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Cancer Research

基于声流体微器件的细胞和细胞核可压缩性测量

Published: July 14, 2022
doi:
10.3791/64225
, , ,
1Sino-French Institute of Nuclear Engineering and Technology,Sun Yat-sen University

Summary

这里提出了一个协议，用于构建一个基于声流体微器件的快速且无损的系统来测量细胞或细胞核的可压缩性。考察了上皮-间充质转化或电离辐射后肿瘤细胞力学性能的变化，证明了该方法在科学研究和临床上的应用前景。

Abstract

细胞力学在肿瘤转移、细胞恶性转化和放射敏感性中起重要作用。在这些过程中，研究细胞的机械性能通常具有挑战性。传统的基于接触的测量方法，如压缩或拉伸，容易造成细胞损伤，影响测量精度和后续细胞培养。贴壁状态下的测量也会影响准确性，尤其是在照射后，因为电离辐射会使细胞变平并增强粘附。在这里，已经开发出一种基于声流体方法的细胞力学测量系统。通过记录细胞在声力作用下的运动轨迹可以获得细胞压缩性，可以实现悬浮状态下的快速无损测量。本文详细介绍了芯片设计、样品制备、轨迹记录、参数提取和分析的协议。基于该方法测量了不同类型肿瘤细胞的压缩性。通过调整压电陶瓷的谐振频率和微通道的宽度，也实现了原子核压缩性的测量。结合免疫荧光实验的分子水平验证，比较药物诱导的上皮到间充质转化（EMT）前后的细胞压缩性。此外，揭示了不同剂量X射线照射后细胞压缩性的变化。本文提出的细胞力学测量方法具有通用性和灵活性，在科学研究和临床实践中具有广阔的应用前景。

Introduction

细胞力学特性在肿瘤转移、细胞恶性转化和放射敏感性中起重要作用1，2。为了深入了解细胞力学性能在上述过程中的作用，准确测量细胞力学至关重要，并且测量不应对细胞造成损害，以便进行后续培养和分析。测量过程应尽可能快，否则如果将细胞长时间从培养环境中取出，可能会影响细胞活力。

现有的电池力学测量方法面临一些局限性。一些方法，如磁扭曲细胞术、磁镊和粒子跟踪微流变学，由于将颗粒引入细胞3，4，5 而引起细胞损伤。通过与细胞接触进行测量的方法，如原子力显微镜（AFM），微量移液器抽吸，微收缩和平行板技术，也容易发生细胞损伤，并且通量难以提高6，7，8。此外，电离辐射会使细胞变平并增加其附着力9;因此，有必要测量悬浮液中的全细胞力学。

针对上述挑战，研制了一种基于声流体方法10、11、12、13、14的细胞力学测量系统。通道宽度与声学半波长相匹配，从而在微通道的中线处创建一个驻波节点。在声辐射力的作用下，细胞或标准珠可以移动到声压节点。由于标准磁珠的物理性质（尺寸、密度和可压缩性）是已知的，因此可以确定声能密度。然后，通过记录细胞在声场中的运动轨迹，可以获得细胞的可压缩性。可以实现悬浮状态细胞的无损高通量测量。本文将介绍微流控芯片的设计、系统的建立和测量步骤。已经对各种类型的肿瘤细胞进行了测量，以验证该方法的准确性。通过调节压电陶瓷的共振频率和微通道的宽度，该方法的应用范围已扩展到亚细胞结构（如细胞核）。此外，还研究了不同剂量药物诱导的EMT或X射线照射后细胞压缩性的变化。结果表明，该方法作为研究生化变化与细胞力学性能之间相关性的有力工具具有广泛的适用性。

Protocol

1. 声流体微器件的制造与组装 微流控芯片的制造。设计一个只有一个入口和出口的单通道芯片，如图 1所示。对于测量单元，将微通道的矩形横截面保持在 740 μm 宽和 100 μm 深。为了测量细胞核，将微通道的宽度和深度分别更改为250μm和100μm。 通过反应离子蚀刻 在 硅晶片上制备微通道。通过阳极粘合15用一块透明?…

Representative Results

本文提出了一种基于声流体微器件的快速无损细胞可压缩性测量系统的构建协议，并展示了其在不同情况下测量细胞和细胞核的优势。 图1 显示了微流体通道的示意图。声流体微器件的组件和组件如图 2所示。 图3 显示了测量系统的设置。在声场力的作用下，细胞和粒子将向微通道的中线移动，如图 4A所示?…

Discussion

常用的细胞力学测量方法有AFM、微量移液器抽吸、微流控方法、平行板技术、光学镊子、光学拉伸器和声学方法20。微流体方法可以使用三种方法：微收缩，拉伸流和剪切流。其中，光学担架、光镊、声学方法、拉伸流和剪切流方法都是非接触式测量。与接触式测量相比，非接触式测量可以有效避免接触或局部变形引起的电池损坏问题。光镊对测量环境非常敏感21</s…

Disclosures

The authors have nothing to disclose.

Acknowledgements

本研究得到了中国国家自然科学基金（批准号12075330和U1932165）和中国广东省自然科学基金（批准号2020A1515010270）的支持。

Materials

0.25% trypsin(1x) GIBCO 15050-065
502 glue Evo-bond cyanoacrylate glue
A549 ATCC CCL-185 lung adenocarcinoma
Cytonucleoprotein and cytoplasmic protein extraction kit Beyotime P0027 Contains cytoplasmic protein extraction reagents A and B
Dulbecco’s modified Eagle medium (DMEM)  corning 10-013-CVRC
Fetal Bovine Srum(FBS) AUSGENEX FBS500-S
HCT116 ATCC CCL247 colorectal carcinoma
Heat-resistant glass Pyrex
Leibovitz’s L-15 medium  GIBCO 11415-064
MCF-7 ATCC HTB-22  breast Adenocarcinoma
MDA-MB-231 ATCC HTB-26  breast Adenocarcinoma
Minimum Essential Medium (MEM) corning 10-010-CV
Penicillin-Streptomycin GIBCO 15140-122
Phosphate buffer corning 21-040-cvc
PMSF Beyotime ST506 100mM
Polybead Polystyrene Red Dyed Microsphere  polysciences 15714 The diameter of microshpere is 6.00µm
propidium iodide(PI) Sigma-Aldrich P4170
SYLGARD 184Silicone ELASTOMER Dow-Corning 1673921 Contains prepolymers and curing agents
Trypan Blue Beyotime C0011
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Keywords: Cell MechanicsTumor MetastasisMalignant TransformationRadiosensitivityAcoustofluidic MethodSuspended StatesEpithelial To Mesenchymal TransitionCirculating Tumor CellsCancer DiagnosisPDMSPlasma CleanerPTFE CatheterStainless Steel NeedlePiezoelectric CeramicCyanoacrylate GluePolystyrene Standard Particle SolutionsCell Suspensions
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Fu, Q., Zhang, Y., Huang, T., Liu, Y. Measurement of the Compressibility of Cell and Nucleus Based on Acoustofluidic Microdevice. J. Vis. Exp. (185), e64225, doi:10.3791/64225 (2022).
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