寛大にMIN6細胞を供給するためのコロラド大学、ボルダーのクリスティAnseth研究室に感謝します。このプロジェクトの資金は、NSFによって提供されています。

1。 PEGDMの合成と光活性PEGDMのマクロマー溶液の調製<ol><li>硬質プラスチック製のキャップで40mlのガラス製バイアル瓶にPEGの2Gを（線形、10,000 Da）の重量を量る。 </li><li>無水メタクリル酸の約308μL追加し、緩くバイアルをキャップ。 </li><li>電子レンジ標準の国内電子レンジで2分の高オンバイアル。さらに5分間高い上にマイクロ波し、耐熱手袋、徹底的にボルテックスバイアルを身に着けている。 </li><li>簡潔にバイアルは手袋で処理されるのに十分冷まします。それを暴露すると徐々にバイアルを旋回しながら、塩化メチレンを追加。解決策は、必要に応じてサンプルをボルテックス、明確かつ均質に表示されるように十分な追加。 2 mLの - 使用される塩化メチレンの合計量は1.5でなければなりません。 </li><li>コー​​ルド200mLにPEGDMを沈殿させる、ソリューションを滴下してジエチルエーテルを撹拌する。 </li><li>真空ろ過によってPEGDMを収集し、それが乾燥することができます。 </li><li>十分な量のPEGDMを溶かすの脱イオン水（通常100 -150 mL）で。 </li><li>千Daの分子量カットオフ値を持つ透析チューブに5日間脱イオン水に対して解決策を透析。 1日1回水を交換してください。 </li><li>分注し-80℃で適切な容器と凍結にソリューション℃で一晩。精製された白色PEGDMの粉末を得た4日間のソリューションを凍結乾燥。 ° C使用しないときは4で粉を保管してください。 </li><li>光活性PEGDM溶液を調製するには、まず、脱イオン水でイルガキュア2959を溶解して0.5重量％の光開始剤のストック溶液を調製。渦解と暗闇の中で保管してください。 </li><li> 10重量％のPEGDMとハンクス平衡塩類溶液（HBSS）0.025重量％イルガキュア2959溶液を調製します。 PEGDMの溶解を確実にするために徹底的にボルテックス。我々は通常、同時に、このソリューションの1mLのを準備。 </li><li> 1.8 mLのマイクロ遠心チューブに0.2μmのシリンジフィルターを通してそれをフィルタリングすることにより解決策を滅菌する。アルミホイルにチューブを巻きますし、4℃で保存</li></ol> 2。文化、感染症、およびMIN6細胞の凝集<ol><li> MIN6細胞を37で加湿環境で10％FBS、7mmのグルコース、100ユニット/ mLのペニシリン/ストレプトマイシン、及び0.5μg/ mLのamphotercin B℃、5％CO 2を添加したRPMI 1640培地中で培養されています。 </li><li>処理された培養フラスコの表面から細胞を解除するには、37で細胞をリンス、培地を吸引除去° C、カルシウム/マグネシウムを含まないHBSS、HBSSを吸引し、37 ° Cトリプシン- EDTA溶液2 mlを加え、細胞を許可する3分間インキュベートする。 </li><li>しっかりと細胞がフリーノックするフラスコの側面をJARファイルにまとめ、その後、37の8 mLを加え° C培地と精力的に気泡を導入しないように注意しながら上下に細胞懸濁液をピペットで。 </li><li>滅菌15 ml遠心チューブに細胞をピペッティングし、血球計算盤またはプロシージャを数え、他の細胞を用いて細胞数を実行します。 </li><li>メーカーで細胞を感染させる準備にウイルスは、最初に感染される細胞の総数を決定し、感染の希望多重度（MOI）（セル50〜100感染粒子）を達成するために必要なウイルス懸濁液の体積を計算する。 </li><li>慎重に1.8 mLのマイクロ遠心チューブにHBSSのプレ分注し、ボリュームにウイルス懸濁液の適正量をペッティングにより、HBSSでウイルスを希釈する。 HBSSウェルあたりの凝集細胞を50μLが適当である。 </li><li>ピペット6ウェル懸濁培養プレートのウェルに加え40万の細胞を達成するために必要な量をし、そのチューブを上下にそれらをペッティングにより細胞を分散させる。 </li><li>所望のMOIを達成するために各ウェルに希釈したウイルスの適切なボリュームを追加します。 </li><li> 1.7 mLの全量をもたらすために各ウェルに培地を追加。 </li><li>インキュベーター内部のオービタルシェーカー上でプレートを置きます。培地は優しく井戸（〜100 rpm）で周りに洗うように、低設定でシェーカーを設定します。凝集して細胞を許可する24〜36時間のTE。 75〜175μmのおおよその直径を持つ凝集体が形成されるべきである。 </li></ol> 3。 PEGDMの細胞のカプセル化<ol><li>細胞は、分散（ステップ2.4​​）または以下の集約（ステップ2.10）としてカプセル化されることがあります。ヒドロゲル前駆体PEGDMソリューションが液体であるため、細胞が架橋ハイドロゲルの形成に先立って解決する傾向がある。セトリングがそのようなより大きな凝集体と同様に、急速に発生が予想されている場合、それは細胞がゲルの中心面に沈殿するように二つのステップでヒドロゲルを生成するために有益であるかもしれません。分散した細胞の適切なワンステップのハイドロゲルの合成手順が示されている（ 図2）およびツーステップ手順（3.2〜3.9）以下同様に（ 図3）と詳細表示されます。 </li><li>ハイドロゲルは、1 mLのプラスチック製の注射器のオフテーパ状先端をカットして、ラックに端を開く配置することによって形成されるの容器を作成します。 </li><li>光活性PEGDMのピペットで20μLのでボリュームが全体のプランジャーの表面を覆うことを確認してプランジャーの上に注射器の開放端にlution。平坦な液体表面を達成するために小さな単位でプランジャーを調整します。 </li><li>ラックと位置ヒドロゲルの架橋を可能にするために約8分間UVランプ（波長365nm、〜7 MW / cm 2）の下のラックにピペットを置きます。この時間の間に、細胞の調製を開始することができる</li><li>ピペットで1.8 mlのマイクロ遠心チューブに細胞/骨材の希望数を含むボリュームを、キャップ、そして5分間、130 gでチューブを遠心する。 </li><li>マイクロピペッタを使用して、慎重にチューブの先端で細胞ペレットを乱すことなくメディアを取り出してください。メディアのヘッドがペレットに近づくにつれて、それは、細かいを使用して10μLピペットチップ役立ちます。 </li><li>ゆっくりと光活性PEGDMの溶液20μLに細胞ペレットを再懸濁します（凝集体を扱う場合広口ピペットチップを使用）し、目の上に注射器に細胞懸濁液を追加eは、以前にハイドロゲルの部分を架橋。 </li><li>ハイドロゲルの構築を完了するために、UV光のさらに8分に注射器を公開。終了すると、細胞が完全に円筒状のハイドロゲル（ 図3）内にカプセル化されるべきである。 </li><li>シリンジからと簡単にゲルを洗浄するために24ウェルプレートのウェル中のHBSSを1 mLにハイドロゲルを取り出します。希望の条件の下で24ウェルプレートと文化のウェルに培地1 mLにゲルを移す。 </li></ol> 4。低酸素状態の追跡<ol><li>任意の時点で文化、画像中の細胞は低酸素シグナル伝達の開始を追跡するために蛍光顕微鏡を使用して。あなたがマルチウェルプレートの画像ができるイメージングや顕微鏡ステージ上に配置する前に、顕微鏡のスライドにゲルを転送されているものに応じて。 DSレッドのピーク励起は556nmで実現し、発光ピークは586nmで発生します。排出量はかなり激しいですので、退色はを観測していない問題があることがED。蛋白質の生産と最初の信号検出の開始の間のタイムラグは約12時間です。信号は、個々のシグナル伝達の細胞を識別するのに十分な固有のものですが、解像度は細胞内シグナルの局在を決定するには不十分かもしれません。シグナル伝達細胞では、信号は通常は細胞質全体に均一に観察される。これは増加した蛋白質の発現、全体的なタンパク質の蓄積、または遅延タンパク質の成熟に起因するかどうか我々はまだ完全に決定していないのに信号強度はまた、低酸素への長時間曝露と増やすことができます。 </li></ol> 5。代表的な結果 PEGハイドロゲル内にカプセル化されたMIN6凝集体の代表例を図4に示されています。架橋ゲルは、反応が実行された容器の形状を取って、全体に固体になります。滑らかな外側表面を有するゲルは、異物の再の予防を支援するために注入するための望ましいsponse。ゲル内で、細胞の凝集体は完全にマトリックスで囲まれ、均一に良い栄養素の輸送を可能にするために配布される必要があります。 MIN6凝集体における低酸素シグナルの代表画像を図5に描かれている。マーカーのウイルスに感染して同一のMIN6集計は、画像キャプチャの前に44時間を20％O 2（5A）または1％O 2（図5b）のいずれかで培養した。 <img alt="図1" src="/files/ftp_upload/3521/3521fig1.jpg" /> 図1当社の低酸素マーカーのシステムの活動の概略図。 DSレッドDR遺伝子と上流のHREのプロモーターのアデノウイルス挿入がHIF - 1の制御下に蛍光タンパク質の低酸素誘導性の製造を可能にする。 <img alt="図2" src="/files/ftp_upload/3521/3521fig2.jpg" /> 図2 CRにMIN6細胞を分散したのシングルステップのカプセル化のための手続きフローチャートPEGDMハイドロゲルをosslinked。それぞれのゲルの場合は、分散した細胞は光活性PEGDMのマクロマー溶液の40μlの懸濁される。これは、首を切ら1mLの注射器内に配置され、ハイドロゲルは、10〜12分後に365nmのUV光の下で形成される。完了すると、ハイドロゲルのディスクは、注射器から取り出し洗浄し、インキュベーションのための媒体を充填したウェルプレートに配置されます。 <img alt="図3" src="/files/ftp_upload/3521/3521fig3.jpg" /> 図3。架橋PEGDMハイドロゲルに集約されたMIN6細胞のデュアルステップカプセル化のための手続きのフローチャート。それぞれのゲルの場合は、ハーフゲルは最初の8分間の光活性PEGDMのマクロマー溶液の20μLのUV架橋により形成される。 MIN6凝集体は、慎重に前もって形成された半ゲルの上に追加された光活性マクロマーのソリューションの追加の20μLに懸濁されている。完全なゲルが完全に内側にカプセル化された凝集体に紫外線照射の追加の8分によって形成されるゲルの平面。 <img alt="図4" src="/files/ftp_upload/3521/3521fig4.jpg" /> 図4 20倍の倍率下で40μlのハイドロゲルのイメージ。 MIN6集計（〜40万総細胞）は、明確にゲル内に見られている。ハイドロゲルの直径は、（バー= 1mm）の約6ミリメートルである。 <img alt="図5" src="/files/ftp_upload/3521/3521fig5.jpg" /> 図5。PEGDMヒドロゲルに集約されたMIN6細胞のシグナル蛍光低酸素症。 44時間20％O 2でカプセル化し、インキュベーションに置かれた細胞は2％44時間のためのO 2でインキュベートしてカプセル化された細胞は明らかに、ユビキタスな信号を表示しながら低酸素シグナルを（）表示されません。 （バー=100μmの）（B）。

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利害の衝突は宣言されません。

ここで紹介するメソッドは、非生理的条件の最小限の使用とPEGハイドロゲル中の細胞のカプセル化のための迅速かつ簡単な技術を提供しています。 PEGは、その生体適合性と修正を容易にするために非常に有用な封止材料を表します。光活性溶液中でPEGの比率の単純な変化は、例えば、孔径によって、機械などの圧縮率などのプロパティ、およびトランスポートのプロパティを調整することができる。また、PEGを簡単に側鎖の付加によって変更されます。 PEGハイドロゲルは、従って、in vitroでの研究のための有望な臨床デバイスと柔軟なプラットフォームの両方を表す PEG -カプセル化された細胞の低酸素状態を追跡するための方法も提示されている。このメソッドは、低酸素症検出の簡略化のためと目的の細胞を犠牲にする必要性を回避するのに便利です。テクニックは、その私たちを作る様々な条件で細胞のさまざまな種類のに適用してもよい幅広いefulness。例えば、幹細胞の分化のための手がかりとして、低酸素は、幹細胞の微量培養に追跡することができる。しかし、このメソッドは、分散した細胞は、後で集約されているセルのシステムまたはシステムを分散させるために適用することができます。また、蛍光シグナルの検出には大きいか密度の高い組織では難しいかもしれません。

<table border="1"><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（省略可能 </td></tr><tr><td> PEG </td><td>シグマアルドリッチ</td><td> 309028 - 500G </td><td></td></tr><tr><td>メタクリル酸無水物</td><td>シグマアルドリッチ</td><td> 276685 - 100ML </td><td></td></tr><tr><td>マイクロ波</td><td>エマーソン</td><td> MW8784SB </td><td></td></tr><tr><td>ボルテックス</td><td>サイエンティフィックインダストリーズ</td><td> SI - A236 </td><td></td></tr><tr><td>塩化メチレン</td><td>シグマアルドリッチ</td><td> D65100 - 1L </td><td></td></tr><tr><td>ジエチルエーテル</td><td>シグマアルドリッチ</td><td> 346136 - 1L </td><td></td></tr><tr><td>透析チューブ</td><td>スペクトラム</td><td> 132640 </td><td></td></tr><tr><td></td><td>研究所</td><td></td><td></td></tr><tr><td>冷凍庫</td><td></td><td></td><td></td></tr><tr><td&gt;凍結乾燥機</td><td> Labconco </td><td> 7670521 </td><td></td></tr><tr><td>真空ポンプ</td><td>ウェルチ</td><td> 8917Z - 01 </td><td></td></tr><tr><td>イルガキュア2959 </td><td>チバガイギー</td><td> 029891301PS04 </td><td></td></tr><tr><td> HBSS </td><td> Mediatech </td><td> 21から022 - CV </td><td></td></tr><tr><td>シリンジフィルタ</td><td> VWR </td><td> 28145-477 </td><td></td></tr><tr><td> RPMI 1640 </td><td> Mediatech </td><td> * </td><td> *カスタム製剤</td></tr><tr><td> FBS </td><td> PAA研究所</td><td> A15 - 351 </td><td></td></tr><tr><td>ペニシリン - ストレプトマイシン</td><td> Mediatech </td><td> 30から002 - CI </td><td></td></tr><tr><td>アムホテリシンB </td><td> Mediatech </td><td> 30から003 - CF </td><td></td></tr><tr><td>インキュベーター</td><td>サーモサイエンティフィック</td><td> 3597 </td><td> NAPCOシリーズ8000 WJ W / O2抑制</td></tr><tr><td>トリプシンEDTA </td><td> Mediatech </td><td> 25から052 - CI </td><td></td></tr><tr><td>オービタルシェーカー</td><td> VWR </td><td> 12620-926 </td><td></td></tr><tr><td> UVランプ</td><td>三洋電機Denri </td><td> FLR40SBLB / M </td><td> two 40W、365nmのブラックライトブルーUV電球を保持しています</td></tr><tr><td>遠心</td><td>エッペンドルフ</td><td> 5811 000.010 * </td><td> *注文番号。 モデル5810 R </td></tr><tr><td>顕微鏡</td><td>ニコン</td><td> TI - ND6 - PFS </td><td> 556nm励起/ 586nm発光のためのフィルタセットを持つ</td></tr></table>

tracking hypoxic signaling within encapsulated cell aggregates

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored1,2. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host's immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim.
Biologically-derived materials, such as alginate3 and agarose4, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth5 which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible,6,7,8. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures6,7. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells7,9, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue10. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma.
An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact &beta;-cells whose insulin secretory function is highly dependent on oxygen11-13. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15. The virus is stored in 10% glycerol at -80&deg; C as many 150 &mu;L aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010 pfu/mL. 
Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1&alpha; for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.

Watch this Scientific Journal Video about Tracking Hypoxic Signaling within Encapsulated Cell Aggregates at JoVE.com

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

架橋PEGハイドロゲル中の細胞の写真 - カプセル化する方法について説明する。カプセル化されたマウスインスリノーマ（MIN6）集合内の低酸素シグナルが蛍光マーカーシステムを用いて追跡されます。このシステムは、ハイドロゲル骨格と細胞表現型の変化と低酸素シグナル伝達の相関関係にあるセルの連続検査が可能になります。

カプセル化された細胞凝集体の中で低酸素シグナルの追跡

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal ...

tracking-hypoxic-signaling-within-encapsulated-cell-aggregates

Research

JoVE Journal

Bioengineering

10.5K ビュー.  University of South Carolina. 架橋PEGハイドロゲル中の細胞の写真 - カプセル化する方法について説明する。カプセル化されたマウスインスリノーマ（MIN6）集合内の低酸素シグナルが蛍光マーカーシステムを用いて追跡されます。このシステムは、ハイドロゲル骨格と細胞表現型の変化と低酸素シグナル伝達の相関関係にあるセルの連続検査が可能になります。

ビデオ: Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

細胞内小胞の追跡への応用：変形二光子顕微鏡での3d軌道の追跡

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

生物工学

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 ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

のためのツールと​​してのリポソームカプセル化された近赤外蛍光体の蛍光消光<em&gt;インビボ</em&gt;光学イメージング

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

蛍光生体膜フォースプローブ：単一細胞上の受容体 - リガンド速度論の同時定量との結合によって誘導される細胞内シグナル伝達

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Transport Properties of Ibuprofen Encapsulated in Cyclodextrin Nanosponge Hydrogels: A Proton HR-MAS NMR Spectroscopy Study

The chemical cross-linking of &#946;-cyclodextrin (&#946;-CD) with ethylenediaminetetraacetic dianhydride (EDTA) led to branched polymers referred to as cyclodextrin nanosponges (CDNSEDTA). Two different preparations are described with 1:4 and 1:8 CD-EDTA molar ratios. The corresponding cross-linked polymers were contacted with 0.27 M aqueous solution of ibuprofen sodium salt (IP) leading to homogeneous, colorless, drug loaded hydrogels.
The systems were characterized by high resolution magic angle spinning (HR-MAS) NMR spectroscopy. Pulsed field gradient spin echo (PGSE) NMR spectroscopy was used to determine the mean square displacement (MSD) of IP inside the polymeric gel at different observation times td. The data were further processed in order to study the time dependence of MSD: MSD = f(td). The proposed methodology is useful to characterize the different diffusion regimes that, in principle, the solute may experience inside the hydrogel, namely normal or anomalous diffusion. The full protocols including the polymer preparation and purification, the obtainment of drug-loaded hydrogels, the NMR sample preparation, the measurement of MSD by HR-MAS NMR spectroscopy and the final data processing to achieve the time dependence of MSD are here reported and discussed. The presented experiments represent a paradigmatic case and the data are discussed in terms of innovative approach to the characterization of the transport properties of an encapsulated guest within a polymeric host of potential application for drug delivery.

The motion regimes of ibuprofen encapsulated in &#946;-cyclodextrin nanosponges polymer network are investigated using pulsed-field-gradient spin-echo (PGSE) NMR technique. Synthesis, purification, drug loading, implementation of the NMR pulse sequence and data analysis to work out the mean square displacement of the drug at several observation times are described in detail.

シクロデキストリンNanospongeヒドロゲル中のイブプロフェンカプセル化の輸送特性：プロトンHR-MAS NMR分光研究

The chemical cross-linking of &#946;-cyclodextrin (&#946;-CD) with ethylenediaminetetraacetic dianhydride (EDTA) led to branched polymers referred to as ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

細胞外マトリックス内に埋め込まれたエンジニアリング三次元上皮組織

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

液晶ナノ粒子キャリアにおける疎水性貨物カプセル化の標的細胞膜配達

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

マイクロウェルアレイプラットフォーム内での微生物コミュニティ開発の組み立てと追跡

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

三次元培養細胞遊走を特徴付ける傷の環境

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye

Many current therapeutics under development for diseases of the posterior pole of the eye are biologics. These drugs need to be administered frequently, typically via intravitreal injections. Encapsulated cells expressing the biologic of choice are becoming a tool for local protein production and release (e.g., via long-term drug delivery). In addition, encapsulation systems utilize permeable materials that allow diffusion of nutrients, waste, and therapeutic factors into and out of cells. This occurs while masking the cells from the host immune response, avoiding the need for suppression of the host immune system. This protocol describes the use of alginate as a polymer in microencapsulation coupled with the electrospray method as a microencapsulation technique. ARPE-19 cells, a spontaneously arising human RPE cell line, has been used in long-term cell therapy experiments due to its lifetime functionality, and it is used here for encapsulation and delivery of the capsules to mouse eyes. The manuscript summarizes the steps for cell microencapsulation, quality control, and ocular delivery.

Presented here is a protocol for the use of alginate as a polymer in microencapsulation of immortalized cells for long-term delivery of biologics to rodent eyes.

マウスアイへの生物学的製剤の送達のためのカプセル化された細胞技術

Many current therapeutics under development for diseases of the posterior pole of the eye are biologics. These drugs need to be administered frequently, ...