Name: gradient strain chip for stimulating cellular behaviors in cell-laden hydrogel
Uploaded: 2017-08-08T12:30:00.000+00:00
Duration: 13 min 28 s
Description: Watch this Scientific Journal Video about Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel at JoVE.com

このプロジェクトで、大学院学生研究留学プログラム (NSC-101-2917-I-007-010); をサポートされていた生体医用工学プログラム (NSC-101-2221-E-007-032-MY3);国立ナノテクノロジー プログラム (NSC-101-2120-M-007-001-) は、中華民国、台湾の国立科学評議会。著者は、ハイドロゲルとセル封止技術を共有するため教授アリ Khademhosseini、Unal-Gulden Camci、タントラ文献に見ポールとハーバード大学医学部で Ronglih 遼を感謝したいです。

1. GelMA Synthesis
<ol>
	<li>Weigh 10 g of gelatin powder and add it to a glass flask with 100 mL ofDulbecco&#39;s phosphate-buffered saline (DPBS). Put a magnetic stir bar into the flask and place the flask on a stirring hot plate.</li>
	<li>Cover the flask with aluminum foil to avoid water evaporation. Set the hot plate temperature to 50-60 &#176;C and the stirrer at 100 rpm for 1 h to dissolve the gelatin powder well.</li>
	<li>After the gelatin has dissolved, add 8 mL of methacrylic anhydride very slowly (one drop per second) using a pipette. Let it react at 60 &#176;C for 3 h.</li>
	<li>Add pre-warmed DPBS (40 &#176;C) to the flask to a final volume of 500 mL and allow this mix well for 15 min to stop the reaction.</li>
	<li>Meanwhile, cut a dialysis membrane (14 kDa cut-off molecular weight) into several 25 cm-long tubes. Immerse them in deionized (DI) water for 15 min and make a knot to close one end of the dialysis tubes.</li>
	<li>Load the appropriate amount (30 - 60 mL) of the polymer solution into the dialysis tubes and close the other end. Place them in a 5-L plastic beaker with DI water for a week. Renew the DI water twice a day and maintain the solution at 40 - 50 &#176;C during the dialysis process.</li>
	<li>Collect the solution from the tube in a 500-mL glass bottle. Pour ~450 - 500 mL of solution in a 500-mL filter cup (pore size of 0.22 &#181;m) and apply a vacuum to the filter cup to force the solution to pass through the filter membrane for sterilization.</li>
	<li>Transfer the sterilized polymer into several 50-mL sterilized tubes and store them in a -80 &#176;C freezer for 3 - 5 days.</li>
	<li>Freeze-dry the -80 &#176;C polymer for 1 week using a freeze dryer to form GelMA. Store the GelMA in a -80 &#176;C freezer.</li>
</ol>
2. 3-(Trimethoxysilyl)propyl Methacrylate (TMSPMA) Modification
<ol>
	<li>&#160;Cut commercial glass slides into two small pieces (25 mm x 37.5 mm) and immerse them in 0.5 M NaOH solution for 4 h. Wash the slides with a large amount of DI water.</li>
	<li>Place the slide on a rack inside a glass container with 95% ethanol and clean using an ultrasonicator at 43 kHz for 15 min. Air dry the glass slide.</li>
	<li>Immerse the glass slides in 5% TMSPMA in 99.5% ethanol for 1 h.</li>
	<li>Wash the slides in 95% ethanol, air dry the slides, and anneal the TMSPMA coating in an oven at 80 &#176;C for 2 h.</li>
</ol>
3. Chip Fabrication
<ol>
	<li>Take one 2 mm- and one 0.3 mm-thick polymethylmethacrylate (PMMA) plate, apply double-sided tape to one side of the PMMA surface, and release the liner on one side. Leave two 2 mm-thick PMMA plates and one 1 mm-thick plate without double-sided tape.</li>
	<li>Laser-cut a 2 mm-thick PMMA plate without double-sided tape to 42 mm x 30 mm to make the bottom plate. Cut a 2 mm-thick PMMA plate with double-sided tape to make the boundary frame, with outer dimensions of 42 mm x 30 mm and inner dimensions of 37.5 mm x 25 mm.</li>
	<li>Laser-cut the 0.3 mm-thick PMMA plate with double-sided tape into a 12-mm center circle with two 2 mm-wide and 8 mm-long flow channels on opposite sides of the circle (Figure 1a).</li>
	<li>To prepare the PMMA mold for casting the PDMS cover, assemble the three pieces of PMMA components from steps 3.2-3.3 (Figure 1b) using double-sided tape.</li>
	<li>Laser-cut a 2 mm-thick PMMA plate with double-sided tape into a 5 cm x 5 cm piece with a 3 x 3 array of 8.5 mm x 8.5 mm hollow rectangles. Cut another 5 cm x 5 cm piece with a 3 x 3 array of 4-mm hollow circles. Laser-cut a 1 mm-thick PMMA plate without double-sided tape into a 5 cm x 5 cm PMMA bottom plate.</li>
	<li>Prepare the PMMA mold for casting the PDMS plug by assembling the three pieces of PMMA components from step 3.5 (Figure 1c) using double-sided tape.</li>
	<li>Prepare the PDMS cover and PDMS plug by properly mixing 30 g of PDMS elastomer and 3 g of PDMS curing agent; degas the mixture under a vacuum chamber for 1 h.</li>
	<li>Cast 1.8-2.0 g of the mixture into the PMMA mold for the PDMS cover and use the appropriate amount to fill each cavity of the PMMA mold for the PDMS plug. Cast 10 g of uncured PDMS mixture in a blank 10-cm plastic plate. Put these molds in a vacuum chamber to degas for 30 min.</li>
	<li>Cure the PDMS for 2 h at 80 &#176;C. After cooling down the cured PDMS on the mold, detach the PDMS covers and the PDMS plugs from the PMMA molds.</li>
	<li>Punch two holes with diameters of about 3 mm at the ends of flow channels of the PDMS covers.</li>
	<li>Cut the PDMS sheet molded from the 10-cm plastic plate into many 1 cm x 1 cm cubes and punch a 3-mm hole in each PDMS cube. Glue two uncured 1 cm x 1 cm PDMS cubes onto the openings of the PDMS cover (to serve as medium reservoirs and to aid in the curing process of the gradient circular hydrogel patterns) and cure for 1 h at 80 &#176;C.</li>
	<li>Bond the PDMS covers with the two PDMS reservoirs onto a TMSPMA-coated slide by pre-treating the bonding side of the PDMS cover and the TMSPMA-coated slide under an oxygen plasma machine (30 W RF power (high mode) and 600 mTorr compressed air) or a high-frequency electronic corona generator (115 V, 50/60 Hz, 0.35 A) for 90 s of O2 plasma treatment.</li>
	<li>Contact the plasma-treated surface of the PDMS covers and the TMSPMA slide and press them closely for permanent bonding through the formation of an Si-O-Si bond. 
	NOTE: Placing the chip in an oven at 80 &#176;C for 1 h can further enhance the bonding strength.</li>
	<li>After cooling, immerse the chips in 95% ethanol for 15 min and air dry. Then, sterilize the chips under UV irradiation for 1 h and store them in a box wrapped in aluminum foil in the laminar hood.</li>
</ol>
4. Static Gradient Strain on the Cell-laden Hydrogel
<ol>
	<li>Print and cut a piece of photomask, 25 mm x 37.5 mm in size, by printing the layout in Figure 2b on a transparent film. Adjust the printed size of Figure 2b to match the dimension in Figure 2a.</li>
	<li>Prepare 100 mL of DMEM medium with 10% FBS, 1% Pen-Strep, and 250 mL of DPBS in a 37 &#176;C water bath to use as the cell culture medium.</li>
	<li>Weigh 25 mg of freeze-dried GelMA into 0.3 mL of prewarmed (37 &#176;C) cell culture medium in a 1.5-mL black microcentrifuge tube. Put the microcentrifuge tube on a laboratory stirrer/hot plate until the GelMA dissolves in the medium.</li>
	<li>Weigh 50 mg of photoinitiator into 1 ml of DPBS in a microcentrifuge tube and place it in an 80 &#176;C oven for 15 min or until the photoinitiator has dissolved.</li>
	<li>Take 25 &#181;L of the 10% photoinitiator from step 4.4 and add it to the microcentrifuge tube from step 4.3. Pipette several times to mix well.</li>
	<li>Count 3 x 106 NIH 3T3 cells using an automated cell counter. Centrifuge the suspension at 200 x g for 5 min, discard the supernatant, and re-suspend the cells in 175 &#181;L of cell culture medium.</li>
	<li>Add the cell solution from step 4.6 to the microcentrifuge tube from step 4.5 to get a prepolymer cell solution of 5% GelMA, 0.5% photoinitiator, and ~6 x 106 3T3 cells/mL. After mixing, load 100 &#181;L of cell prepolymer in a 100-&#181;L micro syringe.</li>
	<li>Manually align (see Figure 3a) a piece of the photomask onto the bottom slide of the sterilized gradient strain chip and simply fix the position using a small drop of DI water in between. Connect the 100-&#181;L micro-syringe loaded with prepolymer cell solution to the inlet of the chip.</li>
	<li>Place (see Figure 3b) 50 &#181;L of prepolymer cell solution in the flow channel using the micro-syringe and then plug the outlet using a PDMS plug. Inject an extra 40 &#181;L of solution to create a convex bulge in the circular PDMS membrane.</li>
	<li>Move the chip with the photomask (bottom), micro-syringe (inlet), and PDMS plug (outlet) from step 4.9 under a UV lamp (365 nm, 9 mW/cm2) and expose it for 30 - 45 s to crosslink the concentric circular hydrogel in the fluidic chamber.</li>
	<li>Remove the PDMS plug and the micro-syringe to release the liquid pressure (see Figure 3c). Use a 1-mL syringe loaded with prewarmed DPBS to wash out uncrosslinked resins 3 times by flushing from the inlet to the outlet.</li>
	<li>Fill the flow channel with about 100 &#181;L of cell culture medium.</li>
	<li>Place the chip in a sterilized culture dish and culture in a 5% CO2 atmosphere at 37 &#176;C for a week. Refresh the medium every day.</li>
	<li>Take images of three chips on day 0 after 4 h of incubation, as a control group, and three chips on day 3, as the experimental set, using a microscope with a 20X objective. Measure the line width of each hydrogel from line 1 to line 12 using software (e.g., ImageJ) to calculate the compress strains ( Figure 4). 
	NOTE: The elongation percentage is calculated by dividing the value of the line width difference between 40 &#181;L and 0 &#181;L by the line width at 40 &#181;L.</li>
</ol>
5. Cell Staining for Alignment Analysis
<ol>
	<li>Use a syringe to inject 4% paraformaldehyde (PFA) in DPBS at RT into the flow chip for 15 min for the fixation of the cell-laden hydrogel. 
	NOTE: Caution. PFA is toxic and should be handled with care.</li>
	<li>Replace the solution with 0.5% cell membrane permeating solution in DPBS for 10 min to permeabilize the cell membrane at RT.</li>
	<li>PBS wash the samples 3 times with 5- to 10-min interval between washes (using a loaded syringe, as in step 5.1).</li>
	<li>Load 1% BSA solution into the fluidic channel for 45-60 min at RT (using a loaded syringe through the inlet port).</li>
	<li>Add 1.5 mL of methanol into the vial with Alexa Fluor 488 phalloidin to yield a final concentration of 6.6 &#181;M stock solution.</li>
	<li>Take 5 &#181;L of Alexa Fluor 488 phalloidin from step 5.5 and dilute it in 200 &#181;L of DPBS with 0.1% BSA to form a final concentration of 0.165 &#181;M Alexa Fluor 488 phalloidin.</li>
	<li>Add 200 &#181;L of the mixture solution to the fluidic channel using a micropipette and incubate the chip at 37 &#176;C for 45 - 60 min for actin staining. PBS wash (as above) the samples 3 times.</li>
	<li>Prepare 1 &#181;g/mL DAPI in PBS, flow it through the chip, and stain cell nuclei at 37 &#176;C for 5 min.</li>
	<li>Pipette DPBS into the fluidic channel to wash out the staining solution and refill the fluidic chamber with PBS solution to take images with a fluorescence microscope.</li>
	<li>Capture the fluorescent images of the 3T3 cells in the hydrogels using an inverted fluorescence microscope under 40X magnification with a CCD detector and filter sets of ex/em at 488/520 nm and 358/461 nm for Alexa Fluor 488 phalloidin (actin) and DAPI (nucleus), respectively.</li>
</ol>

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本稿でハイドロゲル図形指導及び引張ストレッチ後細胞の配向挙動を比較する単純なアプローチについて報告する.柔軟な PDMS 膜は、同心の円形ヒドロゲルの高さ別の生成のためのドームの曲率を作成します。圧力を解放した後 PDMS 膜は自動的にセンターで最大と外側の境界で最小勾配ひずみ/伸長を形成するマイクロ パターンのヒドロゲルに力を適用します。グラデーションの歪みの形成は柔軟な PDMS 膜と流体チップの処理によって設計されに出席する必要がありますいくつかの重要なパラメーターがあります: (i) 正確に制御、PDMS の厚さの膜はグラデーションのひずみ値を調整することが重要です。膜が厚すぎる場合、セル プレポリマーの最大注入量もは PDMS 膜架橋結合セルを含んだヒドロゲルのグラデーションの高さの適切な凸の曲線を生成することができません。対照的に、薄すぎる PDMS 膜は、ヒドロゲルに十分な力を適用できません。PDMS カバー金型で加硫 PDMS の重量はチップあたり約 1.6 2.0 g であることを確認してください。(ii) 汚染の防止は、流体チップで細胞を含んだゲルの架橋処理中に非常に重要です。インキュベーターで培養、徹底的に流体管内滅菌 PBS で洗浄と 75% 使用したセルの前にチップの表面を拭くためエタノールは汚染問題を避けるために助けることができます。(注意深いべきである iii) 光開始剤の濃度と露光量制御、範囲 ~0.1% - 2% (0.5% をお勧めします)。以上架橋、ハイドロゲルと紫外線照射を過剰摂取は低細胞生存率になります。(パターン化ハイドロゲルの iv) の線幅は大きすぎていけません。それ以外の場合、厚いゲルの栄養交換率は細胞増殖をサポートすることはできません。通常より小さい 300 μ m をお勧めします。2 つのハイドロゲル円間の間隔を変えることができるし、50% のデューティ サイクルをお勧めします。(v) 洗浄またはソリューションと流路を補充中は、気泡の形成の避けるべきであります。チップ ソリューションを軽くピペッティングの泡を削除に役立ちます。
PDMS 変形曲率によって生成されるグラデーションのひずみの概念はグラデーション歪を適用するさらにアップグレードすることができ、生化学的刺激、機能的組織再生に関する多くの研究に恩恵を受けることができますと統合することができます。拡張実験的制御のため、高度な流体システムによる PDMS のプラグで簡単な流体注入モジュールを交換できます。PMMA の金型は、SU 8 微細加工金型またはバルク エッチング シリコン型で置き換えることができます。
このグラデーションひずみチップ円形細胞を含んだヒドロゲルは、外部機械的または電気的機械を使わない 3 D ゲルの静的圧縮力を生成できます。したがって、一連のひずみ条件、外部マシンの操作によって引き起こされる汚染問題のリスクなしで細胞の挙動を調査するため高速スクリーニング プラットフォームを提供します。ただし、PDMS 膜ヒドロゲルの劣化まで株を生成するため、時間制御ひずみ刺激は達成可能なありません。

ネイティブの微小環境を模したブロック素材として、ゲルの細胞外マトリックス (ECM) を含む再細胞の成長をサポートする生体模倣組織足場を構築できます。組織の機能を所有するには、組織のセルの配置は必須要件です。さまざまな 2 D (すなわち表面上で培養した細胞) と 3 D (すなわちハイドロゲルにカプセル化細胞) セルの配置は、培養または細胞またはマイクロ フレキシブル基板上にカプセル化することによって達成されている- またはナノ パターン1。マイクロ アーキテクチャの 3 D セルの配置はより魅力的な微小環境は、ネイティブの組織構築2,3,4に近いです。3 D のセルの配置のための一般的なアプローチは、ハイドロゲル形2,3の幾何学的なキューです。短軸方向に細胞増殖の制限された空間のため細胞はマイクロ パターン化ハイドロゲルの長軸方向に沿って配置を目指しています。別のアプローチは、ストレッチ方向4,5並列セルの配置を達成するためにゲルに引張のストレッチを適用することです。
圧縮ひずみなどの ECM ゲル上生物物理刺激または電気のフィールドは、適切な組織統合、増殖および分化1,2,3の細胞機能を調整できます。多くの研究は、複数機械制御ユニット4,6,7,8,9を使用して、一度に 1 つのひずみ条件を適用することによって細胞の挙動を調査するために行われています。たとえば、機械式ステップ モーターの使用は圧迫や共通のアプローチ7,10されている 3 D セルでカプセル化されたコラーゲンのゲルの上に伸ばし。しかし、そのような制御装置は余分なスペースを必要とする、インキュベーター7,9,11,12の汚染の問題に直面しています。さらに、大型の楽器は、再現性の高い13を提供する正確に制御環境を与えることはできません。
細胞を含んだヒドロゲルは、医用マイクロ スケールで通常用いられることを考えると、同時に 3 D 模倣構造体外2,14,15,16,17,18細胞の挙動を調査するためのひずみ/ストレッチ刺激の範囲を生成する MEMS 技術を組み合わせることが有利です。たとえば、マイクロ流体チップの PDMS 膜を変形するガス圧を使用して、異なる系統9,16に細胞の分化を運転系統に上昇を与えることができます。ただし、クリーン ルームやモーター、ポンプ、バルブ、圧縮ガスのソフトウェア管理の統合で複雑なチップ製造プロセスなど、多くの技術的課題があります。
この作品は、同心の円形ハイドロゲル パターンと柔軟な PDMS 膜を用いた自律的勾配の静的ひずみマイクロ流体チップを取得する単純なアプローチを紹介します。異なり、既存のアプローチのほとんどは、当社のプラットフォームは黄色い部屋の外を作製できるし、孵化の間に外部機器なしの携帯カプセルのヒドロゲルを同心円のグラデーションの系統を自己生成所有しているポータブルと使い捨ての小型デバイスです。ハイドロゲル形状の組み合わせによって影響を受けた 3T3 繊維芽細胞の挙動と 3 日間のグラデーションひずみチップで 3 D の ECM 模倣環境内セルの配置の観察の間に様々 な引張ストレッチ指導の手がかりを示した。

<table><tbody><tr><td>1.5 mL black microcentrifuge tube</td><td>Argos Technologies&amp;nbsp;</td><td>03-391-161</td><td>This one can be replaced with a neutral color of 1.5 mL tube covered with aluminun foil</td></tr><tr><td>10x DPBS</td><td>Sigma-Aldrich</td><td>56064C</td><td></td></tr><tr><td>Alexa Fluor 488 phalloidin&amp;nbsp;</td><td>Invitrogen</td><td>A12379&amp;nbsp;</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A1595</td><td></td></tr><tr><td>Calcein</td><td>Molecular Probe</td><td>C1430</td><td>For labeling viable cells</td></tr><tr><td>CCD</td><td>PCO. Imaging</td><td>Pixelfly qe</td><td></td></tr><tr><td>Cell membrane permeating solution</td><td>Sigma-Aldrich</td><td>X100</td><td>0.5% Triton X-100 for permeating cell membrane</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D8417</td><td>Cell nucleus staining</td></tr><tr><td>Dialysis membrane</td><td>Sigma-Aldrich</td><td>D9527</td><td>Molecular weight cut-off = 14,000</td></tr><tr><td>DMEM</td><td>Gibco</td><td>11995-065</td><td></td></tr><tr><td>Double-side tape</td><td>3M</td><td>8003</td><td></td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH30071.03</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G2500</td><td>gel strength 300, type A, from porcine skin</td></tr><tr><td>High frequency electronic corona generator</td><td>Electro-technic products</td><td>MODEL BD-20</td><td></td></tr><tr><td>Methacrylic Anhydride</td><td>Sigma-Aldrich</td><td>276685</td><td></td></tr><tr><td>Micro syringe</td><td>Hamilton</td><td>80501</td><td>50 &amp;mu;L&amp;nbsp;</td></tr><tr><td>Microscope</td><td>Olympus</td><td>IX71</td><td>Include two filter sets: LF405/LP-B-000 and LF488/LP-C-000 from Semrock</td></tr><tr><td>Oxygen plasma machine</td><td>Harrick plasma</td><td>PDC-001</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>For fixing cell</td></tr><tr><td>PDMS</td><td>DOW CORNING</td><td>Sylgard 184</td><td>Mixture for PDMS chip cast-molding fabrication</td></tr><tr><td>Pen-Strep</td><td>Gibco</td><td>10378-016</td><td>penicillin/streptomycin</td></tr><tr><td>Photoinitiator</td><td>CIBA</td><td>Irgacure 2959</td><td></td></tr><tr><td>Propidium iodide</td><td>Sigma-Aldrich</td><td>P4170</td><td>For labeling dead cells</td></tr><tr><td>Sterile Filtration cup</td><td>Millipore</td><td>SCGPT05RE</td><td></td></tr><tr><td>TMSPMA</td><td>Sigma-Aldrich</td><td>440159</td><td>For hydrogel immobilization</td></tr><tr><td>Ultrasonicator</td><td>Delta</td><td>D150H</td><td>150W, 43kHz</td></tr><tr><td>UV light</td><td>DAIHAN</td><td>WUV-L10</td><td></td></tr><tr><td>Freeze Dryer</td><td>FIRSTEK</td><td>150311025</td><td></td></tr><tr><td>NIH3T3(fibroblast)</td><td>Food Industry Research and Development Institute(FIRDI)</td><td>08C0011</td><td></td></tr><tr><td>MOXI Z Mini Automated Cell Counter</td><td>ORFLO</td><td>MXZ001</td><td></td></tr></tbody></table>

gradient strain chip for stimulating cellular behaviors in cell-laden hydrogel

人工細胞の配置については、組織工学の分野でのホットな話題です。過去の研究のほとんどは、複雑な実験的プロセスおよび汚染の問題に通常関連付けられている質量システム制御、使用して細胞を含んだハイドロゲルのひずみ誘起細胞の単一の位置合わせを調査しました。したがって、この記事では PDMS のプラスチック カバー、UV 透明なガラス基板 3 D ゲルの細胞挙動の刺激のため流体チップを用いた勾配静的ひずみを構築するシンプルなアプローチを提案する.流体の商工会議所でのオーバー ロードの写真パターン形成可能なセル プレポリマーは、カバーの凸曲面 PDMS 膜を生成できます。UV 架橋後湾曲した PDMS 膜とバッファー洗浄、調査細胞の微小環境下で同心円形パターンをさまざまなグラデーションの株の下で動作は外部機器なしの単一の流体チップで自己確立です。NIH3T3 細胞をゲル上 15-65% から様々 なひずみの刺激との連携で、ジオメトリの指導の下で細胞の配置の傾向の変化を観察した後示した。3 日間培養後ハイドロゲル ジオメトリは、細胞が高圧縮ひずみハイドロゲル伸長方向に沿って整列低圧縮ひずみ, セルの配置を支配しました。これらの間は、細胞は、ハイドロゲルの伸長とパターン化ハイドロゲルの幾何学指導の根本的な指導の逸散によるランダムな配置を示した。

完成したグラデーションひずみ刺激チップの各円形のハイドロゲルの機械的変化を比較するためは 0 μ L (図 4 a) の注入量と 40 μ L (図 4 b) では、同じチップの 2 つの各円形のハイドロゲルの線幅をそれぞれ測定しました。各サークルでパーセントの伸びを 0 μ L 注入チップ (図 4 c) に対応するハイドロゲルの線幅で 40 μ L 注入チップの伸びを割ることによって求めた。ヒドロゲルは縦収縮ひずみは横方向の伸張ひずみ量に相当するので非圧縮性材料であります。したがって、40 μ L 注入量とチップのパーセントの伸びを示す 15 65% 伸長圧縮ひずみに変換することができます (補足のファイル 1を参照してください)。
それぞれ、細胞核、DAPI とファロイジン、F アクチンの蛍光染色細胞の配置を分析するため行われました。原子力の方向のデータを提供される DAPI 染色と細胞拡散を評価するために適用されたファロイジン染色します。図 5a-cは、グラデーションひずみチップのセルの配置を示しています。行 1、3T3 細胞は、半径方向に沿って配置されます。ヒドロゲル 7 でセルと一直線になりランダムに、セル、行 12 の円形の方向に沿って。蛍光画像を染色、に従って 12 (ラジアル方向で最も低い伸びハイドロゲル) 行の長い軸配置とセルの配置角度にライン 1 (放射状の方向に伸び最大ヒドロゲル) 3T3 細胞のセルの配置の角度から 90 度シフトが発見されました。
以前研究2,9, 200 μ m ライン パターン ヒドロゲルのセルは、ヒドロゲルの長軸方向に沿って配置する目指しています。しかし、この研究では短軸方向に 200 μ m ゲル上の細長いストレッチ提供に影響を与えるし、ハイドロゲルの負担の割合を制御することにより細胞の配置を支配するもう一つの要因であることを見ました。ライン 1 に 65% 株の根本的な配置は、ヒドロゲルの伸びストレッチがセルの配置を支配することを証明しました。12 行目 15% 株の円形配置は長尺効果がセルの配置を支配したことを証明しました。ハイドロゲル 7 の 40% のひずみ、セルはジオメトリのガイダンスとひずみの効果の中和のためにランダムに配向。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/53715/53715fig1.jpg"> 
図 1.PMMA 母 PDMS シート プラグ加工用金。底板、境界フレームと流路を含む PMMA 金型の(、)で区切られたコンポーネント。両面テープで組立後、 (b)流シートの PMMA 母金型が形成されます。(c)別 PMMA 金型は、PDMS のプラグインのために組み立てられます。赤の番号は、深さを表します。(単位: mm)<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53715/53715fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/53715/53715fig2.jpg"> 
図 2.細胞を含んだハイドロゲル パターン プラスチック フォトマスク設計。三角形の図形 (一番下の行と高さ 6.5 mm に 2 mm) と 2 つの開口部がある新鮮な細胞培養液を供給する流路に接続します。(a)プラスチックのフォトマスクが付いてディメンション。同心円の期間は 400 nm とデューティ サイクルは 50%。中央の円の直径は 2 mm です。 (b)プラスチック透明フィルムにレーザー印刷のラベルなしのフォトマスク レイアウト。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53715/53715fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/53715/53715fig3.jpg"> 
図 3。グラデーション作製プロセス歪 PDMS 流体チップ径方向の細胞を含んだハイドロゲル。(a)プラスチック製フォトマスク整列し、TMSPMA コーティングの流体チャネル チップの下で付きます。プレポリマーの細胞液とマイクロ シリンジ チップの入口に挿入され、流路に合わせて約 50 μ L を注入するために使用します。(b)と流路の出口を閉じて、PDMS プラグし、プレポリマー携帯ソリューションの追加の 40 μ L を注入します。ガラス下部は 30 の UV パターン s フロー チップの同心の円形ハイドロゲルを作製します。液体の圧力は、コンセントを抜いて流路で解放され(c) 、国連架橋混合物、DPBS で洗っています。(d)グラデーション歪みを持つチップは、細胞培養の準備ができている同心円状の細胞を含んだヒドロゲルを適用されます。UV 架橋プロセスで(e)半径に沿ってハイドロゲルの非連続的なグラデーションの高さが形成されています。(コンセントを抜いた後に f) PDMS 膜はフラットになるし、セル カプセル化ハイドロゲルのグラデーション ストレスを適用します。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53715/53715fig3large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/53715/53715fig4.jpg"> 
図 4。純粋なハイドロゲル伸長グラデーション。伸長線幅、グラデーションの電池を封止材なしの純粋なハイドロゲルの割合チップ(a) 0 μ L と 3 日 (対照群) と(b) 40 μ L 注入量をひずみ。(c)伸び率は 40 μ L の線幅で 40 μ L と 0 μ L の線幅差の値を割ることによって計算されます<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53715/53715fig4large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/53715/53715fig5.jpg"> 
図 5.3 日目のグラデーション チップでカプセル化された 3T3 細胞の蛍光アクチン核染色画像。 (C)線 1、7 行目(d-f) 、 (g i)線 12 セル配置方向放射状配置、ランダムな配置、および円形配置をそれぞれ明らかに。緑と青の色は、アクチンと核の汚れをそれぞれ示しています。点線の白い線は、ヒドロゲルの境界を表します。スケール バー: 200 μ m.<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53715/53715fig5large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
補足図 1。ドーム型 PDMS 曲率の計算。H(x): 凸 PDMS 曲線;H0: PDMS ドーム前に、と加工後の最大高低差r:; ドームの半径V: 過注入ボリュームのドームとして PDMS 変形青の領域の。詳細については、補足のファイル 1を参照してください。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53715/Supplementary_Figure_1.tif" target="_blank">この図をダウンロードするここをクリックしてください</a>。
1 の補足ファイル。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53715/Supplementary_File_1-SI.docx" target="_blank">このファイルをダウンロードするここをクリックしてください</a>。

Watch this Scientific Journal Video about Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel at JoVE.com

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

組織工学用セルの配置を調整する同心円状細胞を含んだハイドロゲルの非連続的なグラデーション静的系統を提供する単純なアプローチを紹介します。

細胞を含んだハイドロゲルの刺激細胞挙動のひずみ勾配チップ

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single ...

gradient-strain-chip-for-stimulating-cellular-behaviors-cell-laden

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

7.9K ビュー.  National Tsing Hua University. 組織工学用セルの配置を調整する同心円状細胞を含んだハイドロゲルの非連続的なグラデーション静的系統を提供する単純なアプローチを紹介します。

ビデオ: Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

機械的にダイナミックな細胞培養のための微細加工プラットフォーム

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

生物工学

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

細胞生物学の勾配を生むマイクロ流体デバイス

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

生物学

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

シングルまたは共存化学/電気/せん断応力刺激下で細胞応答を研究するためのマイクロ流体デバイスを設計

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

動的圧縮で軟性細胞を刺激するマイクロ流体プラットフォーム

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

化学的集団移動のためのマイクロ流体学と統合された牽引顕微鏡

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

ライブ顕微鏡イメージング下での3Dヒドロゲルの制御株

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Microfluidic Model to Mimic Initial Event of Neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

新血管新生初期事象を模倣する微発性モデル

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

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 ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

マイクロパターントラクション顕微鏡のパターン生成

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

内皮細胞に対する組織剛性とせん断応力の影響の研究

In Vitro Model Integrating Substrate Stiffness and Flow to Study Endothelial Cell Responses

We present an innovative in vitro model aimed at investigating the combined effects of tissue rigidity and shear stress on endothelial cell (EC) function, which are crucial for understanding vascular health and the onset of diseases such as atherosclerosis. Traditionally, studies have explored the impacts of shear stress and substrate stiffness on ECs, independently. However, this integrated system combines these factors to provide a more precise simulation of the mechanical environment of the vasculature. The objective is to examine EC mechanotransduction across various tissue stiffness levels and flow conditions using human ECs. We detail the protocol for synthesizing gelatin methacrylate (GelMA) hydrogels with tunable stiffness and seeding them with ECs to achieve confluency. Additionally, we describe the design and assembly of a cost-effective flow chamber, supplemented by computational fluid dynamics simulations, to generate physiological flow conditions characterized by laminar flow and appropriate shear stress levels. The protocol also incorporates fluorescence labeling for confocal microscopy, enabling the assessment of EC responses to both tissue compliance and flow conditions. By subjecting cultured ECs to multiple integrated mechanical stimuli, this model enables comprehensive investigations into how factors such as hypertension and aging may affect EC function and EC-mediated vascular diseases. The insights gained from these investigations will be instrumental in elucidating the mechanisms underlying vascular diseases and in developing effective treatment strategies.

著者スポットライト:血管メカノバイオロジーにおける内皮細胞応答を研究するための基板剛性と流動条件の統合

We synthesized and characterized a tunable gelatin-based substrate for culturing vascular endothelial cells (ECs) under relevant vascular flow conditions. This biomimetic surface replicates both physiological and pathological conditions, enabling the study of mechanical forces on EC behavior and advancing our understanding of vascular health and disease mechanisms.

内皮細胞応答を研究するための基質の剛性と流れを統合するin vitroモデル

We present an innovative in vitro model aimed at investigating the combined effects of tissue rigidity and shear stress on endothelial cell (EC) function, ...