Experiencing the micro-world - a cell's perspective

在体内，大多数细胞生长与其他邻近细胞密切接触，并与蛋白质和糖的局部基质结合，提供一个有益的微环境。直到最近，大多数研究实验室（包括学术和工业环境）都使用塑料上的二维细胞培养来研究细胞行为，这与体内实际发生的情况有很大的不同，这限制了他们研究的适用性。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，为改进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靶点——仍然是一个“黑盒子”，技术无法在细胞水平上研究这些环境。该提案将“打开这个盒子”，开发迫切需要的技术和方法，以充分探索与单细胞相当或更小的长度尺度的3D细胞培养。目前公认的表征天然和合成基质的方法是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。在样品中生长的细胞所处的基质的信息丢失了。我们的愿景是在所有三个空间维度上对3D细胞培养环境进行成像和表征，在一个延长的时间过程中，在一个多功能仪器上，以便信息可以集成和映射。为了实现这一目标，我们将开发一种微创技术来测量细胞外基质的3D微流变，使用纳米（小于细胞）和微尺寸（与细胞大小相同）的珠子作为局部探针。这些探针将使用光学陷阱保持在矩阵内的固定位置，并使用多平面成像跟踪它们在所有三个空间维度上的布朗运动。从布朗运动的时间分析中提取探针局部细胞外基质的微流变性（粘度和弹性）。为了获得活体3D细胞培养物的深4D （x,y,z，时间）图像，我们将结合光片显微镜与自适应光学（一种校正样品像差的技术，可降低复杂样品的图像质量）。最终的多功能平台将是这4种显微镜技术——光学捕获、多平面成像、光片显微镜和自适应光学——令人兴奋的高潮，能够成像和微机械地感知细胞附近的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，以及它们施加的力和对它们的反应，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针对细胞对来自其局部基质的信号做出异常反应的疾病（如癌症）的治疗方法具有重要意义，为新药设计提供了靶点。我们将在我们的研究中展示如何使用传统的抗癌药物和更创新的疗法，如功能化纳米颗粒。我们预计这项技术将对学术界和工业界都有用（特别是制药行业的药物发现），我们将在整个项目过程中与这些团体密切合作，以确保一旦得到证实，这项技术可以为他们服务。

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.