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

这个项目被支持由研究生学生研究国外程序 (NSC-101-2917-I-007-010);生物医学工程项目 (NSC-101-2221-E-007-032-MY3);和纳米技术国家计划 (NSC-101-2120-M-007-001-) 的侨生，台湾国家科学委员会。作者想要感谢阿里 Khademhosseini，荷兰货币恰姆彻纳尔，阿格保罗教授和哈佛大学医学院嵘利廖分享的水凝胶和单元格的封装技术。

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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作者没有透露。

在本文中，我们报告一个简单的方法来比较水凝胶形状指导和拉伸拉伸后的单元格对齐方式行为。灵活的 PDMS 膜创建用于生成不同高度的同心圆形水凝胶圆顶形的曲率。后释放压力，PDMS 膜自动适用力于微图案水凝胶形成梯度应变延伸，与中心的最大值和最小值外, 边界处。应变梯度的形成由灵活的 PDMS 膜和处理的流控芯片设计的正应参加的几个重要参数: (i) 精确控制厚度的聚二甲基硅氧烷膜是关键要调整应变梯度值。如果膜太厚，甚至最大注射量的细胞预聚体不能在单元格拉丹水凝胶梯度高度交联的聚二甲基硅氧烷膜中生成适当的凸曲线。相比之下，太薄的聚二甲基硅氧烷膜，不能适用水凝胶的足够的力量。请检查未硫化聚二甲基硅氧烷 PDMS 盖模具的重量是大约 1.6-2.0 g 每个芯片。（在流控芯片单元格拉丹水凝胶交联处理期间，ii） 污染防治是非常重要的。在细胞培养中孵化器、 彻底与流体通道中无菌的 PBS 洗涤和使用 75%乙醇擦拭表面的芯片可以帮助避免污染问题。（iii） 光引发剂浓度及紫外线照射的剂量必须小心控制并在范围内的 ~0.1%-2%（推荐 0.5%）。在交联水凝胶和吸食了过量的紫外线照射会导致低细胞活力。（iv） 图案的水凝胶线宽度不应太大。否则，在厚厚的水凝胶中的养分替代率不能够支持细胞增殖。通常，建议使用小于 300 µ m。水凝胶的两个圆之间的间距也可多种多样，并建议 50%工作周期。（v） 在洗涤或加气流道的解决方案时，应避免泡沫的形成。轻轻地吹打在芯片解决方案可以有助于消除气泡。
梯度应变产生的聚二甲基硅氧烷变形曲率的概念可以进一步升级到应用动态梯度菌株，可以结合生化刺激，功能性组织再生的许多研究都可以受惠。简单的流体注入模块与 PDMS 插头可以由任何先进的射流扩展实验控制系统取代。PMMA 模具也可以超微型苏 8 模具或批量蚀刻硅模具所取代。
这个梯度应变片圆形细胞满载水凝胶可以生成静态的压缩力，在没有外部机械或电气机器的 3D 水凝胶。因此，它为研究细胞行为的一系列的应变条件，而没有风险的外部机器操作引起的污染问题提供快速筛选平台。然而，时间控制应变刺激不是可以实现的因为 PDMS 膜生成株直到退化的水凝胶。

作为模仿一个本机的微环境块材料，含细胞外基质 (ECM) 水凝胶可以重新生成仿生支架支持细胞的生长。拥有组织的职能，组织单元格对齐方式是一项基本要求。各种 （即，细胞表面） 2D 和 3D （即，封装在水凝胶的细胞） 培养或封装细胞中或与微型柔性衬底上取得了单元格对齐方式-或纳米模式1。在微体系结构的三维单元格对齐方式是更有吸引力，因为微环境是更接近于自然组织构造2，，34。三维单元格对齐方式的一个常用方法是水凝胶形状2，3的几何提示。由于细胞增殖在短轴方向的受限空间，细胞目的沿微图案的水凝胶的长轴方向对齐。另一种方法是适用于凝胶来实现单元格对齐方式平行于拉伸方向4，5拉抻。
生物物理刺激对 ECM 的水凝胶，如压缩应变或电场的作用，能调节细胞功能的适当组织一体化、 增殖和分化1，，23。很多研究已经通过使用多个机械控制单位4，6，7，，89一次应用一个应变条件探讨细胞行为。例如，机械步进电动机使用挤压或拉伸对 3D 封装细胞胶原凝胶一直共同的办法7，10。然而，这种控制的设备需要额外空间并面临孵化器7，9，，1112中受污染的问题。此外，大型仪器不能精确的控制环境，以提供高重复性13。
考虑到单元格拉丹水凝胶通常受雇在微尺度生物医学应用，它有利于结合 MEMS 技术来生成一个范围的应变/拉伸刺激同时探讨细胞行为在 3D 仿生构造体外2，14，15，16，，1718。例如，利用气体压力变形聚二甲基硅氧烷膜在微流控芯片可以引起不同品系，开车去不同的谱系9，16细胞分化。然而，有许多技术难题，如在一个干净的房间和软件控制集成的电机、 泵、 阀门和压缩的气体的复杂的芯片制造工艺。
在这项工作，我们演示了一个简单的方法来获得自我维持的梯度静态应变微流控芯片采用同心圆形水凝胶模式和灵活的 PDMS 膜。不同于大多数现有的方法，我们的平台是便携式和一次性使用的微型设备，可以制作黄色房间外面并拥有自生梯度菌株对同心细胞封装水凝胶，无需外部机械设备孵化期间。3T3 成纤维细胞细胞行为受水凝胶形状组合和期间的观察 3 天内 3D ECM 拟态环境梯度应变片在单元格对齐方式显示出各种拉伸弹力的指导线索。

<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

人工指导细胞对齐方式是在组织工程领域的热点话题。大部分的先前的研究已经进行单个单元格拉丹的水凝胶应变诱导细胞对准使用复杂的实验过程和质量控制系统，这是通常与污染问题。因此，在本文中，我们提出一种构建流控芯片用塑料的聚二甲基硅氧烷盖和紫外透明的玻璃基板，刺激的 3D 水凝胶的细胞行为的静态应变梯度的简单方法。超载的照片; 细胞预聚物中射流室可以生成凸曲线的 PDMS 膜在封面上。紫外光交联后通过弯曲的聚二甲基硅氧烷膜和缓冲区洗，调查细胞微环境下的同心圆形优缺点行为下个变种的梯度是自建在单一的流控芯片，没有外部文书。观察到的几何在指导下，在合作与应变刺激，相差 15-65%的水凝胶细胞对齐趋势变化后论证了 NIH3T3 细胞。后 3 天孵化，水凝胶几何主导下低的压应变，单元格对齐方式哪里细胞沿水凝胶伸长率高的压缩应变下的方向排列。这些，之间细胞细胞随机对齐方式由于耗散的激进指导的水凝胶伸长和图案的水凝胶的几何指导。

若要比较每个循环的水凝胶在完成的应变梯度刺激芯片之间的机械变化，我们分别测量中的两个相同的芯片，具有 0 微升 (图 4a) 注射量和 40 µ L (图 4b)，每个循环水凝胶的线条宽度。%伸长率每个圆圈都伸长 40 µ L 注射芯片中的除以相应的水凝胶在 0 微升注射芯片 (图 4 c) 的线宽。水凝胶是一种不可压缩的材料，所以垂直订约应变将相当于横向伸长应变。因此，芯片与 40 µ L 注射量的百分比理论伸长值的修正显示 15 65%伸长，可以转换为压缩应变 （见补充文件 1）。
荧光染色细胞核和 F-肌动蛋白与 DAPI 和鬼笔，分别进行了分析的细胞的对齐方式。DAPI 染色提供了关于核定位数据和鬼笔染色用于评估细胞传播。图 5a-c显示单元格对齐方式中梯度应变片。在第 1 行，3T3 细胞沿径向方向排列。在凝胶 7，细胞随机，对齐和单元格对齐沿圆周方向在第 12 行。根据荧光染色图像，发现了带长轴对齐 （水凝胶最低伸长率在径向方向） 12 行中的单元格的对齐方式角度 90 ° 转向视角下的单元格对齐方式的 3T3 细胞 （伸长率最高的水凝胶在径向方向） 1 号线。
在以前研究2，9，在 200-µ m 线图案水凝胶的细胞目的对齐沿长轴方向的水凝胶。然而，在这个研究中，我们观察到 200 微米水凝胶在短轴方向拉长伸展动作所产生的影响和支配的细胞的对齐方式通过控制应变对水凝胶的百分比的另一个因素。1 号线 65%应变，激进的对齐方式证明水凝胶伸长拉伸占主导地位的单元格对齐方式。对于 15%应变第 12 行，圆对齐方式证明了长轴效应占主导地位的单元格对齐方式。水凝胶 7 40%应变，细胞随机排列由于中立化几何指导和应变效应。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/53715/53715fig1.jpg"> 
图 1.聚二甲基硅氧烷薄板和插头制作 PMMA 母亲模具。(a)分离的组分的 PMMA 模具，包括底板、 边框架和流道。装配后用双面胶带，形成了(b)流量表 PMMA 母亲模具。(c)另一个 PMMA 模具组装而成的聚二甲基硅氧烷堵塞。红色数字表示深度。(单位: mm)<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53715/53715fig1large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/53715/53715fig2.jpg"> 
图 2.单元格拉丹水凝胶优缺点塑料光掩模设计。有两个开口三角形形状 （2 毫米在底线和高度 6.5 毫米） 连接到流渠道供应新鲜细胞培养基。(a)塑料光掩模标记与维度。同心圆段是 400 毫微米和占空比是 50%。中心圆的直径为 2 毫米 (b)光掩模布局，没有塑料的透明薄膜激光打印的标签。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53715/53715fig2large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/53715/53715fig3.jpg"> 
图 3。制造工艺的渐变应变沿径向方向的单元格拉丹水凝胶在 PDMS 流控芯片。(a)塑料光掩膜与对齐，并根据芯片在一起，TMSPMA 涂覆的流体通道。与预聚体的细胞液微注射器是插入到芯片的入口和用于注入约 50 µ L 填补流通道。(b)流道出口关闭与 PDMS 插头和注入额外 40 µ L 的预聚体的细胞液。玻璃底部是 UV 图案为 30 s 制作同心圆形水凝胶流芯片中的。(c)液体的压力被释放在流道中拔出插座和联合国交联混合物被淘汰掉的新方案。(d)与静态应变梯度的芯片适用准备细胞培养的同心单元格拉丹的水凝胶。在紫外交联过程中，形成了(e)水凝胶沿半径非连续梯度高度。（f） 后拔出插座，PDMS 膜变得平坦和梯度压力对细胞封装的水凝胶。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53715/53715fig3large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/53715/53715fig4.jpg"> 
图 4。纯净水凝胶伸长率梯度。伸长的线宽和无细胞封装在渐变中的纯净水凝胶的百分比应变芯片第 3 天与(a) 0 微升 （对照组） 和(b) 40 µ L 注射量。(c)除以 40 µ L 的线宽线宽度差 40 µ L 和 0 µ L 之间的值计算得出伸长率是<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53715/53715fig4large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/53715/53715fig5.jpg"> 
图 5.荧光的肌动蛋白核染色图像的 3T3 细胞中天 3 上的梯度芯片的封装。单元格对齐方向(g-i)第 12 行，第 1 行(c) 、 (d f)一行 7 人，揭示径向对齐、 随机对齐和圆形的对齐方式，分别。绿色和蓝色颜色分别显示肌动蛋白和核的污渍。白色虚线表示水凝胶的边界。条码秤: 200 µ m.<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53715/53715fig5large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
补充的图 1。的圆顶状的聚二甲基硅氧烷曲率计算。H(x): 凸的聚二甲基硅氧烷曲线;H0: 聚二甲基硅氧烷圆顶变形; 前后最大高度差异: 半径圆顶;五: 蓝色的区域，这将导致一个圆顶的聚二甲基硅氧烷变形过度的注射量。有关详细信息请参阅补充文件 1 。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53715/Supplementary_Figure_1.tif" target="_blank">请点击这里下载此图。</a>
补充文件 1。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53715/Supplementary_File_1-SI.docx" target="_blank">请点击这里下载此文件。</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

这篇文章介绍了一个简单的方法，对提供非连续梯度静态菌株对同心单元格拉丹水凝胶来调节单元格对齐方式为组织工程。

在单元格拉丹水凝胶中刺激细胞行为的应变梯度芯片

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 次观看.  National Tsing Hua University. 这篇文章介绍了一个简单的方法，对提供非连续梯度静态菌株对同心单元格拉丹水凝胶来调节单元格对齐方式为组织工程。

视频: 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.

微机械动态细胞培养平台

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

科研

生物工程

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.

一个渐变产生的微流体装置，细胞生物学

 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.

设计微流体装置用于研究细胞反应在单人或共存化工/电气/剪切应力刺激

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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.

微流体平台，用于用动态压缩刺激软骨细胞

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.

牵引显微镜与微流体集成，用于化学集体迁移

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.

实时显微镜成像下的 3D 水凝胶控制应变

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.

模仿神经血管化初始事件的微流体模型

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.

机械性刺激软骨细胞琼脂糖凝胶

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.

微图案牵引显微镜的图案生成

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

研究组织刚性和剪切应力对内皮细胞的影响

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.

作者聚焦：整合基质刚度和流动条件以研究血管力学生物学中的内皮细胞反应

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.

整合基质刚度和流动性的体外模型以研究内皮细胞反应

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