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

描述(由申请人提供)：对细胞如何达到并维持给定的大小知之甚少。这种缺乏理解的主要原因是与细胞大小测量不精确、细胞同步产生的伪影和总体平均有关的技术限制。我们打算开发一种用于细胞大小调整的微型系统(MCS)，它将在一次飞跃中克服这三个技术限制。MCS将通过连续的模块使细胞循环：i)保持培养液中的pH、营养和气体平衡，ii)允许添加刺激物，iii)测量来自分子报告器的细胞荧光，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，将有可能对这些细胞进行超过24小时的监测。这些细胞将以单一的文件流动，从而允许跟踪单个细胞。环路内发生的细胞分裂将被识别，然后两个子细胞将被剪切流分开并单独跟踪。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。对于哺乳动物细胞来说，已经被广泛研究的G1期的分子生物学与尚未发现的G1期的基本生理学之间存在着显著的差距。目前还不清楚细胞的生长和大小如何与G1期的两个关键生理转变：限制点和退出G1期(G1/S转变)相联系。此外，G1相中大多数分子事件的相对顺序和相关性尚不清楚。凭借其连续测量生长、细胞周期时间和蛋白质的能力，MCS可能成为受欢迎的单细胞实验通用平台。例如，MCS可以使已知的分子事件(如活细胞中的绿色荧光蛋白融合和固定细胞中的免疫染色所报告的)与限制点、G1/S转变、生长曲线、细胞周期时间以及相互关联。在静止细胞的情况下，不知道大小是否保持得很好，或者是否表现出了正确的生长或自噬。MCS还有许多其他用途。例如，对细胞质量的精确测量可以帮助研究自噬，自噬的程度可能与细胞死亡决定有关。作为细胞新陈代谢的敏感指标，个体生长曲线也可以用来读出药物的靶向或非靶点效应。我们打算开发一种用于细胞大小调整的微型系统(MCS)，它将在一次飞跃中克服技术限制，这些限制扼杀了对细胞生物学中的一个经典问题的研究：细胞如何控制自己的大小。MCS将通过连续的模块使细胞循环，这些模块测量单个细胞的质量、密度和荧光，并提供营养和刺激。这些细胞将以单一的文件流动，从而允许跟踪单个细胞。环路内发生的细胞分裂将被识别，然后两个子细胞将被剪切流分开并单独跟踪。

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.