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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

在这里,我们描述了不同的多细胞球体形成方法,以执行后续多参数活细胞显微镜检查。使用荧光寿命成像显微镜 (FLIM)、细胞自发荧光、染色染料和纳米颗粒,展示了分析活三维 (3D) 癌症和干细胞衍生球状体中细胞代谢、缺氧和细胞死亡的方法。

Abstract

多细胞肿瘤球体是一种流行的 3D 组织微聚集体模型,用于复制肿瘤微环境、测试和优化药物治疗以及在 3D 环境中使用生物和纳米传感器。它们的易于生产、可预测的大小、生长以及观察到的营养和代谢物梯度对于概括 3D 生态位样细胞微环境非常重要。然而,球状体的异质性及其生产方法的可变性会影响整体细胞代谢、活力和药物反应。考虑到大小、可变性、生物制造需求以及在干细胞和癌细胞生物学中用作 体外 3D 组织模型的要求,这使得选择最合适的方法变得困难。特别是,球状体的产生会影响它们与定量活体显微镜的兼容性,例如光学代谢成像、荧光寿命成像显微镜 (FLIM)、使用纳米传感器监测球状体缺氧或活力。这里介绍了许多常规的球状体形成方案,突出了它们与活体宽场、共聚焦和双光子显微镜的兼容性。还介绍了使用多重自荧光 FLIM 以及使用各种类型的癌症和干细胞球体的后续成像到分析管道。

Introduction

多细胞球状体代表一组 3D 组织模型,由细胞自聚集获得,呈球形。它们广泛用于在体外模拟细胞间和细胞间基质相互作用,并在多种癌症和干细胞衍生构建体中复制 3D 环境。采用多种技术来减少细胞粘附并促进聚集。这些方法包括依赖于表面张力1 的悬滴法;细胞附着排斥方法,如超低附着板、微模具和微孔 2,3;基于声波的方法4;流动诱导聚集方法(旋转瓶、生物反应器和微流体装置)5;磁性粒子辅助形成6 以及使用促进聚集的合成和基于 ECM 的基质和支架 7,8,9

在癌症研究、开发和新药疗法的验证中,球状体是一种有吸引力的模型,因为它们能够概括营养物质、废物和 O2 的空间扩散限制梯度,通常会导致形成坏死核心,这是实体瘤的典型特征10,11。根据动....

Protocol

1. 多细胞球体的产生

  1. 细胞培养
    注:细胞培养物可从美国典型培养物保藏中心 (ATCC)、Lonza、Sigma-Aldrich 或其他供应商处收集。ATCC 提供所有必需的处理信息,包括首选生长培养基、传代培养程序、生物安全水平、生长速率和 STR 谱。在这里,将人结肠癌细胞系 HCT116 的 500 个细胞/球体用于补充有 10% FBS 和 1 mM 丙酮酸钠的 McCoy 5A 培养基 (VWR, 392-0420) 中。对于每天监测的长?.......

Representative Results

选择合适的球体形成方法
所选的球状体形成方法可以极大地影响球状体的大小、形状、细胞密度、活力和药物敏感性(图 2)。以前,比较了多种高通量(SphericalPlate 5D、实验室制造的微模具和微组织模具)和“中等通量”低附着(Biofloat 和 Lipidure 涂层的 96 孔板)方法对球体活力和氧合的影响29

在这里,不同的形成方.......

Discussion

多细胞球状体正在成为肿瘤和干细胞生态位微环境研究、药物发现和生物制造“组织构建块”开发的首选方法。球状体的异质性内部结构、营养物质梯度和氧合可以在相对简化和可访问的环境中模拟体内组织和肿瘤的梯度。由于需要更高的方法透明度26,28 和标准化71,这些协议有望帮助研究人员为实时定量荧光寿命成像显微镜实.......

Disclosures

没什么可透露的。

Acknowledgements

这项工作得到了根特大学特别研究基金 (BOF) 资助 (BOF/STA/202009/003;BOF/IOP/2022/058)、佛兰德斯研究基金会(FWO,I001922N)和欧盟,fliMAGIN3D-DN Horizon Europe-MSCA-DN No. 101073507。

....

Materials

NameCompanyCatalog NumberComments
0.05% Trypsin-EDTAGibco25300-054Also available from Sigma
10 mL serological pipetsVWR612-3700Similar products are also available from Sarstedt, Corning, VWR and other companies
12 well cell-culture plates, sterileGreiner bio-one665-180Similar products are also available from Sarstedt, Corning and other companies.
12 Well Chamber slide, removableIbidi81201Also available from Grace Bio-Labs, ThermoFisher Scientific and others
15 mL centrifuge tubesNerbe plus02-502-3001Similar products are also available from Sarstedt, Corning, VWR and other companies
3D Petri Dish micromoldsMicrotissueZ764000-6EA
6 well cell-culture plates, sterileGreiner bio-one657160Similar products are also available from Sarstedt, Corning, VWR and other companies
70% ethanolChemLabCL02.0537.5000
BiofloatSarstedt83.3925.400Commercial available coated 96-well plate for spheroid formation
Calcein Green-AMTebubioAS-89201Apply in dilution 1:1000
CellSens Dimension softwareOlympusversion 3
Collagen from human placenta, type IVSigmaC5533For the preparation of 0.07 mg/mL Collagen and 0.03 mg/mL Poly-D-lysine coated microscopy dishes
Confocal FLIM MicroscopeLeica MicrosystemsN/AStellaris 8 Falcon inverted microscope with white-light laser, HyD X detectors, climate / T control chamber (OkoLab), 25x/0.95 W objective
D(+)-GlucoseMerck8342Prepare 1 M stock solution, 1:100 for preparation of imaging medium (final concentration 10 mM)
Dulbecco's modified Eagle's medium (DMEM), phenol red-, glucose-, pyruvate- and glutamine-freeSigma-AldrichD5030-10X1LFor preparation of imaging medium
Fetal Bovine Serum (FBS)Gibco10270-098Also available from Sigma. Needs to be heat-inactivated before use.
HEPES (1M)Gibco15630-080Dilution 1/100 for preparation of imaging medium (final concentration 10 mM)
Human colon cancer cells HCT116ATCC
ImageJNIHversion 1.54f
Leica Application Suite X (LAS X)Leica Microsystemsversion 4.6.1.27508
L-glutamineGibco25030Also available from Sigma. Apply in dilution 1:100.
Lipidure-CM5206AmsbioAMS.52000034GB1G
McCoy's 5A, need addition of 1 mM Sodium Pyruvate and 10 mM HEPESVWR392-0420Standard growth medium for HCT116 cells
micro-patterned 3D-printed PDMS stampsN/AN/AProvided by the Centre for Microsystems Technology, Professor Dr. Jan Vanfleteren, Ghent University
NaClChemlabCL00.1429.100
Neubauer couting chamberFisher Scientific15980396
O2 probes: MMIR1N/AN/AFull characterization, validation and some applications can be found at: https://www.biorxiv.org/content/10.1101/2023.12.11.571110
v1
PBSFisher scientificGibco18912014Dissolve PBS tablet in 500 mL of distilled water.
Pen Strep :Penicillin (10,000 U/mL) / streptomycin (10,000 μg/mL) 100x solutionGibco15140-122Also available from Sigma. Apply in dilution 1:100.
Poly-D-lysineSigmaP6407-5mgFor the preparation of 0.07 mg/mL Collagen and 0.03 mg/mL Poly-D-lysine coated microscopy dishes
Propidium IodideSigma-Aldrich25535-16-4Cell death staining, use 1 µg/mL at 1h incubation
PVDF syringe filter 0.22 µmNovolabA35149Similar products are also available from Sarstedt, Corning, VWR and other companies
Sodium pyruvate (100 mM)Gibco11360-070Dilution 1/100 for preparation of imaging medium (final concentration 1mM)
SphericalPlate 5D 24-wellKugelmeiersSP5D-24W
sterile petridishGreiner bio-one633181Similar products are also available from Sarstedt, Corning, VWR and other companies
Tissue culture flask (25 cm² )VWR734-2311Similar products are also available from Sarstedt, Corning, VWR and other companies
Tissue culture flask (75 cm²)VWR734-2313Similar products are also available from Sarstedt, Corning, VWR and other companies
U-bottom 96-well plateVWR10062-900Similar products are also available from Sarstedt, Corning, Greiner Bio-one and other companies
Ultrapure AgaroseInvitrogen (Life Technologies)16500-500Other types of Agarose such as Agarose low melting point (A-9414, Sigma), Agarose for routine use (A-9539, Sigma)
Widefield fluorescence inverted microscopeOlympusN/AInverted fluorescence microscope IX81, with motorised Z-axis control, CoolLED pE4000 (16 channels, 365-770 nm), ORCA-Flash4.0LT (Hamamatsu) cMOS camera, glass warming plate Okolab, CellSens Dimension v.3 software and air objectives 4x/0.13 UPlanFLN and 40x/0.6 LUCPlanFLN. (Optional, for high-resolution imaging) 60x/1.0 LUMPLFLN water

References

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