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。

1. 多细胞球体的产生
<ol><li>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?...

用于基于 FLIM 的代谢和缺氧分析的多细胞球状体形成

没什么可透露的。

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...

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。根据动物研究的 3R 原则（替代、还原和精炼），这些更可靠、更复杂的体外模型挑战了广泛使用动物模型的需求（食品和药物管理局 [FDA] 现代化法案 2.012）。除了癌症，球状体还可用于干细胞研究。例如，多能干细胞具有形成拟胚体 （EB） 的能力，可用于诱导多能干细胞 （iPSC） 分化为难以直接从患者那里获得的特殊细胞类型，例如神经前体细胞13 或卵巢颗粒细胞13,14。此外，EB 的形成通常是开发更复杂的类器官模型的第一步，例如神经15、视网膜16、心脏17、肝脏18、胃19 和肠道类器官20。在为实验选择合适的球状体形成方法时，应考虑包括大小、重现性、通量和下游应用在内的因素。
与 2D 培养相比，3D 培养的复杂性增加会导致更高的变异性。营养成分21、介质蒸发22、粘度23、pH 控制24、球状体形成方法，甚至培养时间25,26 等因素都可能导致获得不同形态、大小、活力和不同化学抗性的球状体27,28.最近的研究表明，球体氧梯度并不总是静态的，并且受形成方法、球体大小和细胞外粘度的影响，从而影响球体异质性29。为了提高微球的可重复性和数据可访问性，我们开发了 MISpheroID 知识库26，将细胞系、培养基、形成方法和微球大小确定为可重现结果的最小信息。因此，对多种高通量（SphericalPlate 5D、实验室制造的微模具和微组织模具）和低附着方法（即 Biofloat 和 Lipidure 涂层的 96 孔板，无支架和基于支架）进行了详细比较（图 1 和表 1），包括孔大小（给定最大球体尺寸的估计）、使用的耗材、准备时间以及在不将球体运输到显微镜培养皿的情况下监测球体的可能性。 后者支持长期研究，而使用高通量方法生产的球体通常会导致终点实验。除 5DspheriPlate 的网格外，所有方法都不会带来不需要的自发荧光，因此可以直接在显微镜中使用。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66845/66845fig01.jpg"> 图 1：球体形成方法解释。 高通量方法，例如 SphericalPlate 5D，它在板中集成了专利微孔，而实验室生产的微模具和 MicroTissue 模具使用印章在琼脂糖（蓝色）中制作多个微孔。Lipidure （Amsbio） 和 Biofloat （Sarstedt） 等低附着板使用非粘附涂层，可抑制细胞表面粘附并促进细胞自聚集。 <a href="https://www.jove.com/files/ftp_upload/66845/66845fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td>
			<td>5D 球形板</td>
			<td>自产微模</td>
			<td>微组织</td>
			<td>低附着方法</td>
		</tr><tr><td>球状体数量/孔</td>
			<td>750</td>
			<td>1589</td>
			<td>81</td>
			<td>1</td>
		</tr><tr><td>直径井</td>
			<td>90 微米</td>
			<td>400 微米</td>
			<td>800 微米</td>
			<td>1 毫米</td>
		</tr><tr><td>培养体积</td>
			<td>1 毫升</td>
			<td>5 毫升</td>
			<td>1 毫升</td>
			<td>200 微升</td>
		</tr><tr><td>其他耗材</td>
			<td>/</td>
			<td>7 mL 3% 琼脂糖</td>
			<td>500 μL 2% 琼脂糖</td>
			<td>/ *</td>
		</tr><tr><td>准备时间</td>
			<td>10 分钟</td>
			<td>2 小时 + 3 天培养基适应</td>
			<td>0.5 小时 + 15 分钟培养基适应</td>
			<td>10–30 分钟 + 1 小时干燥</td>
		</tr><tr><td>监测</td>
			<td>是的</td>
			<td>不**</td>
			<td>是的</td>
			<td>是的</td>
		</tr><tr><td>自发荧光</td>
			<td>是的</td>
			<td>不</td>
			<td>不</td>
			<td>不</td>
		</tr><tr><td>可 重用</td>
			<td>不</td>
			<td>是的</td>
			<td>是的</td>
			<td>不**</td>
		</tr><tr><td rowspan="2">成本</td>
			<td rowspan="2">€€</td>
			<td rowspan="2">€</td>
			<td rowspan="2">€€€€</td>
			<td>€€€€€： 涂层和 Matrigel</td>
		</tr><tr><td>€€：商用 96 孔板</td>
		</tr><tr><td colspan="3">*一些细胞系需要添加 ECM（即 2%–5% Matrigel）以形成紧凑的球状体。</td>
			<td></td>
			<td></td>
		</tr><tr><td colspan="5">**涂层可重复使用，直到用完。但是，每个板会消耗少量介质，并且灰尘会随着时间的推移而积累。经常需要过滤器消毒。</td>
		</tr></tbody></table>表 1：多种球体形成方法的比较29.“监测”：无需转移到显微镜培养皿中即可监测球体的能力。€： 0-50€， €€： 50-150€ ， €€€： 150-500€ ， €€€€： >500€
荧光显微镜能够直接监测球状体内的关键生物学方面，包括细胞死亡、活力、增殖、代谢、粘度，甚至机械特性30。荧光寿命成像显微镜 （FLIM） 为研究荧光探针在其（微）环境中的相互作用 31,32,33,34 提供了额外的定量维度，从而可以根据不同的发射寿命35,36 分辨重叠的发射光谱以及基于内在细胞自发荧光探测细胞代谢。因此，烟酰胺腺嘌呤二核苷酸磷酸 （NAD（P）H）、黄素单核苷酸 （FMN）、黄素腺嘌呤二核苷酸 （FAD）、原卟啉 IX 等广泛使用的细胞自发荧光化合物可以用单光子和双光子 FLIM 测量，并作为葡萄糖分解代谢、氧化磷酸化 （OxPhos） 的内在“传感器”，并提供细胞氧化还原状态的一般概述。NAD（P）H 存在于游离细胞质中，或以蛋白质结合的线粒体形式存在37,38。同样，FAD 的氧化态是荧光的，游离形式的寿命更长。NAD（P）H 和 FAD 显微成像通常涉及双光子激发的 FLIM，旨在防止样品光损伤39。通常，“光学代谢成像”FLIM 可以与基于染料的探针、基因编码的生物传感器、磷光寿命成像显微镜 （PLIM） 和基于比率强度的测量结合使用，以提供球状体或类器官代谢、氧合、增殖和细胞活力的更完整图像 29,30,31.此外，FLIM 还可以与 Förster 共振能量转移 （FRET） 方法结合使用，以测量与受体紧密接触时供体荧光团的寿命变化，以研究药物与其靶结构域的结合 33,40,41。
通常对采集的 FLIM 图像进行分析，以逐个像素计算寿命。目前，至少有 3 种常用策略用于获得荧光寿命：半定量“快速 FLIM”42（有时称为“tau 感”43,44）、衰变曲线拟合，使用一、二或三指数拟合，以及具有相量变换和相量图分析的“无拟合”方法。根据供应商的不同，可以使用提供的（LAS X、Symphotime、SPCImage 等）或开源软件（例如 FLIMfit45、FLIMJ46 或其他47）来处理测量的 FLIM 数据。通常，供应商提供的软件可用于初步数据分析，而开源解决方案可以使用相量图和 3D 可视化等工具提供更准确的研究。
尽管 FLIM 作为研究球体的方法有用且有吸引力，但可用的实验方案很少，并且在选择最合适的形成方法以成功进行涉及 FLIM 的活体多参数显微镜实验方面普遍缺乏知识。在这里，根据它们的形态、活力和氧合，与最近验证和表征的远红外和近红外 （NIR） 氧传感纳米传感器 （MMIR1） 详细比较了常用的球状体形成方案。阳离子纳米颗粒浸渍了两种报告染料，即参比 O2 不敏感的氮杂-BODIPY（激发波长 650 nm，发射波长 675 nm）和 NIR O2 敏感的金属卟啉 PtTPTBPF（激发波长 620 nm，发射波长 760 nm）。MMIR1 能够在传统荧光显微镜（使用比率分析）或磷光寿命显微镜 （PLIM） 上实时分析氧梯度，而不会引入细胞毒性，并允许稳定的信号、长期监测和多路复用25,29。根据使用染料或纳米传感器染色的需要、球状体通量或细胞类型，可以选择最合适的形成方案。由于球体活力和氧合的研究与癌症和干细胞衍生的球体的研究相关，因此提出的方案还包括 NAD（P）H-FLIM 和 FAD-FLIM 与这些模型的示例和预期典型结果。所提出的成像和分析管道针对最流行的基于时间相关单光子计数的 FLIM 显微镜平台。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>Also available from Sigma</td>
		</tr>
		<tr>
			<td>10 mL serological pipets</td>
			<td>VWR</td>
			<td>612-3700</td>
			<td>Similar products are also available from Sarstedt, Corning, VWR and other companies</td>
		</tr>
		<tr>
			<td>12 well cell-culture plates, sterile</td>
			<td>Greiner bio-one</td>
			<td>665-180</td>
			<td>Similar products are also available from Sarstedt, Corning and other companies.</td>
		</tr>
		<tr>
			<td>12 Well Chamber slide, removable</td>
			<td>Ibidi</td>
			<td>81201</td>
			<td>Also available from Grace Bio-Labs, ThermoFisher Scientific and others</td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Nerbe plus</td>
			<td>02-502-3001</td>
			<td>Similar products are also available from Sarstedt, Corning, VWR and other companies</td>
		</tr>
		<tr>
			<td>3D Petri Dish micromolds</td>
			<td>Microtissue</td>
			<td>Z764000-6EA</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well cell-culture plates, sterile</td>
			<td>Greiner bio-one</td>
			<td>657160</td>
			<td>Similar products are also available from Sarstedt, Corning, VWR and other companies</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>ChemLab</td>
			<td>CL02.0537.5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Biofloat</td>
			<td>Sarstedt</td>
			<td>83.3925.400</td>
			<td>Commercial available coated 96-well plate for spheroid formation</td>
		</tr>
		<tr>
			<td>Calcein Green-AM</td>
			<td>Tebubio</td>
			<td>AS-89201</td>
			<td>Apply in dilution 1:1000</td>
		</tr>
		<tr>
			<td>CellSens Dimension software</td>
			<td>Olympus</td>
			<td>version 3</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen from human placenta, type IV</td>
			<td>Sigma</td>
			<td>C5533</td>
			<td>For the preparation of 0.07 mg/mL Collagen and 0.03 mg/mL Poly-D-lysine coated microscopy dishes</td>
		</tr>
		<tr>
			<td>Confocal FLIM Microscope</td>
			<td>Leica Microsystems</td>
			<td>N/A</td>
			<td>Stellaris 8 Falcon inverted microscope with white-light laser, HyD X detectors, climate / T control chamber (OkoLab), 25x/0.95 W objective</td>
		</tr>
		<tr>
			<td>D(+)-Glucose</td>
			<td>Merck</td>
			<td>8342</td>
			<td>Prepare 1 M stock solution, 1:100 for preparation of imaging medium (final concentration 10 mM)</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium (DMEM), phenol red-, glucose-, pyruvate- and glutamine-free</td>
			<td>Sigma-Aldrich</td>
			<td>D5030-10X1L</td>
			<td>For preparation of imaging medium</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;(FBS)</td>
			<td>Gibco</td>
			<td>10270-098</td>
			<td>Also available from Sigma. Needs to be heat-inactivated before use.</td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td>Dilution 1/100 for preparation of imaging medium (final concentration 10 mM)</td>
		</tr>
		<tr>
			<td>Human colon cancer cells HCT116</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td>version 1.54f</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Application Suite X (LAS X)</td>
			<td>Leica Microsystems</td>
			<td>version 4.6.1.27508</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030</td>
			<td>Also available from Sigma. Apply in dilution 1:100.</td>
		</tr>
		<tr>
			<td>Lipidure-CM5206</td>
			<td>Amsbio</td>
			<td>AMS.52000034GB1G</td>
			<td></td>
		</tr>
		<tr>
			<td>McCoy&#39;s 5A, need addition of 1 mM Sodium Pyruvate and 10 mM HEPES</td>
			<td>VWR</td>
			<td>392-0420</td>
			<td>Standard growth medium for HCT116 cells</td>
		</tr>
		<tr>
			<td>micro-patterned 3D-printed PDMS stamps</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Provided by the Centre for Microsystems Technology, Professor Dr. Jan Vanfleteren, Ghent University</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Chemlab</td>
			<td>CL00.1429.100</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer couting chamber</td>
			<td>Fisher Scientific</td>
			<td>15980396</td>
			<td></td>
		</tr>
		<tr>
			<td>O2 probes: MMIR1</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Full characterization, validation and some applications can be found at: https://www.biorxiv.org/content/10.1101/2023.12.11.571110 
			v1</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Fisher scientific</td>
			<td>Gibco18912014</td>
			<td>Dissolve PBS tablet in 500 mL of distilled water.</td>
		</tr>
		<tr>
			<td>Pen Strep :Penicillin (10,000 U/mL) / streptomycin (10,000 &#956;g/mL) 100x solution</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Also available from Sigma. Apply in dilution 1:100.</td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P6407-5mg</td>
			<td>For the preparation of 0.07 mg/mL Collagen and 0.03 mg/mL Poly-D-lysine coated microscopy dishes</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Sigma-Aldrich</td>
			<td>25535-16-4</td>
			<td>Cell death staining, use 1 &#181;g/mL at 1h incubation</td>
		</tr>
		<tr>
			<td>PVDF syringe filter 0.22 &#181;m</td>
			<td>Novolab</td>
			<td>A35149</td>
			<td>Similar products are also available from Sarstedt, Corning, VWR and other companies</td>
		</tr>
		<tr>
			<td>Sodium pyruvate (100 mM)</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td>Dilution 1/100 for preparation of imaging medium (final concentration 1mM)</td>
		</tr>
		<tr>
			<td>SphericalPlate 5D 24-well</td>
			<td>Kugelmeiers</td>
			<td>SP5D-24W</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile petridish</td>
			<td>Greiner bio-one</td>
			<td>633181</td>
			<td>Similar products are also available from Sarstedt, Corning, VWR and other companies</td>
		</tr>
		<tr>
			<td>Tissue culture flask (25 cm&#178; )</td>
			<td>VWR</td>
			<td>734-2311</td>
			<td>Similar products are also available from Sarstedt, Corning, VWR and other companies</td>
		</tr>
		<tr>
			<td>Tissue culture flask (75 cm&#178;)</td>
			<td>VWR</td>
			<td>734-2313</td>
			<td>Similar products are also available from Sarstedt, Corning, VWR and other companies</td>
		</tr>
		<tr>
			<td>U-bottom 96-well plate</td>
			<td>VWR</td>
			<td>10062-900</td>
			<td>Similar products are also available from Sarstedt, Corning, Greiner Bio-one and other companies</td>
		</tr>
		<tr>
			<td>Ultrapure Agarose</td>
			<td>Invitrogen (Life Technologies)</td>
			<td>16500-500</td>
			<td>Other types of Agarose such as Agarose low melting point (A-9414, Sigma), Agarose for routine use (A-9539, Sigma)</td>
		</tr>
		<tr>
			<td>Widefield fluorescence inverted microscope</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td>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</td>
		</tr>
	</tbody>
</table>

production and multi-parameter live cell fluorescence lifetime imaging microscopy (flim) of multicellular spheroids

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。

Multicellular spheroids represent a group of 3D tissue models obtained by the self-aggregation of cells and exhibiting a spherical shape. They are widely used to mimic cell-cell and cell-matrix interaction in vitro and to reproduce&#160;a 3D context within a multitude of cancer and stem cell-derived constructs. Several techniques are employed to reduce cell attachment and promote the aggregation. These include the hanging-drop method relying on the surface tension1; cell attachment repelling methods such as ultra-low attachment plates, micro-molds, and microwells2,3; acoustic wave-based approach4; flow-induced aggregation methods (spinner flasks, bioreactor, and microfluidic devices)5; magnetic particles-assisted formation6 and use of the aggregation-promoting synthetic and ECM-based matrices and scaffolds7,8,9.
In cancer research, development, and validation of new drug therapies, spheroids are an attractive model due to their ability to recapitulate the spatial diffusion-limited gradients of nutrients, waste products, and O2, often leading to the formation of a necrotic core, typical to the solid tumors10,11. These more reliable and sophisticated in vitro models challenge the need for extensive use of animal models (Food and Drug Administration [FDA] Modernization Act 2.012), according to the 3Rs principle of animal research (replacement, reduction, and refinement). In addition to cancer, spheroids find their application in stem cell research. For instance, pluripotent stem cells have the capacity to form embryoid bodies (EB), which can be used for the differentiation of induced pluripotent stem cells (iPSCs) towards specialized cell types that are challenging to obtain directly from patients, such as neural precursor cells13 or ovarian granulosa cells13,14. Furthermore, the formation of an EB is often the first step in the development of more complex organoid models, e.g., neural15, retinal16, cardiac17, liver18, stomach19, and intestinal organoids20. Factors including size, reproducibility, throughput, and downstream applications should be considered when choosing an appropriate spheroid formation method for the experiments.
The increased complexity of 3D culture can lead to higher variability compared to 2D culture. Factors such as nutrient composition21, media evaporation22, viscosity23, pH control24, spheroid formation method, and even the time in the culture25,26 can result in obtaining spheroids of varying morphology, sizes, viability, and different chemoresistance27,28. Recent research demonstrated that spheroid oxygen gradients are not always static and are affected by the formation method, spheroid size, and extracellular viscosity, affecting the spheroid heterogeneity29. To improve reproducibility and data accessibility on spheroids, the MISpheroID knowledge base has been developed26, identifying cell line, culture medium, formation method, and spheroids size as the minimal information for a reproducible result. Therefore, a detailed comparison was made of multiple high-throughput (SphericalPlate 5D, lab-made micromolds, and Microtissue molds) and low attachment methods (i.e., Biofloat and Lipidure-coated 96-well plates, both scaffold-free and scaffold-based) (Figure 1 and Table 1), including the well size (given an estimation of the maximum spheroid size), consumables used, preparation time and the possibility of monitoring spheroids without transporting them to microscopy dishes. The latter enables long-term studies, whereas spheroids produced with high-throughput methods often result in endpoint experiments. All methods except for the grids of the 5DspheriPlate do not bring unwanted autofluorescence, hereby enabling their direct use in microscopy.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66845/66845fig01.jpg" /> 
Figure 1: Spheroid formation methods explained. High-throughput methods such as the SphericalPlate 5D, which has integrated patented microwells in the plate, while the lab-produced micromolds and the MicroTissue molds use stamps to make multiple microwells in agarose (blue). Low-attachment plates such as Lipidure (Amsbio) and Biofloat (Sarstedt) use a non-adherent coating inhibiting cell-surface adhesion and promoting cell self-aggregation. <a href="https://www.jove.com/files/ftp_upload/66845/66845fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>5D SpheriPlate</td>
			<td>Self-produced micromolds</td>
			<td>Microtissue</td>
			<td>Low attachment methods</td>
		</tr>
		<tr>
			<td>Number of spheroids/well&#160;</td>
			<td>750</td>
			<td>1589</td>
			<td>81</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Diameter well</td>
			<td>90 &#181;m</td>
			<td>400 &#181;m</td>
			<td>800 &#181;m</td>
			<td>1 mm</td>
		</tr>
		<tr>
			<td>Culture volume</td>
			<td>1 mL</td>
			<td>5 mL</td>
			<td>1 mL</td>
			<td>200 &#181;L</td>
		</tr>
		<tr>
			<td>Other consumables</td>
			<td>/</td>
			<td>7 mL of 3% agarose</td>
			<td>500 &#181;L of 2% agarose</td>
			<td>/ *</td>
		</tr>
		<tr>
			<td>Preparation time</td>
			<td>10 min</td>
			<td>2 h + 3 days media adaptation</td>
			<td>0.5 h + 15 min media adaptation</td>
			<td>10&#8211;30 min + 1 h drying</td>
		</tr>
		<tr>
			<td>Monitoring</td>
			<td>Yes</td>
			<td>No**</td>
			<td>Yes</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>Autofluorescent</td>
			<td>Yes</td>
			<td>No</td>
			<td>No</td>
			<td>No</td>
		</tr>
		<tr>
			<td>Reusable</td>
			<td>No</td>
			<td>Yes</td>
			<td>Yes</td>
			<td>No**</td>
		</tr>
		<tr>
			<td rowspan="2">Cost</td>
			<td rowspan="2">&#8364;&#8364;&#160;</td>
			<td rowspan="2">&#8364;</td>
			<td rowspan="2">&#8364;&#8364;&#8364;&#8364;</td>
			<td>&#8364;&#8364;&#8364;&#8364;: Coating and Matrigel</td>
		</tr>
		<tr>
			<td>&#8364;&#8364;: Commercial 96-well plate</td>
		</tr>
		<tr>
			<td colspan="3">*Some cell lines need addition of&#160; ECM (i.e. 2%&#8211;5% Matrigel) to form compact spheroids.&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="5">**The coating is reusable until depleted. However, each plate will consume a small amount of media and dust can accumulate over time. Filter sterilization is regularly needed.&#160;</td>
		</tr>
	</tbody>
</table>
Table 1: Comparison of multiple spheroid formation methods29. &#34;Monitoring&#34;: the ability to monitor spheroid without the need for transfer to a microscopy dish. &#8364;: 0-50&#8364;, &#8364;&#8364;: 50-150&#8364; , &#8364;&#8364;&#8364;: 150-500&#8364; , &#8364;&#8364;&#8364;&#8364;: &#62;500&#8364;
Fluorescence microscopy enables direct monitoring of the key biological aspects within spheroids, including cell death, viability, proliferation, metabolism, viscosity, and even mechanical properties30. Fluorescence lifetime imaging microscopy (FLIM) provides an additional quantitative dimension for studying fluorescent probe interactions within their (micro)environment31,32,33,34, allowing resolving the overlapping emission spectra according to different emission lifetimes35,36 and probing cell metabolism based on intrinsic cellular autofluorescence. Thus, such widespread cellular autofluorescent compounds as nicotinamide adenine dinucleotide phosphate (NAD(P)H), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), protoporphyrin IX, and others can be measured with one- and two-photon FLIM and serve as intrinsic &#39;sensors&#39; of glucose catabolism, oxidative phosphorylation (OxPhos) and provide a general overview of the cell redox state. NAD(P)H exists in free cytoplasmic, or in protein-bound mitochondrial forms37,38. Similarly, the oxidized state of FAD is fluorescent with a longer lifetime of the free form. NAD(P)H and FAD microscopies usually involve two-photon excited FLIM, aiming at preventing sample photodamage39. Frequently, &#39;optical metabolic imaging&#39; FLIM can be combined with the use of dye-based probes, genetically encoded biosensors, phosphorescence lifetime imaging microscopy (PLIM), and ratiometric intensity-based measurements in order to provide a more complete picture of spheroid or organoid metabolism, oxygenation, proliferation and cell viability29,30,31. In addition, FLIM can also be combined with F&#246;rster resonance energy transfer (FRET) method to measure the lifetime variation of the donor fluorophore when in close contact with the acceptor to investigate the binding of a drug with its target domain33,40,41.
The acquired FLIM images are typically analyzed to calculate the lifetime pixel-by-pixel. Currently, there are at least 3 common strategies used to obtain fluorescence lifetime: semi-quantitative &#39;fast FLIM&#39;42 (sometimes referred to as &#39;tau sense&#39;43,44), decay curve fitting, using one-, two- or three-exponential fitting, and &#39;fitting-free&#39; approach with phasor transformation and phasor plot analysis. Depending on the vendor, either provided (LAS X, Symphotime, SPCImage, etc.) or open-source software (e.g., FLIMfit45, FLIMJ46, or others47) can be used to handle measured FLIM data. Typically, vendor-provided software is useful for preliminary data analysis, while open-source solutions can provide for more accurate studies using, e.g., phasor plots and 3D visualization.
Despite the usefulness and attractiveness of FLIM as a method for studying spheroids, very few experimental protocols are available, and there is a general lack of knowledge in choosing the most appropriate formation method for successful live multiparametric microscopy experiments involving FLIM. Here, a detailed comparison of commonly used spheroid formation protocols is presented based on their morphology, viability, and oxygenation with the recently validated and characterized far-red and near-infra-red (NIR) oxygen-sensing nanosensor (MMIR1). The cationic nanoparticle is impregnated with two reporter dyes, the reference O2-insensitive aza-BODIPY (excitation 650 nm, emission 675 nm) and the NIR O2-sensitive metalloporphyrin, PtTPTBPF (excitation 620 nm, emission 760 nm). The MMIR1 enables real-time analysis of oxygen gradients on a conventional fluorescence microscope (using ratiometric analysis) or phosphorescence lifetime microscope (PLIM) without introducing cellular toxicity and allowing for stable signals, long-term monitoring, and multiplexing25,29. Depending on the need to stain with dyes or nanosensors, spheroid throughput, or cell type, the most appropriate formation protocol can be chosen. Since the studies of spheroids viability and oxygenation are relevant for studies of cancer and stem cell-derived spheroids, the presented protocols also include examples and expected typical results of NAD(P)H-FLIM and&#160;FAD-FLIM with these models. The presented imaging and analysis pipelines target the most popular time-correlated single photon counting-based FLIM microscopy platforms.

作者聚焦：使用荧光寿命成像显微镜标准化用于代谢和氧合分析的球状体形成方法

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）

In this work, we present and compare spheroid formation methods that can be used for analysis of cell metabolism and oxygen distribution using live cell microscopy. In recent years, fluorescence lifetime imaging microscopy has been extensively used to investigate metabolic biomarkers like NADPH and FAD in live cells, and several fluorescent nanoparticles have been developed to multiplex such measurements with imaging of cell and tissue oxygenation. Multicellular spheroids, organoids, and organ-on-a-chip can replicate a complex, in-vivo-like microenvironment, minimizing the need in animal research.For spheroid production, we show different formation methods, can span from low to high throughput. They also highlight their optical accessibility, compatibility with fluorescence lifetime imaging microscope, and the possibility of including extracellular matrix components. So, even though 3D in vitro models provide better context in comparison to 2D cultures, their high variability, low reproducibility, and incomplete experimental reporting remain a problem.Parameters such as the spheroid size, the nutrient composition, the extracellular viscosity, and even spheroid formation methods can all lead to increased cellular heterogeneity. With this protocol, we aim to harmonize and standardize the spheroid production methods, highlighting key aspects important for life-continuous and multiparametric analysis of spheroids using FLIM microscopy.

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...

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。

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug ...

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JoVE Journal

Bioengineering

Production and Multi-Parameter Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM) of Multicellular Spheroids

391 Views.  Ghent University. In this work, we present and compare spheroid formation methods that can be used for analysis of cell metabolism and oxygen distribution using live cell microscopy. In recent years, fluorescence lifetime imaging microscopy has been extensively used to investigate metabolic biomarkers like NADPH and FAD in live cells, and several fluorescent nanoparticles have been developed to multiplex such measurements with imaging of cell and tissue oxygenation. Multicellular spheroids, organoids, and orga...

Video: Author Spotlight: Standardizing Spheroid Formation Methods for Metabolic and Oxygenation Analysis Using Fluorescence Lifetime Imaging Microscopy