酿酒酵母单细胞复制衰老过程中表型分析的高性能微流控三维光电表征方法研究

Saccharomyces cerevisiae, is an ideal model organism to study the mechanism of aging. Although the phenotypic dynamics during yeast aging significantly impact cellular longevity, methodologies of cell aging research are still under developing to obtain high-throughput, high-accuracy, and three-dimensional (3D) phenotypic dynamics of cells during yeast replicative aging. Herein, we propose a microfluidic method, which enables high-throughput single-cell immobilization, culturing, and dissection, as well as provides high-accuracy measurements of cellular phenotypes with an integrated 3D optical-electronic method. Thus, cellular phenotypic characteristics about cell volume, cell cycle and growth rate during the entire life of yeast (from new born to dead) can be gained, and the correlation between these characteristics and yeast replicative lifespan can be established. .Novelties of this project, in detail, consists of: (1) a microfluidic yeast-dissection device, which features a baby machine to produce virgin cells, reliable single-cell immobilization and culturing, efficient dissection of yeast buds, and complete recording of cell replicative lifespan; (2) a combined method between high-resolution optical imaging and single-cell electrical impedance spectroscopy to perform 3D real-time dynamic recording of cellular phenotypic characteristics during yeast replicative aging, and to obtain cellular phenotypic dynamics about cell volume, cell cycle and growth rate with high accuracy; (3) analyzing the variation of phenotypic dynamics along the replicative aging of S. cerevisiae, and uncovering the correlation mechanisms between these phenotypic characteristics and yeast replicative aging and, thereby conducting a powerful methodology for further study of cell aging.

酿酒酵母是研究生物体衰老机制的主要模式细胞，酵母细胞表型特征与其复制寿命密切相关。现有技术手段无法为酵母细胞复制衰老的研究提供高通量、高精准性、三维动态的细胞表型特征参数信息。本项目拟提出一种微流控方法，进行高性能的酵母单细胞捕获、培养、解剖、以及对细胞表型特征的精准三维光电表征，获得细胞从新生到死亡完整复制衰老过程中的细胞体积、细胞周期、生长速率及其动态变化等信息，建立其与酵母复制寿命之间的相关性。具体包括：①研制微流控解剖芯片，实现新生细胞生产单元的集成、稳定的细胞捕获培养、高效的子细胞去除、完整的复制寿命检测；②结合高分辨率光学显微成像与电阻抗谱单细胞检测技术，实现对细胞表型特征的三维光电实时检测，获得精准的细胞体积、细胞周期、生长速率等动态信息；③分析并揭示酿酒酵母细胞表型特征参数及其动态变化与细胞复制衰老之间的影响规律，进而为细胞衰老机制的深入研究提供一种高效的方法。

针对高性能酿酒酵母单细胞捕获、培养、解剖的微流控方法，以及对细胞复制寿命的精准三维光电表征和细胞表型分析的研究目标，本项目主要设计研制了用于酿酒酵母细胞复制寿命及衰老过程光、电信号分析的微流控芯片及外部电学系统，实现了细胞表型特征的参数提取和复制寿命的完整追踪，并分析了两者的关联及影响。具体包括：①研制了用于高通量酿酒酵母细胞单细胞捕获-培养-解剖的微流控芯片，实现了稳定的细胞捕获培养、高效的子细胞去除、完整的复制寿命检测；②开发了一套细胞复制衰老分析和表型特征参数提取的软件系统。针对高通量酿酒酵母光学成像数据，实现了细胞周期、细胞截面积/体积以及复制寿命的自动化提取分析；③构建了一套高通量单细胞复制寿命电阻抗检测系统，开发了微电极阵列与酵母单细胞捕获芯片集成的微加工工艺，研制了电阻抗检测的控制、检测电路及实时交互控制软件系统，实现了单细胞时序宽频电阻抗特性检测；④实现了细胞表型特征参数及其动态变化与复制衰老的关联性研究。本项目揭示了酿酒酵母细胞表型特征参数及其动态变化与细胞复制衰老之间的影响规律，作为一套通用型平台将有助于推进细胞衰老机制的深入研究。