High throughput monitoring of mass, density and fluorescence of single cells

描述（由申请人提供）：对细胞如何达到并保持给定大小的理解很少。这种理解的缺乏主要是由于与细胞大小的不精确测量、来自同步细胞的伪影和群体平均相关的技术限制。我们打算开发一种用于细胞大小测量的微系统（MCS），它将在一个飞跃中克服这三个技术限制。MCS将使细胞循环通过连续模块，i）维持培养基中的pH值、营养物和气体平衡，ii）允许添加刺激物，iii）测量来自分子报告物的细胞荧光，以及iv）测量质量和质量密度，从而测量体积。大约100个细胞将通过该系统，并且可以对这些细胞进行超过24小时的监测。细胞将在单个文件中流动，允许跟踪单个细胞。将识别环内发生的细胞分裂，然后通过剪切流分离两个子细胞并单独跟踪。细胞生长和G1期之间的关系是理解细胞周期的核心，对肿瘤学有重要的影响。对于哺乳动物细胞，G1期的分子生物学和G1期的基本生理学之间存在着显著的差距，G1期的分子生物学已经被广泛研究，而G1期的基本生理学还没有被广泛研究。目前还不了解细胞生长和大小如何与G1期的两个关键生理转变：限制点和退出G1期（G1/S转变）。此外，G1期大部分分子事件的相对顺序和依赖性尚不清楚。凭借其连续测量生长、细胞周期时间和蛋白质的能力，MCS可能成为单细胞实验的通用平台。例如，MCS可以使已知的分子事件（如活细胞中的GFP融合和固定细胞中的免疫染色所报告的）与限制点、G1/S转换、生长曲线、细胞周期时间以及彼此相关。在静止细胞的情况下，不知道是否完全保持大小或是否表现出生长或自噬的校正事件。MCS还有许多其他用途。例如，精确测量细胞质量可以帮助研究自噬，其程度可能与细胞死亡决定有关。作为细胞代谢的敏感指标，个体生长曲线也可用作药物的靶向或脱靶效应的读数。我们打算开发一种用于细胞大小测量的微系统（MCS），它将在一次飞跃中克服技术限制，这些限制阻碍了对细胞生物学中一个经典问题的研究：细胞如何控制它们的大小。MCS将使细胞通过连续的模块循环，这些模块测量单细胞质量、密度和荧光，并提供营养和刺激。细胞将在单个文件中流动，允许跟踪单个细胞。将识别环内发生的细胞分裂，然后通过剪切流分离两个子细胞并单独跟踪。

DESCRIPTION (provided by applicant): There is little understanding of how cells achieve and then maintain a given size. This dearth of understanding is due primarily to technical limitations related to imprecise measures of cell size, artifacts from synchronizing cells, and population averaging. We intend to develop a microsystem for cell sizing (MCS) that will - in a single leap - overcome these three technical limitations. The MCS will circulate cells through consecutive modules that i) maintain the pH, nutrient, and gas balance in the medium, ii) allow for the addition of stimuli, iii) measure cell fluorescence from molecular reporters, and iv) measure mass and mass density, and thus volume. Approximately 100 cells will pass through the system and it will be possible to monitor these cells for more than 24 hours. The cells will flow in single file, allowing individual cells to be tracked. Cell divisions that occur within the loop will be identified and the two daughter cells will then be separated by shear flow and tracked individually. The relationship between cell growth and G1 phase is central to understanding the cell cycle and has important ramifications for oncology. For mammalian cells, remarkable gaps exist between the molecular biology of G1 phase, which has been extensively explored, and the basic physiology of G1 phase, which has not. There is currently no understanding of how cell growth and size interface with the two key physiological transitions of G1 phase: the Restriction Point and the exit from G1 phase (G1/S transition). Furthermore, the relative order and dependency of most of the molecular events in G1 phase is not known. By virtue of its capability to continuously measure growth, cell cycle time, and proteins, the MCS could become a popular general platform for single cell experimentation. For instance, the MCS could enable known molecular events (as reported by GFP fusions in live cells and immunostaining in fixed cells) to be correlated to the Restriction Point, G1/S transition, growth curve, cell cycle time, and each other. In the case of quiescent cells, it is not known if size is maintained perfectly or if corrective episodes of growth or autophagy are exhibited. There are many additional uses of the MCS. For instance, precise measurements of cell mass could aid the study of autophagy, the extent of which may be related to cell death decisions. As a sensitive indicator of cell metabolism, individual growth curves could also be used as a read-out for the on-target or off-target effects of drugs. We intend to develop a microsystem for cell sizing (MCS) that will - in a single leap - overcome technical limitations that have stifled research into a classic problem in cell biology: how cells control their size. The MCS will circulate cells through consecutive modules that measure single cell mass, density and fluorescence, and provide nutrients and stimuli. The cells will flow in single file, allowing individual cells to be tracked. Cell divisions that occur within the loop will be identified and the two daughter cells will then be separated by shear flow and tracked individually.