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Bioengineering

实时显微镜成像下的 3D 水凝胶控制应变

Published: December 4th, 2020

DOI:

10.3791/61671

1Department of Biomedical Engineering, Faculty of Engineering, Tel-Aviv University, 2Department of Materials Science and Engineering, Faculty of Engineering, Tel-Aviv University, 3School of Mechanical Engineering, Faculty of Engineering, Tel-Aviv University, 4Center for the Physics and Chemistry of Living Systems, 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。

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。

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....

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1. 解决方案准备（将提前执行）

  1. 纤维蛋白原标签
    注意：只有当需要分析纤维蛋白凝胶的变形时，才需要标记步骤。对于细胞实验，可以使用无标签的凝胶。
    1. 在 50 mL 离心管中加入 38 微升 10 毫克/mL 的超新星酯荧光染料（溶解在 DMSO 中）至 1.5 mL 15 毫克/mL 纤维素溶液（摩尔比为 5：1），并在室温下放置摇床 1 小时。之后，将管子放在离心机中3分钟，温度为800 x g（ 室温）。

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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.......

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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?.......

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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/10.1007/s10439-019-02426-7

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NameCompanyCatalog NumberComments
Alexa Fluor 546 carboxylic acid, succinimidyl esterInvitrogenA20002
Cell Medium (DMEM High Glucose)Biological Industries01-052-1AAdd 10% FBS, 1% PNS, 1% L-Glutamine, 1% Sodium Pyruvate
Cover Slip #1.5Bar-Naor Ltd.BN72204-3022×40 mm
DIMETHYL SULPHOXIDE 99.5% GC DMSOSigma-Aldrich Inc.D-5879-500 ML
Dulbecco's Phosphate-Buffered SalineBiological Industries02-023-1A
EVICEL Fibrin Sealant (Human)Omrix Biopharmaceuticals3902Fibrinogen: 70 mg/mL, Thrombin: 800-1200 IU/mL
Fibrinogen BufferN/ARecipe for 1L: 7g NaCl, 2.94g trisodium citrate dihydrate, 9g glycine, 20g arginine hydrochloride & 0.15g calcium chloride dihydrate. Bring final volume to 1L with PuW (pH 7.0-7.2)
Fluorescent micro-beads FluoSpheres (1 µm)InvitrogenF8820Orange (540/560)
Provided as suspension (2% solids) in water plus 2 mM sodium azide
High-Temperature Silicone RubberMcMaster-Carr3788T41580 µm-thick
E = 1.5 Mpa
Poisson Ratio = 0.48
Tensile Strength = 4.8 MPa
Upper limit of stretch = +300% engineering strain
HiTrap desalting column 5 mL (Sephadex G-25 packed)GE Healthcare17-1408-01
HIVAC-G High Vacuum Sealing CompoundShin-Etsu Chemical Co., Ltd.HIVAC-G 100
ImageJ FIJI software39National Institute of Health, Bethesda, MDVersion 1.8.0_112
Microcontroller (Adruino Uno + Adafruit Motorshield v2.3)Arduino/AdafruitArduino-DK001/Adafruit-1438
MicroVL 21R CentrifugeThermo Scientific75002470
ParafilmBemisPM-996
Primovert Light MicroscopeCarl Zeiss Suzhou Co., Ltd.491206-0011-000
SCyUS CAD (Solidworks)Dassault SystèmesN/A
SCyUS Code37N/AN/A
Servomotor - TowerPro SG-5010Adafruit155
SL 16R CentrifugeThermo Scientific75004030For 50 mL tubes
Sterile 10 cm non-culture platesCorning430167
Thrombin bufferN/ARecipe 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)
Trypsin EDTA Solution B (0.25%), EDTA (0.05%)Biological Industries03-052-1B
USB Cable (Type B Male to Type A Male)N/AN/A
Zeiss LSM 880 Confocal MicroscopeCarl Zeiss AG2811000417
ZEN 2.3 SP1 FP3 (black)Carl Zeiss AGRelease Version 14.0.0.0

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