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。

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。

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...

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

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。

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）

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

production-multi-parameter-live-cell-fluorescence-lifetime-imaging

Research

JoVE Journal

Bioengineering

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

621 次观看.  Ghent University. 在这项工作中，我们提出并比较了可用于使用活细胞显微镜分析细胞代谢和氧分布的球状体形成方法。近年来，荧光寿命成像显微镜已被广泛用于研究活细胞中的代谢生物标志物，如 NADPH 和 FAD，并且已经开发了几种荧光纳米颗粒，用于将此类测量与细胞和组织氧合成像进行多重检测。多细胞球状体、类器官和器官芯片可以复制复杂的、类似体内的?...

视频: 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

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

在活细胞中的荧光寿命成像分子转子

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

科研

生物工程

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

从快速荧光成像在分子扩散法对活细胞的膜在商业显微镜

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Lab-on-a-CD Platform for Generating Multicellular Three-dimensional Spheroids

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the living body than two-dimensional cell culture. In this study, we fabricated an electrical motor-driven lab-on-a-CD (compact disc) platform, called a centrifugal microfluidic-based spheroid (CMS) culture system, to create three-dimensional (3D) cell spheroids implementing high centrifugal force. This device can vary rotation speeds to generate gravity conditions from 1 x g to 521 x g. The CMS system is 6 cm in diameter, has one hundred 400 &#956;m microwells, and is made by molding with polydimethylsiloxane in a polycarbonate mold premade by a computer numerical control machine. A barrier wall at the channel entrance of the CMS system uses centrifugal force to spread cells evenly inside the chip. At the end of the channel, there is a slide region that allows the cells to enter the microwells. As a demonstration, spheroids were generated by monoculture and coculture of human adipose-derived stem cells and human lung fibroblasts under high gravity conditions using the system. The CMS system used a simple operation scheme to produce coculture spheroids of various structures of concentric, Janus, and sandwich. The CMS system will be useful in cell biology and tissue engineering studies that require spheroids and organoid culture of single or multiple cell types.

We present a motor-powered centrifugal microfluidic device that can cultivate cell spheroids. Using this device, spheroids of single or multiple cell types could be easily cocultured under high gravity conditions.

用于生成多细胞三维球体的 CD 实验室平台

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

实时显微镜成像下的 3D 水凝胶控制应变

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

将3D多细胞乳房球体直接生物打印到内皮网络

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue ...

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented. Thus, characterization and standardization of spheroid phenotype are required to ensure the reproducibility and transparency of the research results. The analysis of average spheroid oxygenation and the value of O2 gradients in three dimensions (3D) can be a simple and universal way for spheroid phenotype characterization, pointing at their metabolic activity, overall viability, and potential to recapitulate in vivo tissue microenvironment. The visualization of 3D oxygenation can be easily combined with multiparametric analysis of additional physiological parameters (such as cell death, proliferation, and cell composition) and applied for continuous oxygenation monitoring and/or end-point measurements. The loading of the O2 probe is performed during the stage of spheroid formation and is compatible with various protocols of spheroid generation. The protocol includes a high-throughput method of spheroid generation with introduced red and near-infrared emitting ratiometric fluorescent O2 nanosensors and the description of multi-parameter assessment of spheroid oxygenation and cell death before and after bioprinting. The experimental examples show comparative O2 gradients analysis in homo- and hetero-cellular spheroids as well as spheroid-based bioprinted constructs. The protocol is compatible with a conventional fluorescence microscope having multiple fluorescence filters and a light-emitting diode as a light source.

The protocol describes high-throughput spheroid generation for bioprinting using the multi-parametric analysis of their oxygenation and cell death on a standard fluorescence microscope. This approach can be applied to control the spheroids viability and perform standardization, which is important in modeling 3D tissue, tumor microenvironment, and successful (micro)tissue biofabrication.

经济实惠的氧气显微镜辅助多细胞球体生物制造

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

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）

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）