Name: controlled strain of 3d hydrogels under live microscopy imaging
Uploaded: 2020-12-04T13:00:00.000+00:00
Duration: 7 min 41 s
Description: Watch this Scientific Journal Video about Controlled Strain of 3D Hydrogels under Live Microscopy Imaging at JoVE.com

此处包含的一些数字经版权许可中心许可已修改:斯普林格自然， 生物医学工程年鉴。用均匀的 z 轴菌株应变 3D 水凝胶，同时实现实时显微镜成像，A. Roitblat Riba，S. 纳坦，A. 科莱尔，H. 拉什金，O. 柴切扬，A. 莱斯曼，版权所有© （2019）。
https://doi-org.bdigitaluss.remotexs.co/10.1007/s10439-019-02426-7

作者没有什么可透露的。

本文介绍的方法和协议主要基于我们之前由罗伊特布拉特·里巴等人进行的研究。 
与现有方法相比，该方法的主要优点包括有可能从其周长中拉紧非常柔软的 3D 水凝胶（弹性 Modulus +100 Pa），并在 实时 对焦成像下进行。其他方法通常限制其在 Z轴中应用应变场的能力，并且无法在拉伸时提供原位高放大显微镜图像，这主要是由于从样品到目标镜头的工作距离限制。大多数可用系统基于 2D 基板拉伸，这在模仿生理组织环境方面是有限的。
该系统的局限性包括凝胶聚合后纤维密度的潜在不一致均匀性（图7A），特别是当使用大凝胶体积或复杂的几何形状时。这种非同质性可导致凝胶中的不均匀菌株和纤维对齐。此外，相对深 的 Z节（>30 μm）的光学限制可能导致光纤方向分析错误。
3D 拉伸协议中的关键步骤包括以下 （i） 确保样品凝胶已实现对硅胶内几何切口内壁的完全粘附，无气泡或撕裂，尤其是在Z方向沿线。厚度至少为 100μm（Eq.1）的样品的完整粘附和连续性对于在拉伸之前将样品可靠地拉伸到所需的应变（ii）正确将样品连接到SCyUS设备至关重要。有必要使用 3D 打印夹具 （图 5A）将硅胶条连接到支架和非弹性织物长度扩展器上，并验证所有部件均在线连接，确保在凝胶中对称地诱导应变。这也适用于设备主轴（图 6B-C）（iii）的正确加载，仔细捕捉样品的参考（拉伸前）位置。如果在样品已经处于紧张状态时拍摄参考图像（例如，由于此步骤之前服务器的附件），则所有应变测量将不准确。
未来可能的应用和对所提交协议的修改包括:
（一） 循环拉伸系统可应用于嵌入式水凝胶的细胞。由于电机的动态运动由微控制器软件编码，因此任何循环配置文件都可以进行编程，仅受系统中使用的电机的分辨率和旋转速度的限制（Roitblat Riba 等人提供的更多详细信息等41）。在这项工作中，我们证明了纤维蛋白凝胶可以在动态拉伸下维持粘附（表1）。虽然需要进一步测试，但循环加载的应用将深入了解水凝胶和嵌入式细胞的动态响应。
（二） 可以修改硅胶切口的几何形状和体积。在当前协议中，我们只关注圆形几何形状中的凝胶。在这种情况下，内部菌株在整个凝胶中相对均匀，正如我们的FE模拟（图13）所预测的那样。但是，如果通过修改硅胶的切口部分来考虑其他凝胶几何形状，梯度就会出现。例如，"钻石"形状的切口可能导致凝胶菌株的梯度，因为沿拉伸方向切口的初始长度会逐渐变化。这种凝胶应变控制方法特别吸引人，因为不同形状的切口相对来说比较小。此外，硅胶切口的厚度和面积可以很容易地改变，从而允许对凝胶体积进行调整，从几微升的微型体积到与 mm-cm 比例凝胶尺寸相关的较大体积。此外，可以在同一硅胶条中加入几个切口，允许同时拉伸多个凝胶。
（三） 细胞可以嵌入水凝胶中。在将细胞嵌入拉伸的水凝胶中时，应考虑一些挑战。嵌入在 3D 水凝胶中的大多数细胞在矩阵上应用了相当大的牵引力，并随着时间的推移使基质降解。这可能导致凝胶的全球收缩和消化，这可能导致凝胶在长期潜伏时与硅胶切出的内壁断开。应对这种情况的一个可能方法是使用低细胞浓度，并将细胞实验的时间限制在凝胶收缩和降解最少的间隔内。我们以前已经证明，在静态拉伸下，在10%的静态拉伸下培育纤维细胞的可能性，在5小时内，没有任何证据表明凝胶撕裂或与硅胶切口41断开。
（四） 此设置还应与其他类型的水凝胶一起测试，特别是合成的，如聚丙烯酰胺 （PAA） 或聚乙二醇 （PEG） 基水凝胶，这些水凝胶通常用于细胞培养。应检查这些水凝胶对硅胶切口的自然粘附，如果发现不足以进行凝胶拉伸，则应使用鼓励粘附的技术。应考虑使用生物胶水48、49或化学处理50、51、52到切口表面。

组织对机械力的反应是多种生物功能的组成部分，包括基因表达1、细胞分化2和组织改造3。此外，细胞外基质（ECM）的力诱发变化，如纤维对齐和密度化，可以影响细胞行为和组织形成4，5，6。ECM的纤维网状结构具有耐人寻味的机械特性，如非线性弹性、非擦合变形和塑料变形7、8、9、10、11、12。这些特性影响细胞及其周围微环境对外部机械力的反应13、14。了解 ECM 和组织对机械力的反应将推动组织工程领域以及更精确的计算和理论模型的开发取得进展。
最常见的机械拉伸样本的方法都集中在细胞载2D基材上，以探索对细胞行为的影响。例如，这些包括将菌株应用于多晶硅氧烷（PDMS）基板，以及分析与拉伸方向15、16、17、18、19相关的细胞重新定向角度。然而，研究3D细胞嵌入式水凝胶对外部拉伸的反应的方法更为有限，这种情况更密切地模仿组织微环境。与3D矩阵20相比，3D拉伸方法的进步尤为重要，因为细胞在2D基材上的行为不同。这些行为包括细胞重新调整，蛋白质表达水平，和迁移模式21，22，23。
允许3D样品拉伸的方法和设备包括市售的24、25、26、27、28和为实验室研究开发的方法和设备29。这些方法使用可拆解硅胶管30，多井室31，夹子26，32，生物反应器11，33，罐34，35，36，和磁铁37，38。有些技术会产生局部变形的3D水凝胶，例如从凝胶5中的两个单点拔针，而另一些技术允许凝胶16的大部分变形。此外，这些系统大多侧重于分析X-Y平面中的应变场，而Z方向的应变场信息有限。此外，这些设备中只有少数能够进行微观原位成像。原位高放大成像（例如共聚焦显微镜）的主要挑战是，从目标透镜到样品的几百微米工作距离有限。允许在拉伸过程中进行实时成像的设备牺牲了Z轴应变的均匀性，或者相对复杂且难以在其他实验室39，40中复制。
这种伸展3D水凝胶的方法允许在活体对焦显微镜期间进行静态或周期性单轴应变。拉伸设备（称为"智能环状单轴拉伸器 - SCyUS"）采用 3D 打印部件和低成本硬件构建，可在其他实验室中轻松复制。连接到设备的是一个市售的硅橡胶，其中心有几何切口。水凝胶组件聚合以填充切口。在聚合过程中，生物水凝胶，如纤维蛋白或胶原蛋白，自然粘附在切口的内壁上。使用SCyUS，硅胶条是非近似拉伸，转移控制菌株到嵌入式3D水凝胶41。
与其他现有方法相比，该系统允许将功能和优势的独特组合。首先，该系统允许从其外围单轴拉伸厚的 3D 软水凝胶（>100 微米厚，<1 kPa 刚度），在整个水凝胶中 Z- 同质变形。这些水凝胶太软，不能被传统的拉伸技术抓住和拉伸。其次，拉伸设备可以很容易地在其他实验室复制，因为 3D 打印可供研究人员使用，设计中使用的电子产品成本低廉。第三，也许也是最吸引人的特征，硅胶条中切口的几何形状和大小可以很容易地操纵，允许可调谐应变梯度和边界条件，以及使用各种样品体积，下至几微升。
所提出的协议包括将纤维蛋白凝胶成直径约2毫米的圆盘，在0.5毫米厚的硅橡胶条中，在活体共焦显微镜下进行单轴拉伸。以下详细讨论了测量和分析几何切口上的菌株、水凝胶中形成的内部菌株以及各种拉伸操作后产生的纤维对齐的实验程序。最后，讨论了将细胞嵌入水凝胶中并使其暴露在受控外部拉伸的可能性。

<table><tbody><tr><td>Alexa Fluor 546 carboxylic acid, succinimidyl ester</td><td>Invitrogen</td><td>A20002</td><td></td></tr><tr><td>Cell Medium (DMEM High Glucose)</td><td>Biological Industries</td><td>01-052-1A</td><td>Add 10% FBS, 1% PNS, 1% L-Glutamine, 1% Sodium Pyruvate</td></tr><tr><td>Cover Slip #1.5</td><td>Bar-Naor Ltd.</td><td>BN72204-30</td><td>22&amp;times;40 &lt;em&gt;mm&lt;/em&gt;</td></tr><tr><td>DIMETHYL SULPHOXIDE 99.5% GC DMSO</td><td>Sigma-Aldrich Inc.</td><td>D-5879-500 ML</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline</td><td>Biological Industries</td><td>02-023-1A</td><td></td></tr><tr><td>EVICEL Fibrin Sealant (Human)</td><td>Omrix Biopharmaceuticals</td><td>3902</td><td>Fibrinogen: 70 mg/mL, Thrombin: 800-1200 IU/&lt;em&gt;mL&lt;/em&gt;</td></tr><tr><td>Fibrinogen Buffer</td><td>N/A</td><td></td><td>Recipe for 1L: 7g NaCl, 2.94g trisodium citrate dihydrate, 9g glycine, 20g arginine hydrochloride &amp;amp; 0.15g calcium chloride dihydrate. Bring final volume to 1L with PuW (pH 7.0-7.2)</td></tr><tr><td>Fluorescent micro-beads FluoSpheres (1 &lt;em&gt;&amp;micro;m&lt;/em&gt;)</td><td>Invitrogen</td><td>F8820</td><td>Orange (540/560)&lt;br/&gt; Provided as suspension (2% solids) in water plus 2 mM sodium azide</td></tr><tr><td>High-Temperature Silicone Rubber</td><td>McMaster-Carr</td><td>3788T41</td><td>580 &lt;em&gt;&amp;micro;m&lt;/em&gt;-thick&lt;br/&gt; E = 1.5 Mpa&lt;br/&gt; Poisson Ratio = 0.48&lt;br/&gt; Tensile Strength = 4.8 MPa&lt;br/&gt; Upper limit of stretch = +300% engineering strain</td></tr><tr><td>HiTrap desalting column 5 &lt;em&gt;mL&lt;/em&gt; (Sephadex G-25 packed)</td><td>GE Healthcare</td><td>17-1408-01</td><td></td></tr><tr><td>HIVAC-G High Vacuum Sealing Compound</td><td>Shin-Etsu Chemical Co., Ltd.</td><td>HIVAC-G 100</td><td></td></tr><tr><td>ImageJ FIJI software&lt;sup&gt;39&lt;/sup&gt;</td><td>National Institute of Health, Bethesda, MD</td><td>Version 1.8.0_112</td><td></td></tr><tr><td>Microcontroller (Adruino Uno + Adafruit Motorshield v2.3)</td><td>Arduino/Adafruit</td><td>Arduino-DK001/Adafruit-1438</td><td></td></tr><tr><td>MicroVL 21R Centrifuge</td><td>Thermo Scientific</td><td>75002470</td><td></td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM-996</td><td></td></tr><tr><td>Primovert Light Microscope</td><td>Carl Zeiss Suzhou Co., Ltd.</td><td>491206-0011-000</td><td></td></tr><tr><td>SCyUS CAD (Solidworks)</td><td>Dassault Syst&amp;egrave;mes</td><td>N/A</td><td></td></tr><tr><td>SCyUS Code&lt;sup&gt;37&lt;/sup&gt;</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Servomotor - TowerPro SG-5010</td><td>Adafruit</td><td>155</td><td></td></tr><tr><td>SL 16R Centrifuge</td><td>Thermo Scientific</td><td>75004030</td><td>For 50 &lt;em&gt;mL&lt;/em&gt; tubes</td></tr><tr><td>Sterile 10 &lt;em&gt;cm&lt;/em&gt; non-culture plates</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>Thrombin buffer</td><td>N/A</td><td></td><td>Recipe for 1L: 20g mannitol, 8.77g NaCl, 2.72g sodium acetate trihydrate, 24 mL 25% Human Serum Albumin, 5.88g calcium chloride. Bring final volume to 1L with PuW (pH 7.0)</td></tr><tr><td>Trypsin EDTA Solution B (0.25%), EDTA (0.05%)</td><td>Biological Industries</td><td>03-052-1B</td><td></td></tr><tr><td>USB Cable (Type B Male to Type A Male)</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Zeiss LSM 880 Confocal Microscope</td><td>Carl Zeiss AG</td><td>2811000417</td><td></td></tr><tr><td>ZEN 2.3 SP1 FP3 (black)</td><td>Carl Zeiss AG</td><td>Release Version 14.0.0.0</td><td></td></tr></tbody></table>

controlled strain of 3d hydrogels under live microscopy imaging

外力是组织形成、发育和维护的重要因素。这些力的作用通常使用专门的体外拉伸方法进行研究。各种可用系统使用基于 2D 基板的担架，而 3D 技术对软水凝胶应变的可及性则受到更多限制。在这里，我们描述了一种方法，允许软水凝胶从其周长外部拉伸，使用弹性硅胶条作为样品载体。本协议中使用的拉伸系统由 3D 打印部件和低成本电子产品构建，使其简单易于在其他实验室中复制。实验过程从聚合厚（>100微米）软纤维素水凝胶（弹性Modulus+100 Pa）开始，切口位于硅胶条的中心。然后，硅胶构造连接到打印拉伸装置上，并放置在共焦显微镜阶段。在实时显微镜下，拉伸装置被激活，凝胶以各种拉伸量度进行成像。然后，用于图像处理来量化由此产生的凝胶变形，在整个凝胶的 3D 厚度（Z-轴） 中显示相对均匀的菌株和纤维对齐。这种方法的优点包括能够在进行原位显微镜时以3D方式应变极其柔软的水凝胶，以及根据用户的需求自由地操纵样品的几何形状和大小。此外，通过适当的适应，该方法可用于拉伸其他类型的水凝胶（如胶原蛋白、聚丙烯酰胺或聚乙二醇），并允许在更逼真3D条件下分析细胞和组织对外部力量的反应。

图9显示了用于硅胶条的静态增量拉伸数据，该条带有3D纤维蛋白水凝胶，内嵌1μm荧光珠。分析表明硅胶拉伸对切口的几何变化以及凝胶中发达菌株的影响。整个凝胶的Z 堆栈图像用于评估原始圆形切口到椭圆几何形状的变形 （图 9A）。这些图像用于计算ε xx（孔） （方程 2）。在凝胶拉伸过程中放大并手动跟踪多个珠子聚合物（图 9B），允许在中心计算局部凝胶菌株，在轴向εxx（凝胶） 和垂直εyy（凝胶） 方向 （图 9C，D）。轴向菌株相对线性地从硅胶切口边缘传播到凝胶的中心，并且大于压缩垂直菌株（图9D）。
荧光标记凝胶的高放大图像允许观察和量化纤维对齐，由硅胶拉伸诱导。图11显示了典型的未拉伸和相对同向水凝胶（图11A）和高80%切口应变下的水凝胶的代表性放大图像，描绘了在拉伸方向高度对齐的纤维（图11B）。使用 ImageJ 分析纤维重新定向显示，光纤对齐 （NOP） 与外部应变ε之间的线性依赖性高达 40%，在菌株中处于中度饱和度超过 40% （图 11D）。图12显示了不同Z切片中纤维对齐的详细分析。在这里，为每个Z切片（图 12A）及其相应的 NOP（图 12B）计算纤维对齐的直方图，以及切口（图 12C）中每个外部应变幅度的所有Z切片的平均 NOP。随着ε孔的增加，纤维对齐增加，遵循图12C中显示的整体非线性曲线。请注意，凝胶收缩沿Z轴由于泊松效应，代表在图12B中较高的应变较短的线。
由于已证明很难对整个凝胶中的内部菌株进行深入分析，因此，鉴于纤维素（图 13）的机械特性，我们在此提供了在 2D 连续材料上进行的有限元素 （FE） 模拟的初步结果。切口的外部应变为 ~40%，彩色地图表示拉伸诱发的应变场。颜色图的范围从38%-42%，表明凝胶菌株是相对均匀的整个圆形凝胶区域。我们注意到，如果使用其他凝胶几何形状，可能会出现应变梯度，这是未来研究的课题。
为了证明嵌入式纤维素凝胶可以在高应变（0 到 +30%） 和频率（最多 1 Hz）下保持粘附到硅胶条上，我们进行了初步测试（此测试的协议不包括在本工作中），显示凝胶与切口内壁没有分离。测试了三个凝胶，凝胶1，总持续时间约87分钟，凝胶2为60分钟，凝胶3为30分钟（表1）。在这三种情况下，纤维素水凝胶都保持对硅胶的粘附。
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/61671/61671fig11.jpg"> 图11:凝胶纤维对齐以响应外部拉伸。在荧光标记凝胶的中心拍摄的图像，而（A） 未展开和 （B）应用后，ε孔= 80%（C） 平均直方图的纤维方向下增加的ε孔[%] 应变。灰色误差条表示每个测试菌株（D） 平均内基顺序参数 （NOP） 中评估的 z 堆栈中的 40 片之间的差异，作为三种不同凝胶样品的ε孔函数（包括图 11A-C中分析的"Gel 1"）。错误条表示每个拉伸级的Z堆栈中的切片之间的差异。此图已在 Roitblat Riba 等人的许可下进行调整。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61671/61671fig11large.jpg" target="_blank"></a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/61671/61671fig12.jpg"> 图12: 使用定向J44 工具（ImageJ软件）43 （A） 不同 Z切片的光纤方向的 3D 直方图分析。显示的是两个直方图的示例，用于拉伸（ε孔 = 17.6%，顶部）和未拉伸（底部）凝胶。直方图正常化，使曲线下的区域等于 1 （B） 导致 NOP 与 Z深度:该图显示了每个直方图的NOP作为 Z-深度（C）表示NOP作为 ε孔函数的计算。错误条表示相同凝胶中相同 XY 点的单个拉伸量度中所有 Z切片的 NOP 标准偏差。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61671/61671fig12large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/61671/61671fig13v2.jpg"> 图13: 拉伸凝胶的2D有限元素（FE）模拟。凝胶被硅胶载体拉伸（ε孔 ≈40%）。应变场在观察到小梯度时，会呈现最大本金应变值。虚线表示的圆是孔的预应变几何形状，原始长度（lo）是应用拉伸前沿 x 轴的中心长度，拉伸长度（l）是应用拉伸后沿 x 轴的中心长度。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61671/61671fig13v2large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<table border="1" fo:font-size="8px" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td rowspan="10">凝胶#1</td>
			<td>动态应变振幅（频率 1 Hz）</td>
			<td>动态拉伸持续时间（最小）</td>
		</tr>
<tr>
<td rowspan="5">0-37%</td>
			<td>0.1</td>
		</tr>
<tr>
<td>1</td>
		</tr>
<tr>
<td>5</td>
		</tr>
<tr>
<td>15</td>
		</tr>
<tr>
<td>30</td>
		</tr>
<tr>
<td rowspan="3">0-48%</td>
			<td>1</td>
		</tr>
<tr>
<td>5</td>
		</tr>
<tr>
<td>30</td>
		</tr>
<tr>
<td>动态拉伸下的总时间</td>
			<td>87分钟</td>
		</tr>
<tr>
<td rowspan="7">凝胶#2</td>
			<td>动态应变振幅（频率 1 Hz）</td>
			<td>动态拉伸持续时间（最小）</td>
		</tr>
<tr>
<td rowspan="4">0-30%</td>
			<td>0</td>
		</tr>
<tr>
<td>10</td>
		</tr>
<tr>
<td>20</td>
		</tr>
<tr>
<td>30</td>
		</tr>
<tr>
<td>0-50%</td>
			<td>30</td>
		</tr>
<tr>
<td>动态拉伸下的总时间</td>
			<td>60分钟</td>
		</tr>
<tr>
<td rowspan="5">凝胶#3</td>
			<td>动态应变振幅（频率 1 Hz）</td>
			<td>动态拉伸持续时间（最小）</td>
		</tr>
<tr>
<td rowspan="3">0-30%</td>
			<td>10</td>
		</tr>
<tr>
<td>20</td>
		</tr>
<tr>
<td>30</td>
		</tr>
<tr>
<td>动态拉伸下的总时间</td>
			<td>30 分钟</td>
		</tr>
</tbody></table>表1:动态拉伸概念证明的代表性结果。 三个凝胶嵌入硅胶条中，并在不同持续时间内动态拉伸（1 Hz）。菌株的范围从零到各种幅度超过30%。这三种凝胶都成功地将其粘附在硅胶条圆形切口的内壁上。

Watch this Scientific Journal Video about Controlled Strain of 3D Hydrogels under Live Microscopy Imaging at JoVE.com

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

提出的方法涉及嵌入硅橡胶的3D软水凝胶的单轴拉伸，同时允许活体共焦显微镜。演示了外部和内部水凝胶菌株的特征以及纤维对齐。开发的设备和协议可以评估细胞对各种菌株机制的反应。

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 ...

controlled-strain-of-3d-hydrogels-under-live-microscopy-imaging

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

3.4K 次观看.  Tel-Aviv University. 提出的方法涉及嵌入硅橡胶的3D软水凝胶的单轴拉伸，同时允许活体共焦显微镜。演示了外部和内部水凝胶菌株的特征以及纤维对齐。开发的设备和协议可以评估细胞对各种菌株机制的反应。

视频: Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

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

Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

在机械拉伸活细胞成像

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

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

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single strain-induced cellular alignment on a cell-laden hydrogel by using complex experimental processes and mass controlling systems, which are usually associated with contamination issues. Thus, in this article, we propose a simple approach to building a gradient static strain using a fluidic chip with a plastic PDMS cover and a UV transparent glass substrate for the stimulation of cellular behavior in a 3D hydrogel. Overloading photo-patternable cell prepolymer in the fluidic chamber can generate a convex curved PDMS membrane on the cover. After UV crosslinking, through a concentric circular micropattern under the curved PDMS membrane, and buffer washing, a microenvironment for investigating cell behaviors under a variety of gradient strains is self-established in a single fluidic chip, without external instruments. NIH3T3 cells were demonstrated after observing the change in the cellular alignment trend under geometry guidance, in cooperation with strain stimulation, which varied from 15 - 65% on hydrogels. After a 3-day incubation, the hydrogel geometry dominated the cell alignment under low compressive strain, where cells aligned along the hydrogel elongation direction under high compressive strain. Between these, the cells showed random alignment due to the dissipation of the radical guidance of hydrogel elongation and the geometry guidance of the patterned hydrogel.

This article introduces a simple approach to providing non-continuous gradient static strains on a concentric cell-laden hydrogel to regulate cell alignment for tissue engineering.

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 ...

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

使用丙烯酰胺水凝胶的细胞功能的研究矩阵刚度的影响

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

生物学

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.&#160;

We present a procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain a repeatable pattern formation. A cubic device containing two different hydrogels is employed to achieve multi-directional imaging for tissue pattern formation.

用于可重复模式形成的混合凝胶立方体器件中的初始三维单元群集控制

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its ...

High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the effect of applied extracellular tensile strain on cell populations in vitro via biochemical assays, a device has previously been designed which can be fabricated simply and is small enough to fit inside tissue culture incubators, as well as on top of microscope stages. However, the previous design of the polydimethylsiloxane substratum did not allow high-resolution subcellular imaging via oil-immersion objectives. This work describes a redesigned geometry of the polydimethylsiloxane substratum and a customized imaging setup that together can facilitate high-resolution subcellular imaging of live cells while under applied strain. This substratum can be used with the same, earlier designed device and, hence, has the same advantages as listed above, in addition to allowing high-resolution optical imaging. The design of the polydimethylsiloxane substratum can be improved by incorporating a grid which will facilitate tracking the same cell before and after the application of strain. Representative results demonstrate high-resolution time-lapse imaging of fluorescently labeled nuclei within strained cells captured using the method described here. These nuclear dynamics data give insights into the mechanism by which applied tensile strain promotes differentiation of oligodendrocyte progenitor cells.&#160;

Using a previously designed device to apply mechanical strain to adherent cells, this paper describes a redesigned substratum geometry and a customized apparatus for high-resolution single-cell imaging of strained cells with a 100x oil immersion objective.

单轴拉伸应变下活细胞核动力学的高分辨率成像

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the ...

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types&#39; responses to matrix stiffness have been characterized, how adherent cells&#39; susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

基于聚丙烯酰胺的多井格式实验研究细胞外基质硬度对黏附细胞细菌感染的影响

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, ...

免疫与感染

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

使用水凝胶图案化技术控制细胞粘附，用于牵引力显微镜的应用

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

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 ...

Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark of cardiac and musculoskeletal tissues. To study cell response to aligned microenvironments in vitro, several protocols have been developed to generate COL1 matrices with defined fiber alignment, including magnetic, mechanical, cell-based, and microfluidic methods. Of these, microfluidic approaches offer advanced capabilities such as accurate control over fluid flows and the cellular microenvironment. However, the microfluidic approaches to generate aligned COL1 matrices for advanced in vitro culture platforms have been limited to thin "mats" (&lt;40 &#181;m in thickness) of COL1 fibers that extend over distances less than 500 &#181;m and are not conducive to 3D cell culture applications. Here, we present a protocol to fabricate 3D COL1 matrices (130-250 &#181;m in thickness) with millimeter-scale regions of defined fiber alignment in a microfluidic device. This platform provides advanced cell culture capabilities to model structured tissue microenvironments by providing direct access to the micro-engineered matrix for cell culture.

This protocol demonstrates the use of a microfluidic channel with changing geometry along the fluid flow direction to generate extensional strain (stretching) to align fibers in a 3D collagen hydrogel (&lt;250 &#181;m in thickness). The resulting alignment extends across several millimeters and is influenced by the extensional strain rate.

具有长距离纤维排列的微工程3D胶原蛋白水凝胶

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark ...