Experiencing the micro-world - a cell's perspective

体验微观世界——细胞的视角

• 批准号: EP/R035563/1
• 负责人: Amanda Wright
• 金额: $ 76.55万
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FR035563%2F1
• 关键词: Experiencing; micro; world; cell; perspective

In the body, most cells grow in close contact with other neighbouring cells and with a local matrix of proteins and sugars that combine to provide an instructive microenvironment. Until recently, most research labs (in both academic and industrial settings) have used 2D cultures of cells on plastic to study cell behaviour, a significant departure from what is actually happening in vivo that can limit the applicability of their research. However, there has been a recent and dramatic shift away from traditional 2D culture to the use of complex, 3D cultures, that more effectively mimic the micro-environment experienced by cells in vivo. This development impacts directly on fields such as regenerative medicine, drug discovery and cancer research, with significant opportunities for improved in vitro modelling of cell behaviour. Despite these improvements in culture techniques, the interaction of the cells with their local microenvironment - a key target in therapies for cancer, wound healing, and fibrosis etc. - remains a 'black box' with technologies unable investigate these environments at the cell level. This proposal will 'open that box', developing the technology and methodology urgently required to fully explore 3D cell cultures on length scales comparable, or smaller than, single cells. The currently accepted protocol to characterise natural and synthetic matrices, uses a bulk rheometer to produce a single, averaged value of the viscosity and elasticity of the material, destroying the sample in the process. Information about the matrix local to the cells growing inside the samples is lost. Our vision is to image and characterise 3D cell culture environments in all three spatial dimensions, over an extended time course, and on a single multifunctional instrument so that the information can be integrated and mapped. To achieve this we will develop a minimally-invasive technique to measure the 3D micro-rheology of the extracellular matrix using nano- (smaller than the cells) and micro-sized (can be the same size at the cells) beads as local probes. These probes will be held at a fixed position within the matrix using an optical trap and their Brownian motion in all three spatial dimensions tracked using multiplane imaging. The micro-rheology (viscosity and elasticity) of the extracellular matrix local to the probe is extracted from temporal analysis of the Brownian motion. To achieve deep 4D (x,y,z, time) images of live 3D cell cultures, we will combine light sheet microscopy with adaptive optics (a technique for correcting for sample aberrations that reduce image quality deep into complex samples). The final multifunctional platform will be the exciting culmination of these 4 microscopy techniques - optical trapping, multiplane imaging, light sheet microscopy and adaptive optics - capable of imaging and micro-mechanically sensing the 3D environment close to cells. The output from this work will be the innovation required to allow scientists to study how cells interact with their local microenvironment, combining technologies in a way that's not been possible previously, to observe both the cells, and the forces they exert and are responding to, as they grow and move in 3D space over time. The ability to study cell behaviour in this way is of importance for developing therapies for diseases where cells respond abnormally to signals from their local matrix, such as cancer, providing targets for new drug design. We will include a demonstration of how this can work in our study using both traditional anti-cancer drugs and more innovative therapies such as functionalised nanoparticles. We anticipate that the technology will be useful to both academics and industry (particularly drug discovery in the pharmaceutical industry) and we will work closely with these groups throughout the course of this project to ensure that, once proven, this technology can work for them.

在体内，大多数细胞与其他邻近细胞以及蛋白质和糖的局部基质密切接触，这些基质结合联合收割机提供指导性的微环境。直到最近，大多数研究实验室（学术和工业环境）都使用塑料上的2D细胞培养物来研究细胞行为，这与体内实际发生的情况有很大的不同，这可能会限制他们研究的适用性。然而，最近从传统的2D培养到使用复杂的3D培养已经发生了巨大的转变，3D培养更有效地模拟了细胞在体内所经历的微环境。这一发展直接影响到再生医学、药物发现和癌症研究等领域，为改善细胞行为的体外建模提供了重要机会。尽管培养技术有了这些改进，但细胞与其局部微环境的相互作用-癌症，伤口愈合和纤维化等治疗的关键目标-仍然是一个“黑匣子”，技术无法在细胞水平上研究这些环境。该提案将“打开那个盒子”，开发出迫切需要的技术和方法，以充分探索长度尺度与单细胞相当或更小的3D细胞培养。目前公认的天然和合成基质测试方案使用体积流变仪来产生材料粘度和弹性的单一平均值，在此过程中破坏样品。关于在样品内生长的细胞的局部基质的信息丢失。我们的愿景是在所有三个空间维度上，在延长的时间过程中，在单个多功能仪器上对3D细胞培养环境进行成像和建模，以便可以整合和映射信息。为了实现这一目标，我们将开发一种微创技术，使用纳米（小于细胞）和微米（可以与细胞大小相同）珠作为局部探针来测量细胞外基质的3D微流变学。这些探针将使用光阱保持在基质内的固定位置，并且使用多平面成像跟踪它们在所有三个空间维度中的布朗运动。从布朗运动的时间分析中提取探针局部的细胞外基质的微观流变学（粘度和弹性）。为了实现活3D细胞培养物的深度4D（x，y，z，时间）图像，我们将联合收割机与自适应光学（一种用于校正样品像差的技术，该技术会降低复杂样品的图像质量）相结合。最终的多功能平台将是这4种显微镜技术的令人兴奋的高潮-光学捕获，多平面成像，光片显微镜和自适应光学-能够成像和微机械感测靠近细胞的3D环境。这项工作的成果将是所需的创新，使科学家能够研究细胞如何与其局部微环境相互作用，以一种以前不可能的方式结合技术，观察细胞以及它们施加和响应的力量，随着时间的推移，它们在3D空间中生长和移动。以这种方式研究细胞行为的能力对于开发疾病的治疗方法非常重要，这些疾病的细胞对来自其局部基质的信号反应异常，例如癌症，为新药设计提供了靶点。我们将在我们的研究中使用传统抗癌药物和功能化纳米颗粒等更具创新性的疗法来演示这一点。我们预计该技术将对学术界和工业界（特别是制药行业的药物发现）都有帮助，我们将在整个项目过程中与这些团体密切合作，以确保一旦得到证明，该技术可以为他们工作。

项目成果

Living Cells as a Biological Analog of Optical Tweezers -- a Non-Invasive Microrheology Approach

活细胞作为光镊的生物模拟——一种非侵入性微流变学方法

• DOI: 10.48550/arxiv.2211.14189
• 发表时间: 2022
• 作者: Hardiman W

Compressional stress stiffening & softening of soft hydrogels - how to avoid artefacts in their rheological characterisation

压缩应力硬化

• DOI: 10.1039/d3sm00077j
• 发表时间: 2023
• 期刊: Soft Matter
• 影响因子: 3.4
• 作者: Ferraro R

Microrheology reveals microscale viscosity gradients in planktonic systems.

• DOI: 10.1073/pnas.2011389118
• 发表时间: 2021-01-05
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Guadayol Ò;Mendonca T;Segura-Noguera M;Wright AJ;Tassieri M;Humphries S
• 通讯作者: Humphries S