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Summary
Abstract
Introduction
Protocol
Results
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Acknowledgements
Materials
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생체공학

实时显微镜成像下的 3D 水凝胶控制应变

Published: December 04, 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

Summary

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。

Abstract

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。

Introduction

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。

Protocol

1. 解决方案准备（将提前执行） 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。 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）。</l…

Representative Results

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…

Discussion

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?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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

Materials

Alexa Fluor 546 carboxylic acid, succinimidyl ester Invitrogen A20002
Cell Medium (DMEM High Glucose) Biological Industries 01-052-1A Add 10% FBS, 1% PNS, 1% L-Glutamine, 1% Sodium Pyruvate
Cover Slip #1.5 Bar-Naor Ltd. BN72204-30 22×40 mm
DIMETHYL SULPHOXIDE 99.5% GC DMSO Sigma-Aldrich Inc. D-5879-500 ML
Dulbecco's Phosphate-Buffered Saline Biological Industries 02-023-1A
EVICEL Fibrin Sealant (Human) Omrix Biopharmaceuticals 3902 Fibrinogen: 70 mg/mL, Thrombin: 800-1200 IU/mL
Fibrinogen Buffer N/A Recipe 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) Invitrogen F8820 Orange (540/560)
Provided as suspension (2% solids) in water plus 2 mM sodium azide
High-Temperature Silicone Rubber McMaster-Carr 3788T41 580 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 Healthcare 17-1408-01
HIVAC-G High Vacuum Sealing Compound Shin-Etsu Chemical Co., Ltd. HIVAC-G 100
ImageJ FIJI software39 National Institute of Health, Bethesda, MD Version 1.8.0_112
Microcontroller (Adruino Uno + Adafruit Motorshield v2.3) Arduino/Adafruit Arduino-DK001/Adafruit-1438
MicroVL 21R Centrifuge Thermo Scientific 75002470
Parafilm Bemis PM-996
Primovert Light Microscope Carl Zeiss Suzhou Co., Ltd. 491206-0011-000
SCyUS CAD (Solidworks) Dassault Systèmes N/A
SCyUS Code37 N/A N/A
Servomotor – TowerPro SG-5010 Adafruit 155
SL 16R Centrifuge Thermo Scientific 75004030 For 50 mL tubes
Sterile 10 cm non-culture plates Corning 430167
Thrombin buffer N/A 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)
Trypsin EDTA Solution B (0.25%), EDTA (0.05%) Biological Industries 03-052-1B
USB Cable (Type B Male to Type A Male) N/A N/A
Zeiss LSM 880 Confocal Microscope Carl Zeiss AG 2811000417
ZEN 2.3 SP1 FP3 (black) Carl Zeiss AG Release Version 14.0.0.0
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Tags

Keywords: 3D HydrogelsLive Microscopy ImagingMechanical CuesBiomechanical ResponsesHydrogel GeometryCell ResponseExtracellular MatrixDisease ProgressionCancer TherapiesFibrin GelFibrinogenThrombinPBSStretching DeviceSilicone Strip
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Kolel, A., Roitblat Riba, A., Natan, S., Tchaicheeyan, O., Saias, E., Lesman, A. Controlled Strain of 3D Hydrogels under Live Microscopy Imaging. J. Vis. Exp. (166), e61671, doi:10.3791/61671 (2020).
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