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Ricerca sul cancro

三维肿瘤球体的纵向形态和生理监测

Published: February 09, 2019
doi:
10.3791/59020
, , , , , , , , , , , , ,
1Department of Electrical and Computer Engineering,Lehigh University, 2Department of Mechanical Engineering,Lehigh University, 3Department of Biomedical Engineering,Southern University of Science and Technology, 4Department of Technology R&D,Nexcelom Bioscience LLC, 5Department of Bioengineering,Lehigh University, 6Center for Photonics and Nanoelectronics,Lehigh University

Summary

光学相干断层扫描 (oct) 是一种三维成像技术, 用于监测和表征多细胞肿瘤球体的生长动力学。证明了采用体素计数法精确定量肿瘤球体的体积, 并基于固有光学衰减对比度对球体中无标签的死组织检测进行了验证。

Abstract

肿瘤球体已被开发为癌症研究和抗癌药物发现中的三维细胞培养模型。然而, 目前, 由于光穿透、荧光染料扩散和荧光检测的有限, 利用明亮场或荧光检测的高通量成像模式无法解决肿瘤球体的整体三维结构。深度解析。最近, 我们的实验室展示了光学相干断层扫描 (oct) 的应用, 一种无标签和非破坏性的三维成像模式, 在96孔板中对多细胞肿瘤球体进行纵向表征。oct 能够获得高度约为600μm 的肿瘤球体的三维形态和生理信息。本文展示了一种高通量 oct (ht-oct) 成像系统, 该系统可自动扫描整个多井板, 并获得肿瘤球体的三维 oct 数据。我们描述了 HT-OCT 系统的细节和协议中的构造指南。从三维 oct 数据中, 可以用三维渲染和正交切片来可视化球体的整体结构, 根据肿瘤球体的大小和体积的形态信息来描述肿瘤球体的纵向生长曲线, 并监测肿瘤球体的生长情况。基于光学固有衰减对比度的肿瘤球体中的死细胞区域。我们表明, HT-OCT 可以作为一种高通量的成像方法进行药物筛选, 并对生物磨料样品进行表征。

Introduction

癌症是世界上第二大死因1。开发针对癌症的药物对患者至关重要。然而, 据估计, 90% 以上的新抗癌药物在开发阶段失败, 因为缺乏疗效和意外毒性在临床试验2。部分原因是使用了简单的二维细胞培养模型进行复合筛选, 这些模型为药物发现的以下阶段提供了化合物疗效和毒性预测值有限的结果2,3 个,4. 最近, 已经开发出三维 (3d) 肿瘤球体模型, 为抗癌药物发现3、4、5 提供临床相关的生理和药理数据。,6,7,8,9,10, 11,12, 13,14, 15,16,17,18,19,20,21,22,23, 24,25。由于这些球体可以模仿肿瘤在体内的组织特异性特性, 如营养和氧梯度、缺氧核心以及耐药19, 使用这些模型有可能缩短药物发现的时间,降低投资成本, 更有效地为患者带来新药。评估三维肿瘤球状发育复合疗效的一个关键方法是监测治疗9,26下的球体生长和复发。要做到这一点, 定量定性的肿瘤形态, 涉及其直径和体积, 高分辨率成像方式, 是必不可少的。

传统的成像方式, 如明亮场、相位对比 7、9、22、24和荧光显微镜8、9、16、 18,22可以提供测量球体直径, 但不能解决球体在三维空间中的整体结构。造成这些限制的因素很多, 包括探测光在球体中的穿透;荧光染料扩散到球体中;由于强吸收和散射, 从激发荧光染料内部或球体相对表面发出荧光信号;这些成像方式的深度分解性。这通常会导致不准确的体积测量。球体坏死核心的发育类似于体内肿瘤中的坏死 6、10、15、19、25.这种病理特征不可能在二维细胞培养19、25、27、28 中复制。球体尺寸大于 500μm, 在球体 6, 10 中可以观察到三层同心结构, 包括增殖细胞的外层、中间的静止细胞层和坏死的核心 ,15,19,25, 由于缺乏氧气和营养。活细胞和死细胞荧光成像是标记坏死核边界的标准方法。然而, 同样, 这些荧光染料和可见光的穿透阻碍了探测坏死核的潜力, 以监测其实际形状的发展。

另一种三维成像方式, 光学相干断层扫描 (oct) 被引入到表征肿瘤球体。oct 是一种生物医学成像技术, 能够从生物组织 29、30、31、32、33中高达 1-2 毫米的深度获取无标签、非破坏性的3d 数据,34。oct 采用低相干干涉测量法检测来自样品不同深度的反向散射信号, 并在横向和纵向以微米级空间分辨率提供重建的深度分辨图像。oct 已被广泛应用于眼科35,36,37和血管造影38,39。以往的研究已经使用 oct 观察了基底膜基质 (如matrigel) 中体外肿瘤球体的形态, 并评估了它们对光动力疗法40,41的反应。最近, 我们的团队建立了一个高通量 oct 成像平台, 系统地监测和量化三维肿瘤球体在多井板42的生长动力学。基于固有光学衰减对比度, 采用体素计数法和无标签坏死组织检测法, 对三维肿瘤球体进行了精确体积定量。本文详细介绍了 oct 成像平台是如何构建和应用于获取肿瘤球体的高分辨率三维图像的。描述了三维肿瘤球体生长动力学的逐步定量分析, 包括对球体直径和体积的精确测量。并提出了基于固有光学衰减对比度的 oct 对坏死组织区域进行无损检测的方法。

Protocol

1. 细胞的制备 从合格的供应商处获取细胞系。注: 验证感兴趣的细胞系的细胞在培养基中或在基板 (基底膜基质, 如 matrigel) 的帮助下可以形成球体。查看文献9或执行一轮预实验进行检查。 按照细胞线供应商提供的特定程序解冻冷冻细胞。一般程序可在其他地方找到43。 在25厘米2培养瓶中培养1-2 个通道的细胞。然后, 这些细胞?…

Representative Results

96孔板中球体的高通量光学相干层析成像 图 3显示了在第3天用 hct 116 肿瘤球体对96孔板进行 HT-OCT 扫描的结果。整个板块的顺序扫描从右下角井 (h12) 开始。图 3b显示了 HT-OCT 系统软件实现的流程图。在收集和处理了一个球体数据后, 板块会移动到下一个井, 等待 ~ 2秒才能让球体休息, 并?…

Discussion

肿瘤活动与其形态结构高度相关。与监测二维细胞培养的特征生长曲线类似, 跟踪三维肿瘤球体的生长曲线也是描述不同细胞系长期球状生长行为的常规方法。值得注意的是, 我们可以通过分析直接反映在生长曲线上的肿瘤降解或肿瘤再生来描述药物反应。因此, 定量评价三维肿瘤球体, 包括大小和体积, 得出生长曲线, 对于肿瘤球体的表征和复合效果的评价具有重要意义。目前, 基于明亮场、相位?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作得到了 nsf 的支持, 该基金提供了 idbr (dbi-1455613)、pfi/air-tt (iip-1640707)、nih 授予 r21ey0266380、r15eb019704 和 r01eb025209 和 le高兴大学启动基金。

Materials

Custom Spectral Domain OCT imaging system Developed in our lab
Superluminescent Diode (SLD) Thorlabs SLD1325 light source
2×2 single mode fused fiber coupler, 50:50 splitting ratio AC Photonics WP13500202B201
Reference Arm
Lens Tube Thorlabs
Adapter Thorlabs
Collimating Lens Thorlabs AC080-020-C
Focusing Lens Thorlabs
Kinematic Mirror Mount Thorlabs
Mirror Thorlabs
1D Translational Stage Thorlabs
Continuous neutral density filter Thorlabs
Pedestrial Post Thorlabs
Clamping Fork Thorlabs
Sample Arm
Lens Tube Thorlabs
Adapter Thorlabs
Collimating Lens Thorlabs AC080-020-C
Galvanometer Thorlabs
Relay Lens Thorlabs AC254-100-C two Relay lens to make a telescope setup
Triangle Mirror Mount Thorlabs
Mirror Thorlabs
Objective Mitutoyo
Pedestrial Post Thorlabs
Clamping Fork Thorlabs
Polarization Controller Thorlabs
30mm Cage Mount Thorlabs
Cage Rod Thorlabs
Stage
3D motorized translation stage Beijing Mao Feng Optoelectronics Technology Co., Ltd. JTH360XY
2D Tilting Stage
Rotation Stage
Plate Holder 3D printed
Spectrometer
Lens Tube Thorlabs
Adapter Thorlabs
Collimating Lens Thorlabs AC080-020-C
Grating Wasatch G = 1145 lpmm
F-theta Lens Thorlabs FTH-1064-100
InGaAs Line-scan Camera Sensor Unlimited SU1024-LDH2
Name Company Catalog Number Comments
Cell Culture Component
HCT 116 Cell line ATCC CCL-247
Cell Culture Flask SPL Life Sciences 70025
Pipette Fisherbrand 14388100
Pipette tips Sorenson Bioscience 10340
Gibco GlutaMax DMEM Thermo Fisher Scientific 10569044
Fetal Bovine Serum, certified, US origin Thermo Fisher Scientific 16000044
Antibiotic-Antimycotic (100X) Thermo Fisher Scientific 15240062
Corning 96-well Clear Round Bottom Ultra-Low Attachment Microplate Corning 7007
Gibco PBS, pH 7.4 Thermo Fisher Scientific 10010023
Gibco Trypsin-EDTA (0.5%) Thermo Fisher Scientific 15400054
Forma Series II 3110 Water-Jacketed CO2 Incubators Thermo Fisher Scientific 3120
Gloves VWR 89428-750
Parafilm Sigma-Aldrich P7793
Transfer pipets Globe Scientific 138080
Centrifuge Eppendorf 5702 R To centrifuge the 15 mL tube
Centrifuge NUAIRE AWEL CF 48-R To centrifuge the 96-well plate
Microscope Olympus
Name Company Catalog Number Comments
Histology & IHC
Digital slide scanner Leica Aperio AT2 Obtain high-resolution histological images
Histology Service Histowiz Request service for histological and immunohistological staining of tumor spheroid
Name Company Catalog Number Comments
List of Commerical OCTs
SD-OCT system Thorlabs Telesto Series
SD-OCT system Wasatch Photonics WP OCT 1300 nm
Name Company Catalog Number Comments
Software for Data Analyses
Basic Image Analysis NIH ImageJ Fiji also works.
3D Rendering Thermo Fisher Scientific Amira Commercial software. Option 1
3D Rendering Bitplane Imaris Commercial software. Option 2. Used in the protocol
OCT acquisition software custom developed in C++.
Stage Control Beijing Mao Feng Optoelectronics Technology Co., Ltd. MRC_3 Incorporated into the custom OCT acquisition code
OCT processing software custom developed in C++. Utilize GPU. Incorporated into the custom OCT acquisition code.
Morphological and Physiological Analysis custom developed in MATLAB

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Tags

3D Tumor SpheroidsOptical Coherence Tomography3D Cell CultureAnti-cancer Drug DiscoveryCell SeedingCell SuspensionUltra-low Attachment PlateCentrifugationCell ConcentrationCell MonitoringIncubationMedium RefreshOCT System Construction

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Huang, Y., Zou, J., Badar, M., Liu, J., Shi, W., Wang, S., Guo, Q., Wang, X., Kessel, S., Chan, L. L., Li, P., Liu, Y., Qiu, J., Zhou, C. Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography. J. Vis. Exp. (144), e59020, doi:10.3791/59020 (2019).
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