1。プライマリ関節軟骨細胞の単離動物の関節の関節面から収穫10月15日全層軟骨スライス（ローカルabbatoirから得られた骨格成熟した牛の中手·指関節など ）。 <ol><li> 37℃で2時間、ハムF-12（w / v）の中の0.5％プロテアーゼ20mlの100mmペトリ皿とインキュベート℃の場所軟骨スライスハムF-12培地で3回リンスした後、0.15％コラゲナーゼ20mlで37℃で一晩インキュベートハムF-12培地（℃） </li><li>きれいな皿に200メッシュのスクリーンフィルタを介して細胞懸濁液をろ過する。ハムF-12 5mlでフィルターを洗って皿に追加します。 50 mlコニカルチューブに細胞懸濁液を移す。ハムF-12 10mlのペトリ皿を洗い、コニカルチューブに追加します。 </li><li>室温で6から8分間600から800 rcfでチューブを遠心分離します。 </li><li>目を動かさないように確保し、上清を吸引し電子細胞ペレットとハムF-12培地40mlに再懸濁する。 </li><li> 6月8日分の600から800 rcfでチューブを遠心分離します。 </li><li>合計3回の手順1.4と1.5を2回以上繰り返します。前に三回目の遠心分離に、攪拌を介してペレットを再懸濁し、細胞をカウントするための500μlのアリコートを得る。 </li><li>血球計および倒立光学顕微鏡とトリパンブルー排除法9を使用して生細胞数を数えます。 </li><li>上清を吸引除去し、所望の播種密度（カプセル化後の10×10 6細胞/ mlの最終濃度のために、例えば 20×10 6細胞/ ml）を倍増させるハムF-12培地の体積でペレットを再懸濁する。 </li></ol> 2。軟骨細胞 - アガロースハイドロゲルのカプセル<ol><li>ヒート0.8グラムオートクレーブ滅菌タイプVIIアガロース溶解°Cまで、120に設定したホットプレート上に滅菌1X PBS（pH7.4）を20mlのインチ一度溶解し、℃より高い温度として60に熱を下げるウィルlは、細胞の生存に影響を与えます。 </li><li> 50 mlコニカルチューブでは、徹底的にアガロース（最終2％アガロースヒドロゲルのための例えば、4％アガロース懸濁液）の二重所望の濃度で細胞懸濁液の等量を混ぜる。彼らは細胞の生存に影響を与え、疲労寿命を構築することができるような大きな気泡の生成を避ける。 </li><li>大きな気泡の生成を回避しながら、直径60mmのペトリ皿に注意深くアリコート軟骨細胞 - アガロース混合物10mlを、。 </li><li>ゲル化するまで室温で30分間放置する。 </li><li>必要に応じて4ミリメートル生検パンチを使用して（通常は20から30）は、多くの軟骨アガロースハイドロゲルをサンプルとして取得します。 </li><li>マイクロメーターを用いて代表的なサンプルの物理的な大きさ（直径と高さ）を測定し、記録します。構造物の高さが約5​​ mmでなければなりません、試料高さの分散が2％以上であった領域から得られたすべてのサンプルを破棄します。 </li><li>新鮮な60ミリメートルペトリ皿とサプリメントにサンプルを転送完全培地10ミリリットル（20mMのHEPES、100μg/ mlのアスコルビン酸、および2倍の抗生物質/抗真菌剤とハムF-12培地中で、例えば 20％のウシ胎児血清）を持つ。 </li></ol> 3。軟骨アガロースゲルの機械的刺激と放射性<ol><li>圧縮リグの金属部品をオートクレーブ。 70％エタノール（一晩）と紫外線（少なくとも15分間）でプラスチック部品を使用して滅菌する。 </li><li> 24穴培養プレートのウェル内に、転倒鉗子を使用して、場所12滅菌したプラスチック製リテーニングリング（コンストラクト径より大きい内径を持つ）（ただし、n = 6、検体およびコントロール）から構造物を維持する。 </li><li>ピンセットを用いて、慎重に止め輪の各1を確保し、ペトリ皿から24ウェルプレートに軟骨細胞 - アガロースヒドロゲルを転送します。 </li><li>完全培地を400μl（20mMのHEPESとハムF-12培地中、例えば 20％のFBS、100μg/ mlのアスコルビン酸を移し、各サンプルウェルに）抗生物質/抗真菌剤の2倍。 </li><li>滅菌した圧縮リグ（ 図1）を構築し、止めネジでプラテンを固定します。 24ウェル培養プレートに圧縮リグを取り付けます。 </li><li>プラテンハイドロゲルコンタクト（ゼロひずみ状態）を確立するためにセットスクリューを解放し、再固定します。 </li><li>マッハ1微小機械試験システムに組み立て圧縮リグをロードします。セットネジで位置決めピンとロックを介して垂直ステージへの安全なリグ。間隔治具を外します。 </li><li>周波数（ 例えば 1 Hz）と持続時間またはサイクル数（60分などの時間間隔まで）、所望の振幅（サンプルの元の高さに基づいて、 例えば、10％のひずみ振幅）で動的圧縮荷重を適用します。 </li><li>機械的な刺激が完了すると、間隔治具を交換する位置決めピン止めねじを緩め、マッハ1マイクロメカニカル試験システムから圧縮リグや培養プレートを取り外します。 </lI&gt; <li>放射性標識細胞のヒドロゲル所望の同位体（軟骨細胞特有のコラーゲン合成および[35 S] -硫黄プロテオグリカン合成のためのもの [3 H] -プロリンタンパク質標識）の5μCiのあるメディアを補完することにより構造物。 </li><li> 37℃で24時間、放射性標識された構造物をインキュベート℃、5％CO 2で10。 </li><li>放射性標識されたハイドロゲルの構築物を収穫し、法人格のない任意の同位体を除去するために1X PBS（pH7.4）中で3回すすいでください。パパイン（20 mM酢酸アンモニウム、40μg/ mlの、65℃で1mMのethyldiaminetetraacetic酸および2mMジチオスレイトール℃で72時間の場合）を使用してダイジェストのサンプル。 </li><li>ピコグリーン色素アッセイ11とで設立された同位体の活動、12,13直後パパイン消化を数え、β-液体シンチレーションによって、DNA含量に正規化を用いてDNAコンテンツに関するアッセイダイジェスト。 </li></ol>注：放射性物質の購入·処分（廃棄物の同位体とアイテム接触放射性物質になってきたかもしれない）（および/または政府）の政策や放射性物質の安全な取り扱いと廃棄のための手順、関連制度に従わなければなりません。 4。代表的な結果ウシ関節軟骨細胞-アガロースハイドロゲル構造体（10×10 6細胞/ mlカプセル化された細胞を2％アガロース）を機械的に20から60分（または1200〜3,600サイクルの1 Hzの周波数で10％の圧縮歪の振幅で刺激した）及び放射性同位元素の取り込みによってDNA含量および細胞外マトリックス合成についてアッセイした。 DNAや細胞外マトリックスの合成が用量依存的に影響を受けました。刺激の60分は、（ 図2）は効果を示さなかったのに対し、DNA量は、刺激の20〜30分と有意（p &lt;0.01）の結果として重要な35％の減少を示した。軟骨特異的コラーゲンとプロテオグリカン合成（[3 H] -プロリンとによって決定されるそれぞれ[35 S] -硫黄混入は）観察可能な効果を示さない刺激の60分（ 図3）と、刺激の20〜30分と有意（p &lt;0.01）に対応して約60％の大幅な増加を示した。 <img src="/files/ftp_upload/4229/4229fig1.jpg" alt="図1" /> 。軟骨アガロースコンストラクトを刺 ​​激するための特注の動的圧縮リグの図1の回路図左 ：組み立て済みのリグ右 ：個々の構成要素を示す分解図。 <img src="/files/ftp_upload/4229/4229fig2.jpg" alt="図2" /> 図2：軟骨細胞-アガロースのDNA含量の変化は、機械的に20から60分（平均±SEM、N = 6/groupを意味する）は1Hzで10％の圧縮ひずみ振幅下で刺激ヒドロゲル。 図3：軟骨細胞-アガロースの細胞外マトリックス合成の変化は（コラーゲンおよびプロテオグリカン）機械的に20から60分（平均±SEM、N = 6/groupを意味する）は1Hzで10％の圧縮ひずみ振幅下で刺激ヒドロゲル。

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<html>細胞播種アガロースヒドロゲルへの制御された機械的刺激を加えるための記載された方法は、軟骨代謝に及ぼす動的圧縮力の影響への直接調査を行うことが可能。保持リングと一緒にカスタム·テスト·リグを使用することによって、サンプルの転倒の潜在的な問題を回避するための構成体の横方向の制約を提供した。セットのクルーによって保護死ん加重ローディングプラテンを使用す...

<table border="1"><tbody><tr><td> 試薬や機器の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td>ハムF-12 </td><td>サーモフィッシャーサイエンティフィック</td><td> SH3001002 </td><td></td></tr><tr><td>コラゲナーゼ</td><td>シグマアルドリッチジャパン（株） </td><td> C0130 </td><td></td></tr><tr><td>プロテアーゼ</td><td>シグマアルドリッチジャパン（株） </td><td> P5147 </td><td></td></tr><tr><td>ウシ胎仔血清</td><td>シグマアルドリッチジャパン（株） </td><td> F1051 </td><td></td></tr><tr><td>アスコルビン酸塩</td><td>シグマアルドリッチジャパン（株） </td><td> A4034 </td><td></td></tr><tr><td>抗生物質/抗真菌薬</td><td>シグマアルドリッチジャパン（株） </td><td> A5955 </td><td></td></tr><tr><td> HEPES </td><td> Bioshopカナダ株式会社</td><td> HEP001 </td><td></td></tr><tr><td>トリパンブルー</td><td>シグマアルドリッチジャパン（株） </td><td> 93595 </td><td></td></tr><tr><td>ライヘルトブライトライン 血球計</td><td> Hausser科学</td><td> 1490 </td><td></td></tr><tr><td>クワント-iTのピコグリーン</td><td>インビトロジェン</td><td> P7589 </td><td></td></tr><tr><td>パパイヤラテックスからのパパイン</td><td>シグマアルドリッチジャパン（株） </td><td> P3125 </td><td></td></tr><tr><td>酢酸アンモニウム</td><td>シグマアルドリッチジャパン（株） </td><td> A1542 </td><td></td></tr><tr><td> Ethyldiaminetetraacetic酸</td><td>シグマアルドリッチジャパン（株） </td><td> E9884 </td><td></td></tr><tr><td> DL-ジチオスレイトール</td><td>シグマアルドリッチジャパン（株） </td><td> 43819 </td><td></td></tr><tr><td>低融点アガロース、VII型</td><td>シグマアルドリッチジャパン（株） </td><td> A9045 </td><td></td></tr> &lt;TR&gt; <td>メッシュスクリーン（200）フィルタ</td><td>シグマアルドリッチジャパン（株） </td><td> S4145 </td><td></td></tr><tr><td>マッハ1マイクロメカニカルテスター</td><td> Biomomentum株式会社</td><td> V500cs </td><td></td></tr><tr><td>圧縮ローディングジグ</td><td>特注の</td><td></td><td>同様の製品はBiomomentum社によって供給することができる</td></tr><tr><td>ファルコン24ウェル培養プレート</td><td>サーモフィッシャーサイエンティフィック</td><td> B353047 </td><td></td></tr><tr><td> β - 液体シンチレーションカウンター</td><td>ベックマン·コールター</td><td> LS6500 </td><td></td></tr><tr><td> [3H]プロリン</td><td>パーキンエルマー</td><td> NET323005MC </td><td></td></tr><tr><td> [35S]硫黄</td><td>パーキンエルマー</td><td> NEX041005MC </td><td></td></tr></tbody></table>

mechanical stimulation of chondrocyte-agarose hydrogels

機械的傷害や変形性関節症などの疾患による劣化によって破損時の関節軟骨は、限られた修復能力に苦しんでいる。この欠点を改善するために、いくつかの医療介入が開発されている。そのような方法の一つは、組織工学軟骨損傷した領域を再浮上することですが、設計された組織は、典型的には、長期的な存続可能性に疑問を投げかけ、ネイティブ軟骨の生化学的特性と耐久性を欠いている。これは、注入材料を保護するために、周囲の組織に頼らず、小焦点欠陥の修理に軟骨組織工学の応用を制限します。開発組織の特性を改善するために、機械的な刺激は、軟骨細胞外マトリックスの合成と同様に設計された組織の結果として得られる機械的特性を向上させるために利用される一般的な手法です。機械的な刺激は、組織への力は、生体内で経験したものと類似の構築に適用されます。この機械的な環境は、部分的には、天然組織1,2の開発と保守を調節していることを前提としている。軟骨組織工学における機械的刺激の最も一般的に適用されたフォームは1Hz 1,3の周波数で約5〜20％の生理的な歪みにおける動的圧縮です。いくつかの研究では、動的圧縮の影響を調査して、それが最終的に開発された4月8日組織の機能的特性に影響を与える、軟骨細胞代謝と生合成にプラスの効果を有することが示されています。本稿では、機械的に、動的圧縮下で軟骨細胞 - アガロースハイドロゲル構造体を刺激し、生化学的および放射性同位体のアッセイを通じて生合成の変化を分析するための方法を示す。また、この方法は容易に機械的刺激の結果として細胞応答におけるどんな潜在的に誘発された変化を評価するために変更することができます。

Watch this Scientific Journal Video about Mechanical Stimulation of Chondrocyte-agarose Hydrogels at JoVE.com

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

軟骨細胞による軟骨細胞外基質の生合成は、機械的刺激を印加することによって影響を受けることができます。このメソッドは、3Dコンストラクトおよび軟骨代謝の誘導された変化の評価にカプセル化された軟骨細胞への動的圧縮歪みを適用する方法について説明します。

軟骨アガロースゲルの機械的刺激

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

mechanical-stimulation-of-chondrocyte-agarose-hydrogels

Research

JoVE Journal

Biology

11.6K ビュー.  Queen's University. 軟骨細胞による軟骨細胞外基質の生合成は、機械的刺激を印加することによって影響を受けることができます。このメソッドは、3Dコンストラクトおよび軟骨代謝の誘導された変化の評価にカプセル化された軟骨細胞への動的圧縮歪みを適用する方法について説明します。

ビデオ: Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Ex vivo Mechanical Loading of Tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37&deg;C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

A new in vitro system for simultaneously loading four tendons in culture is described.

腱の生体外で機械的負荷

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

生物学

Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain

The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated phenomena such as bone remodeling, muscular hypertrophy, and smooth muscle cell plasticity. However, the forces involved are often extremely complex and difficult to monitor and control in vivo. To better investigate the effects of mechanical forces on cells, we have developed an in vitro method for applying uniaxial cyclic tensile strain to adherent cells cultured on elastic membranes. This method utilizes a custom-designed bioreactor with a motorized cam-rotor system to apply the desired force. Here we present a step-by-step video protocol demonstrating how to assemble the various components of each "stretch chamber", including, in this case, a silicone membrane with micropatterned topography to orient the cells with the direction of the strain. We also describe procedures for sterilizing the chambers, seeding cells onto the membrane, latching the chamber into the bioreactor, and adjusting the mechanical parameters (i.e. magnitude and rate of strain). The procedures outlined in this particular protocol are specific for seeding human mesenchymal stem cells onto silicone membranes with 10 &micro;m wide channels oriented parallel to the direction of strain. However, the methods and materials presented in this system are flexible enough to accommodate a number of variations on this theme: strain rate, magnitude, duration, cell type, membrane topography, membrane coating, etc. can all be tailored to the desired application or outcome. This is a robust method for investigating the effects of uniaxial tensile strain applied to cells in vitro.

It is widely understood that mechanical forces in the body can influence cell differentiation and proliferation. Here we present a video protocol demonstrating the use of a custom-built bioreactor for delivering uniaxial cyclic tensile strain to stem cells cultured on flexible micropatterned substrates.

繰返し一軸性歪みを用いた幹細胞の機械的刺激

The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

組織工学のための3Dハイドロゲルの細胞のカプセル化

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

集積光と原子間力顕微鏡による研究機械的刺激に対する細胞応答を生きる

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

細胞単層に機械的刺激によって誘発されるカルシウム波の伝播：ウシ角膜内皮細胞の例

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

DNA架橋ポリアクリルアミドハイドロゲルの調製

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. It has been discovered and rigorously studied using the patch-clamp technique for almost three decades. Its basic role of translating tension applied to the cell membrane into permeability response makes it a strong candidate to function as a mechanoelectrical transducer in artificial membrane-based biomolecular devices. Serving as building blocks to such devices, droplet interface bilayers (DIBs) can be used as a new platform for the incorporation and stimulation of MscL channels. Here, we describe a micropipette-based method to form DIBs and measure the activity of the incorporated MscL channels. This method consists of lipid-encased aqueous droplets anchored to the tips of two opposing (coaxially positioned) borosilicate glass micropipettes. When droplets are brought into contact, a lipid bilayer interface is formed. This technique offers control over the chemical composition and the size of each droplet, as well as the dimensions of the bilayer interface. Having one of the micropipettes attached to a harmonic piezoelectric actuator provides the ability to deliver a desired oscillatory stimulus. Through analysis of the shapes of the droplets during deformation, the tension created at the interface can be estimated. Using this technique, the first activity of MscL channels in a DIB system is reported. Besides MS channels, activities of other types of channels can be studied using this method, proving the multi-functionality of this platform. The method presented here enables the measurement of fundamental membrane properties, provides a greater control over the formation of symmetric and asymmetric membranes, and is an alternative way to stimulate and study mechanosensitive channels.

Bacterial mechanosensitive channels can be used as mechanoelectrical transducers in biomolecular devices. Droplet interface bilayers (DIBs), cell-inspired building blocks to such devices, represent new platforms to incorporate and stimulate mechanosensitive channels. Here, we demonstrate a new micropipette-based method of forming DIBs, allowing the study of mechanosensitive channels under mechanical stimulation.

液滴界面二重層中に細菌機械受容イオンチャネルの取り込みおよび刺激のための多機能、マイクロピペットベースの方法

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

三次元ハイドロゲルでイメージングをFRET

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

キイロタマホコリカビ急性の機械的刺激に対する応答の評価

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

ポルシン卵母細胞カプセル化のためのタンパク質分解アルギン酸ヒドロゲルおよび疎水性マイクロバイオリアクター

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...