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）
August 9th, 2024
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。
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、类似体内的微环境，从而最大限度地减少动物研究的需求。
对于球状体生产，我们展示了不同的形成方法，可以从低通量到高通量。它们还强调了它们的光学可及性、与荧光寿命成像显微镜的兼容性以及包含细胞外基质成分的可能性。因此，尽管与 2D 培养相比，3D 体外模型提供了更好的背景，但它们的高变异性、低重现性和不完整的实验报告仍然是一个问题。
球体大小、营养成分、细胞外粘度，甚至球体形成方法等参数都可能导致细胞异质性增加。通过该协议，我们的目标是协调和标准化球体生产方法，突出了使用 FLIM 显微镜对球体进行生命连续和多参数分析的重要关键方面。
概述
本文介绍了多种形成多细胞球体的方法，用于使用活细胞显微镜分析细胞代谢和氧气分布。该研究强调了使用荧光寿命成像显微镜（FLIM）来研究活细胞中的代谢生物标志物。
关键研究组成部分
科学领域
- 神经科学
- 细胞生物学
- 活细胞成像
背景
- 多细胞球体和类器官复制类似于体内的微环境。
- 荧光寿命成像显微镜用于研究代谢生物标志物。
- 高变异性和低重复性是3D体外模型中的挑战。
- 不同的球体形成方法可以用于各种应用。
研究目的
- 比较活细胞显微镜下的球体形成方法。
- 分析3D模型中的细胞代谢、缺氧和细胞死亡。
- 改善对多细胞系统中氧气分布的理解。
使用的方法
- 荧光寿命成像显微镜（FLIM）
- 细胞自发荧光分析
- 使用染色染料和荧光纳米粒子
- 不同的球体形成技术
主要结果
- 展示了分析3D球体中细胞代谢的方法。
- 显示了球体与FLIM在代谢研究中的兼容性。
- 强调了细胞外基质成分在球体形成中的重要性。
- 解决了3D培养的重复性和变异性的挑战。
结论
- 不同的球体形成方法可以增强活细胞分析。
- FLIM为3D模型中的代谢过程提供了宝贵的见解。
- 这些方法的持续发展可以减少对动物模型的依赖。
常见问题
Executive Industry Relevance
Standardized production and multi-parameter FLIM analysis of multicellular spheroids address critical challenges in modeling tumor microenvironments for drug discovery. Harmonizing spheroid formation and live-cell imaging workflows enhances predictive confidence in metabolic and hypoxia-related readouts, supporting robust early-stage decision-making. This approach strengthens translational continuity and reduces biological risk across oncology and stem cell R&D portfolios.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of metabolic pathways and oxygen gradients in physiologically relevant 3D models.
- Supports functional target validation by quantifying NADPH and FAD biomarkers in live spheroids.
- Facilitates biological de-risking through multiplexed metabolic and hypoxia measurements.
- Improves predictive confidence for target engagement and pathway modulation in complex systems.
Screening & Assay Development
- Prepares reproducible 3D spheroid models compatible with widefield, confocal, and two-photon FLIM platforms.
- Standardizes assay conditions to reduce variability and enhance quantitative output reliability.
- Enables scalable, high-throughput screening of compound effects on cell metabolism and viability.
- Supports robust evaluation of bio- and nanosensor integration for multiplexed readouts.
Translational & Preclinical Research
- Aligns in vitro metabolic and hypoxia profiles with disease-relevant tumor microenvironments.
- Provides continuity from discovery through preclinical validation by minimizing animal model reliance.
- Enables risk-adjusted advancement decisions based on quantitative, multiparametric data.
- Facilitates translational biomarker development using live-cell FLIM outputs.
Pipeline & Workflow Integration
This harmonized workflow integrates from early discovery through lead identification and preclinical validation, leveraging standardized spheroid production and FLIM analytics.
- Discovery Biology: Supports hypothesis testing and pathway clarification in 3D, in vivo-like contexts.
- Screening: Delivers reproducible, quantitative metabolic and hypoxia readouts for compound triage.
- Analytics: Provides multiplexed fluorescence lifetime measurements for robust condition comparison.
- Translational Research: Bridges in vitro findings to preclinical models by recapitulating tumor microenvironment features.
- Enterprise Reuse: Establishes a reusable platform for diverse oncology and stem cell R&D programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in 3D model systems.
- Operational Value: Enhances standardization, reproducibility, and scalability of live-cell imaging workflows.
- Strategic Value: Improves go/no-go decisions and capital efficiency by providing robust, quantitative data.
- Portfolio Impact: Enables risk-adjusted prioritization and advancement of candidates with validated biological relevance.
Implementation Considerations
- Requires expertise in 3D cell culture, live-cell microscopy, and FLIM analytics.
- Demands access to widefield, confocal, or two-photon FLIM instrumentation and compatible analysis software.
- Necessitates cross-team standardization of spheroid production and imaging protocols.
- May require adaptation for different cell types, extracellular matrices, or sensor integration.
- Variability in spheroid size and composition can impact reproducibility and data interpretation.
Biopharma FAQs
Chapters in this video
0:00
Introduction
1:51
High‐Throughput Generation of Multicellular Spheroids from Human Cancer Cell Line for Live 3D Imaging
4:15
Assessing the Impact of Spheroid Formation Methods in 3D Cell Culture Systems Using Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM)
