High accuracy optical growth assay of 3D cellular systems

项目摘要 哺乳动物细胞的生长调节被描述为“细胞生物学中最后一个未解决的大问题之一”。 生物学”。准确测量单细胞生长速率的能力一直是研究的主要障碍。 回答这个问题。从临床的角度来看，细胞生长动力学的基本理解以及它是如何 通过疾病和治疗的调节将允许更有针对性的药物开发。 近年来，生物医学工程师对多学科工作产生了浓厚的兴趣， 科学家们的愿景是开发人体器官功能、解剖学和疾病的3D离体组织模型。 这些3D细胞系统可互换地称为类器官、器官型或球状体（球形 类器官）。类器官在适当的条件下自组装，即，当相关组件，如 细胞外基质（ECM）蛋白。类器官被很好地记录，以更好地概括方面 体内器官功能和人类疾病的关系。共聚焦是分析这类系统的常用工具 固定标本的（荧光）显微镜检查。然而，这种方法并没有揭示结构信息， 最重要的是，在延时成像方面受到限制。存在一个临界 需要以高对比度的无标记模式揭示亚细胞结构，这允许动态的，非 破坏性成像同时，定量类器官及其细胞组分的干质量将有助于 告知基本器官功能和疾病，治疗和不治疗。 尽管有这种迫切的需求，一个统一的，易于使用的方法来测量单个细胞的生长速度， 缺乏3D结构。直到最近，评估单细胞生长曲线的最先进方法仍然是 使用库尔特计数器测量大量细胞的体积，结合仔细的 数学分析对于相对简单的细胞，如大肠杆菌（E.大肠杆菌），传统显微镜 技术也被用来详细评估增长。在这种类型的方法中，假设 体积是质量的一个很好的替代;然而，这个假设并不总是有效的，例如，由于 渗透压的变化。 我们建议开发一种用于2D细胞群以及3D类器官的实用干质量测定法， 基于我们实验室开发的一种新的成像方法：空间光干涉显微术 梯度光干涉显微镜（GLIM）用于3D类器官。SLIM/GLIM 利用了通过活细胞累积的光学相位延迟是线性的这一事实， 与细胞的干质量（非水含量）成比例。由于其特殊的干涉 根据这一原理，GLIM显著抑制了多次散射，因此能够成像厚的 标本如类器官/球状体。该项目旨在优化和翻译复合材料 SLIM/GLIM技术转化为可被研究人员广泛采用的细胞生长测定仪器 在研究和制药市场上。

Project Summary Growth regulation of mammalian cells has been described as "One of the last big unsolved problems in cell biology". The ability to measure accurately the growth rate of single cells has been the main obstacle in answering this question. From a clinical perspective, the basic understating of cell growth kinetics and how it is modulated by disease and treatment will allow for more targeted drug development. In recent years, there has been a significant interest in multidisciplinary work by biomedical engineers and scientists with a vision of developing 3D ex vivo tissue models of human organ function, anatomy, and disease. These 3D cellular systems are referred interchangeably as organoid, organotypic, or spheroid (spherical organoid). Organoids self-assemble under proper conditions, i.e., when relevant components, such as extracellular matrix (ECM) proteins, are present. Organoids are well documented to better recapitulate aspects of in vivo organ function and human disease. The common tool for analysis of such systems has been confocal (fluorescence) microscopy of fixed specimens. However, this approach does not reveal structural information in the center of the construct and, most importantly, is limited in terms of time-lapse imaging. There is a critical need for revealing subcellular structures in label-free mode with high contrast, which allows for dynamic, non- destructive imaging. At the same time, quantifying the dry mass of the organoid and its cellular components will inform on the basic organ function and disease, with and without treatment. Despite this critical need, a unified, easy-to-use methodology to measure the growth rate of individual cells and 3D constructs is lacking. Until recently, the state-of-the-art method to assess a single cell growth curve was using Coulter counters to measure the volume of a large number of cells, in combination with careful mathematical analysis. For relatively simple cells such as Escherichia coli (E. coli), traditional microscopy techniques have also been used to assess growth in great detail. In this type of method the assumption is that volume is a good surrogate for mass; however, this assumption is not always valid, for example due to variations in osmotic pressure. We propose to develop a practical dry mass assay for 2D cell populations, as well as 3D organoids, based on a novel imaging method developed in our laboratory: Spatial Light Interference Microscopy (SLIM) for 2D cultures and Gradient Light Interference Microscopy (GLIM) for 3D organoids. SLIM/GLIM takes advantage of the fact that optical phase delay accumulated through a live cell is linearly proportional to the dry mass (non-aqueous content) of the cell. Due to its particular interferometric principle, GLIM significantly suppresses multiple scattering and, as result, is capable of imaging thick specimens such as organoid/spheroids. The project aims to optimize and translate the composite SLIM/GLIM technology into a cell growth assay instrument that can be broadly adopted by researchers in both the research and pharma markets.