High accuracy optical growth assay of 3D cellular systems

3D 细胞系统的高精度光学生长测定

• 批准号: 10330571
• 负责人: Rashid Bashir
• 金额: $ 45.82万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10330571
• 关键词: 3-Dimensional; Adopted; Algorithms; Anatomy; Basic Science; Biological Assay; Biomedical Engineering; Cell Therapy; Cells; Cellular biology; Clinic; Clinical; Clinical Research; Collaborations; Coulter counter; Disease; Drug Targeting; Escherichia coli; Extracellular Matrix Proteins; Fluorescence; Fluorescence Microscopy; Growth; Image; Individual; Interference Microscopy; Kinetics; Label; Laboratories; Letters; Light; Mammalian Cell; Measurement; Measures; Methodology; Methods; Microscope; Microscopy; Multimodal Imaging; Neoplasm Metastasis; Nuclear; Optics; Organ; Organoids; Osmotic Pressure; Performance; Phase; Population; Process; Quantitative Microscopy; Regulation; Research; Research Personnel; Specimen; Subcellular structure; System; Techniques; Technology; Thick; Time; Tissue Model; Translating; Variant; Vision; Work; base; biomedical scientist; cell growth; drug development; human disease; human model; imaging modality; in vivo; instrument; interest; mathematical analysis; multidisciplinary; novel; screening; targeted treatment; tool

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.

项目摘要 哺乳动物细胞的生长调控被描述为“细胞中最后未解决的重大问题之一” 准确测量单个细胞的生长速度的能力一直是 回答这个问题。从临床角度来看，对细胞生长动力学及其如何 受疾病和治疗的调节，将允许更有针对性的药物开发。 近年来，生物医学工程师和研究人员对多学科工作产生了浓厚的兴趣 科学家的愿景是开发人体器官功能、解剖学和疾病的3D体外组织模型。 这些3D细胞系统可互换地称为有机体、器型或球体(球形 有机化合物)。有机化合物在适当的条件下自组装，即当相关组件，如 细胞外基质(ECM)蛋白的存在。有机化合物被很好地记录下来，以更好地概括各方面 体内器官功能和人类疾病的关系。分析这类系统的常用工具是共焦 固定样品的(荧光)显微镜。但是，此方法不会在 在结构的中心，最重要的是，在时间推移成像方面有限。有一个关键的问题 需要在无标记模式下以高对比度显示亚细胞结构，这允许动态、非 破坏性成像。同时，量化有机物质及其细胞成分的干质量将 告知基本器官功能和疾病，包括治疗和不治疗。 尽管这一迫切需要，一种统一的、易于使用的方法来测量单个细胞的生长速度和 目前还缺乏3D构造。直到最近，评估单细胞生长曲线的最先进方法是 使用库尔特计数器测量大量细胞的体积，并结合仔细 数学分析。对于相对简单的细胞，如大肠杆菌(E.coli)，传统显微镜 技术也被用来非常详细地评估增长。在这种方法中，假设是 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。 我们建议开发一种实用的2D细胞群体以及3D有机物的干质量分析方法， 基于我们实验室开发的一种新的成像方法：空间光干涉显微镜 (SLIM)用于2D培养和梯度光干涉显微镜(GLIM)用于3D有机物。纤细/纤细 利用通过活细胞积累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)成正比。由于其特殊的干涉测量 原理上，GLIM显著抑制多次散射，因此能够成像厚 有机体/球体等标本。该项目旨在优化和翻译复合材料 将SLIM/GLIM技术转化为可被研究人员广泛采用的细胞生长分析仪器 在研究和制药市场都是如此。

项目成果

Network science characteristics of brain-derived neuronal cultures deciphered from quantitative phase imaging data

• DOI: 10.1038/s41598-020-72013-7
• 发表时间: 2020-09
• 期刊: Scientific Reports
• 影响因子: 4.6
• 作者: Chenzhong Yin;Xiongye Xiao;Valeriu Balaban;M. Kandel;Y. J. Lee;G. Popescu;P. Bogdan

Cell-to-cell influence on growth in large populations

• DOI: 10.1364/boe.10.004664
• 发表时间: 2019-09-01
• 期刊: BIOMEDICAL OPTICS EXPRESS
• 影响因子: 3.4
• 作者: Kandel, Mikhail E.;Lu, Wenlong;Popescu, Gabriel
• 通讯作者: Popescu, Gabriel

Epi-illumination gradient light interference microscopy for imaging opaque structures

• DOI: 10.1038/s41467-019-12634-3
• 发表时间: 2019-10-16
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Kandel, Mikhail E.;Hu, Chenfei;Popescu, Gabriel
• 通讯作者: Popescu, Gabriel

Engineering geometrical 3-dimensional untethered in vitro neural tissue mimic

工程几何三维无束缚体外神经组织模拟

• DOI: 10.1073/pnas.1916138116
• 发表时间: 2019
• 期刊: Proceedings of the National Academy of Sciences
• 作者: Pagan-Diaz, Gelson J.;Ramos-Cruz, Karla P.;Sam, Richard;Kandel, Mikhail E.;Aydin, Onur;Saif, M. Taher;Popescu, Gabriel;Bashir, Rashid
• 通讯作者: Bashir, Rashid