Experiencing the micro-world - a cell's perspective

在体内，大多数细胞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在一起，提供一个有益的微环境。直到最近，大多数研究实验室(无论是在学术还是工业环境中)都在使用塑料上的2D细胞培养来研究细胞行为，这与体内实际发生的情况严重背道而驰，这可能会限制他们的研究的适用性。然而，最近发生了戏剧性的转变，从传统的2D培养转向使用复杂的3D培养，这种培养更有效地模拟了细胞在体内经历的微环境。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，但细胞与其局部微环境的相互作用--癌症、伤口愈合和纤维化等治疗中的关键靶点--仍然是一个“黑匣子”，技术无法在细胞水平上研究这些环境。这项提议将“打开那个盒子”，开发出在与单个细胞相当或更小的长度尺度上全面探索3D细胞培养所迫切需要的技术和方法。目前被接受的表征天然和合成基质的协议使用散装流变仪来产生材料的粘度和弹性的单一平均值，在此过程中破坏样品。有关生长在样本内的细胞的局部基质的信息会丢失。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，在单一的多功能仪器上对3D细胞培养环境进行图像和特征描述，以便可以集成和绘制信息。为了实现这一点，我们将开发一种微创技术，使用纳米(比细胞小)和微米(细胞大小可以相同)的珠子作为局部探针来测量细胞外基质的3D微流变学。这些探测器将被固定在基质中的固定位置，使用光学陷阱，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了探针局部细胞外基质的微流变学(粘性和弹性)。为了获得活的3D细胞培养的深度4D(x，y，z，时间)图像，我们将结合光片显微镜和自适应光学(一种用于校正深度到复杂样本的图像质量的样本像差的技术)。最终的多功能平台将是这4种显微技术-光学陷阱、多平面成像、光片显微镜和自适应光学-的激动人心的顶峰，能够成像和以微机械方式感知细胞附近的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，观察细胞及其施加和响应的力，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，对于开发细胞对局部基质信号做出异常反应的疾病的治疗方法具有重要意义，例如癌症，为新药设计提供靶点。我们将在我们的研究中包括如何使用传统抗癌药物和更多创新疗法(如功能化纳米颗粒)来演示这一点。我们预计这项技术将对学术界和产业界都有用(特别是制药业的药物发现)，我们将在整个项目过程中与这些小组密切合作，以确保一旦得到证实，这项技术可以为他们工作。

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.