用于测定单细胞材料特性的剪切测定方案
Summary
该协议概述了 体外癌性和非癌性细胞系的机械特性的定量。癌细胞和正常细胞力学的保守差异可以作为生物标志物，可能对预后和诊断产生影响。
Abstract
不规则生物力学是癌症生物学的标志，需要广泛研究。电池的机械性能与材料的机械性能相似。细胞对压力和应变的抵抗力、弛豫时间和弹性都是可以得出并与其他类型的细胞进行比较的特性。量化癌细胞（恶性）与正常（非恶性）细胞的机械特性使研究人员能够进一步揭示这种疾病的生物物理基础。虽然已知癌细胞的机械性能始终与正常细胞的机械性能不同，但缺乏从培养细胞中推断出这些特性的标准实验程序。
本文概述了使用流体剪切测定法在 体外量化 单细胞机械性能的程序。该测定背后的原理涉及将流体剪切应力施加到单个细胞上，并随着时间的推移光学监测由此产生的细胞变形。随后使用数字图像相关（DIC）分析表征细胞机械性能，并将适当的粘弹性模型拟合到DIC分析生成的实验数据中。总体而言，此处概述的方案旨在为诊断难以治疗的癌症提供更有效和有针对性的方法。
Introduction
研究癌细胞和非癌细胞之间的生物物理差异提供了新的诊断和治疗机会1。了解生物力学/机械生物学的差异如何影响肿瘤进展和治疗耐药性，将揭示靶向治疗和早期诊断的新途径2。
虽然已知癌细胞的机械性能与正常细胞不同（例如，质膜和核包膜的粘弹性）3,4,5，但缺乏在活细胞中测量这些特性的稳健且可重复的方法6。剪切测定方法用于量化细胞的机械性能，方法是使单个细胞承受流体剪切应力并分析其个体响应和对施加应力的抵抗力3,4,5,7,8,9。尽管已经使用了几种方法和技术来表征单个细胞的机械性能，但这些方法和技术往往会通过以下方式影响细胞材料性能：i）由于压痕深度，复杂的尖端几何形状或与原子力显微镜（AFM）相关的基板刚度而穿孔/损坏细胞膜10,11，ii）在光学捕获期间诱导细胞光损伤12， 13或iii）诱导与微量移液器抽吸相关的复杂应力状态14,15。这些外部效应与细胞粘弹性测量精度的显著不确定性有关6,16,17。
为了解决这些限制，这里描述的剪切测定方法提供了一种高度可控且简单的方法来模拟体内的生理流动，而不会影响过程中的细胞材料特性。该测定中的流体剪切应力表示体内细胞在循环过程中通过肿瘤间质内的液体或血液中的液体所经历的机械应力18,19,20。此外，这些流体应激促进癌细胞中的各种恶性行为，包括进展，迁移，转移和细胞死亡19,21,22,23，这些行为在致瘤细胞和非致瘤细胞之间有所不同。此外，癌细胞改变的机械特征（即，它们通常比同一器官中发现的正常细胞“更软”）使它们能够在敌对的肿瘤微环境中持续存在，侵入周围的正常组织，并转移到远处24,25,26。通过创造一个伪生物环境，使细胞经历生理水平的流体剪切应力，实现了生理相关且对细胞不具有破坏性的过程。细胞对这些施加的流体剪切应力的反应使我们能够表征细胞的机械性能。
本文为广泛研究施加剪切应力下癌细胞和非癌细胞的力学性能和行为提供了一种剪切测定方案。细胞以弹性和粘性的方式响应外力，因此可以理想化为粘弹性材料3。该技术分为：（i）分散的单细胞的细胞培养，（ii）流体剪切应力的受控应用，（iii）细胞行为的 原位 成像和观察（包括抗应力和变形），（iv）细胞应变分析以确定变形程度，以及（v）表征单个细胞的粘弹性特性。通过研究这些机械特性和行为，可以将复杂的细胞力学生物学提炼为可量化的数据。概述该方法的方案允许对各种恶性和非恶性细胞类型进行编目和比较。量化这些差异有可能建立诊断和治疗生物标志物。
Protocol
1. 单细胞剪切测定的准备 细胞培养在含有 2 mL 培养基的 35 mm x 10 mm 培养皿中接种约 50,000 个悬浮单细胞。注意：在接种前涡旋悬浮细胞以分解细胞聚集体。 将细胞在37°C孵育，并允许10至48小时用于细胞附着和完整的细胞骨架蛋白形成。注意：考虑细胞附着的持续时间，以及增殖和生长速率，以确保足够的细胞生长和附着，同时避免细胞聚集。这些参数因细…
Representative Results
剪切测定方案与使用 DIC 和粘弹性模型的变形分析相结合，成功地在体外量化了单个细胞的机械性能。该方法已在人和鼠细胞系上进行了测试，包括正常人乳腺细胞（MCF-10A）3,4,9，转移性较小的三阴性乳腺癌细胞（MDA-MB-468）3，三阴性乳腺癌细胞（MDA-MB-231）3，人骨肉瘤细胞7,8以及最近的胶质母细胞瘤细…
Discussion
剪切测定方法，包括建立一个伪机械生物学环境来模拟细胞与周围机械微环境的相互作用及其对机械应力的反应，已经产生了细胞力学性能目录，其模式显示了癌细胞系中保守的物理异型性3,4,5,7,8.该方法结合了对基本流体力学和物理学的理解，以表征细胞的独特机械性能，?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢伍斯特理工学院Soboyejo小组的先前研究人员，他们首先开创了这项技术：Yifang Cao博士，Jingjie Hu博士和Vanessa Uzonwanne博士。这项工作得到了美国国家癌症研究所（NIH / NCI K22 CA258410至医学博士）的支持。人物是用 BioRender.com 创造的。
Materials
| CELL CULTURE | |||
| .25% Trypsin, 2.21 mM EDTA, 1x[-] sodium bicarbonate | Corning | 25-053-ci | For cellular detachment from substrate in cell culture |
| 15 mL centrifuge tubes | Falcon by Corning | 05-527-90 | |
| 35 mm Petri dishes | Corning | 430165 | |
| 50 mL centrifuge tubes | Falcon by Corning | 14-432-22 | |
| centrifuge | any | For sterile cell culture | |
| Dulbecco's Modification of Eagle's Medium (DMEM) 1x | Corning | 10-013-cv | Or any other media for culturing cells. DMEM was used for culturing U87 cells |
| gloves | any | For sterile cell culture | |
| Heracell Vios 160i CO2 Incubator | Thermo Scientific | 51033770 | For Incubation during cell culture |
| Hood | any | For sterile cell culture | |
| micropipette | any | For sterile cell culture | |
| micropipette tips | any | For sterile cell culture | |
| Microscope | Leica/any | For sterile cell culture | |
| Phosphate Buffered Saline without calcium and magnesium PBS, 1x | Corning | 21-040-CM | |
| pipetman | any | For sterile cell culture | |
| pipette tips | any | For sterile cell culture | |
| Precision GP 10 liquid incubator | Thermo Scientific | TSGP02 | |
| T25 flask | Corning | 430639 | |
| T75 flask | Corning | 430641U | |
| SHEAR ASSAY | |||
| 100 mL beaker | any | For creating DMEM + methyl cellulose viscous shear media | |
| DMEM | Corning | ||
| Flow chamber + rubber gasket | Glycotech | 31-001 | Circular Flow chamber Kit ( for 35 mm tissue culture dishes) |
| Hybrid Rheometer | HR-2 Discovery Hybrid Rheometer | For determination of shear fluid viscosity | |
| magnetic stir bar | any | For creating DMEM + methyl cellulose viscous shear media | |
| magnetic stir plate | any | For creating DMEM + methyl cellulose viscous shear media | |
| methyl cellulose | any | To increase viscosity of DMEM in flow media | |
| Syringe Pump | KD Scientific Geminin 88 plus | 788088 | For programming fluid infusion and withdrawal |
| syringes, tubing, and connectors | For shear apparatus setup | ||
| SOFTWARE | |||
| ABAQUS software | Simulia | ||
| Digitial Image Correlation software | LaVision, Germany | DAVIS 10.1.2 | |
| Imaging software | Leica/any microscope software | ||
| MATLAB | MATLAB | MATLAB_R2020B |
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Cite This Article
Holen, L. J., Onwudiwe, K., Najera, J., Zarodniuk, M., Obayemi, J. D., Soboyejo, W. O., Datta, M. Shear Assay Protocol for the Determination of Single-Cell Material Properties. J. Vis. Exp. (195), e65333, doi:10.3791/65333 (2023).