical micro-sized compartments composed of amphiphilic lipid molecules and can hold various materials, including cells. In this study, a single bacterial cell was encapsulated into 10&#8211;30 &#956;m GVs by the droplet transfer method and the GVs containing bacterial cells were immobilized on a supported membrane on a glass substrate. Our method is useful for observing the real-time growth of single bacteria inside GVs. We cultured Escherichia coli (E. coli) cells as a model inside GVs, but this method can be adapted to other cell types. Our method can be used in the science and industrial fields of microbiology, biology, biotechnology, and synthetic biology.

We demonstrate single-cell culture of bacteria inside giant vesicles (GVs). GVs containing bacterial cells were prepared by the droplet transfer method and were immobilized on a supported membrane on a glass substrate for direct observation of bacterial growth. This approach may also be adaptable to other cells.

巨小胞内部の単細胞レベルにおける細菌細胞培養

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for ...

Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Specific and Rapid Detection of Tilapia Lake Virus

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the aquaculture industry that has resulted in the mass morbidity and mortality of tilapia in many parts of the world. An effective, rapid, and accurate diagnostic assay for TiLV infection is therefore necessary to detect the initial infection and to prevent the spread of the disease in aquaculture farming. In this study, a highly sensitive and practical reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is presented to detect tilapia lake virus in fish tissue. A comparison of the RT-qPCR and RT-LAMP assays of infected samples revealed positive results in 63 (100%) and 51 (80.95%) samples, respectively. Moreover, an analysis of uninfected samples showed that all 63 uninfected tissues yielded negative results for both the RT-qPCR and RT-LAMP assays. The cross-reactivity with five pathogens in tilapia was evaluated using RT-LAMP, and all the tests showed negative results. Both the liver and mucus samples obtained from infected fish showed comparable results using the RT-LAMP method, suggesting that mucus can be used in RT-LAMP as a nonlethal assay to avoid killing fish. In conclusion, the results demonstrated that the presented RT-LAMP assay provides an effective method for TiLV detection in tilapia tissue within 1 h. The method is therefore recommended as a screening tool on farms for the rapid diagnosis of TiLV.

Reverse Transcription Loop-Mediated Isothermal Amplification RT-LAMP Assay for the Specific and Rapid Detection of Tilapia Lake Virus

We present an RT-LAMP assay for the detection of TiLV in tilapia fish using simple instruments over a relatively short period of time compared to conventional RT-PCR techniques. This protocol may help control the epidemic spread of TiLVD, especially in developing countries.

チラピア湖ウイルスの特異的かつ迅速な検出のための逆転写ループ媒介等熱増幅(RT-LAMP)アッセイ

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

P. アフルギノーサ 抗感染性の前臨床評価のための空気・液体界面における気管支上皮細胞とマクロファージの感染3Dコカルチャー

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

模様の作成3D細胞培養のための2つの成分​​のために温度応答性ハイドロゲルリバース金型の印刷

模様の作成3D細胞培養のための2つの成分のために温度応答性ハイドロゲルリバース金型の印刷