多细胞球体的生产和多参数活细胞荧光寿命成像显微镜 （FLIM）

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?.......

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.......

Materials

Name	Company	Catalog Number	Comments
0.05% Trypsin-EDTA	Gibco	25300-054	Also available from Sigma
10 mL serological pipets	VWR	612-3700	Similar products are also available from Sarstedt, Corning, VWR and other companies
12 well cell-culture plates, sterile	Greiner bio-one	665-180	Similar products are also available from Sarstedt, Corning and other companies.
12 Well Chamber slide, removable	Ibidi	81201	Also available from Grace Bio-Labs, ThermoFisher Scientific and others
15 mL centrifuge tubes	Nerbe plus	02-502-3001	Similar products are also available from Sarstedt, Corning, VWR and other companies
3D Petri Dish micromolds	Microtissue	Z764000-6EA
6 well cell-culture plates, sterile	Greiner bio-one	657160	Similar products are also available from Sarstedt, Corning, VWR and other companies
70% ethanol	ChemLab	CL02.0537.5000
Biofloat	Sarstedt	83.3925.400	Commercial available coated 96-well plate for spheroid formation
Calcein Green-AM	Tebubio	AS-89201	Apply in dilution 1:1000
CellSens Dimension software	Olympus	version 3
Collagen from human placenta, type IV	Sigma	C5533	For the preparation of 0.07 mg/mL Collagen and 0.03 mg/mL Poly-D-lysine coated microscopy dishes
Confocal FLIM Microscope	Leica Microsystems	N/A	Stellaris 8 Falcon inverted microscope with white-light laser, HyD X detectors, climate / T control chamber (OkoLab), 25x/0.95 W objective
D(+)-Glucose	Merck	8342	Prepare 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-free	Sigma-Aldrich	D5030-10X1L	For preparation of imaging medium
Fetal Bovine Serum (FBS)	Gibco	10270-098	Also available from Sigma. Needs to be heat-inactivated before use.
HEPES (1M)	Gibco	15630-080	Dilution 1/100 for preparation of imaging medium (final concentration 10 mM)
Human colon cancer cells HCT116	ATCC
ImageJ	NIH	version 1.54f
Leica Application Suite X (LAS X)	Leica Microsystems	version 4.6.1.27508
L-glutamine	Gibco	25030	Also available from Sigma. Apply in dilution 1:100.
Lipidure-CM5206	Amsbio	AMS.52000034GB1G
McCoy's 5A, need addition of 1 mM Sodium Pyruvate and 10 mM HEPES	VWR	392-0420	Standard growth medium for HCT116 cells
micro-patterned 3D-printed PDMS stamps	N/A	N/A	Provided by the Centre for Microsystems Technology, Professor Dr. Jan Vanfleteren, Ghent University
NaCl	Chemlab	CL00.1429.100
Neubauer couting chamber	Fisher Scientific	15980396
O2 probes: MMIR1	N/A	N/A	Full characterization, validation and some applications can be found at: https://www.biorxiv.org/content/10.1101/2023.12.11.571110 v1
PBS	Fisher scientific	Gibco18912014	Dissolve PBS tablet in 500 mL of distilled water.
Pen Strep :Penicillin (10,000 U/mL) / streptomycin (10,000 μg/mL) 100x solution	Gibco	15140-122	Also available from Sigma. Apply in dilution 1:100.
Poly-D-lysine	Sigma	P6407-5mg	For the preparation of 0.07 mg/mL Collagen and 0.03 mg/mL Poly-D-lysine coated microscopy dishes
Propidium Iodide	Sigma-Aldrich	25535-16-4	Cell death staining, use 1 µg/mL at 1h incubation
PVDF syringe filter 0.22 µm	Novolab	A35149	Similar products are also available from Sarstedt, Corning, VWR and other companies
Sodium pyruvate (100 mM)	Gibco	11360-070	Dilution 1/100 for preparation of imaging medium (final concentration 1mM)
SphericalPlate 5D 24-well	Kugelmeiers	SP5D-24W
sterile petridish	Greiner bio-one	633181	Similar products are also available from Sarstedt, Corning, VWR and other companies
Tissue culture flask (25 cm² )	VWR	734-2311	Similar products are also available from Sarstedt, Corning, VWR and other companies
Tissue culture flask (75 cm²)	VWR	734-2313	Similar products are also available from Sarstedt, Corning, VWR and other companies
U-bottom 96-well plate	VWR	10062-900	Similar products are also available from Sarstedt, Corning, Greiner Bio-one and other companies
Ultrapure Agarose	Invitrogen (Life Technologies)	16500-500	Other types of Agarose such as Agarose low melting point (A-9414, Sigma), Agarose for routine use (A-9539, Sigma)
Widefield fluorescence inverted microscope	Olympus	N/A	Inverted 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