Key Laboratory of MEMS of Ministry of Education, Southeast University, Sipailou 2, Nanjing, 210096, Jiangsu, China.
School of Pharmaceutical Science and Technology, Tianjin University, Weijin Road 92, Tianjin, 300072, China.
Anal Bioanal Chem. 2021 Mar;413(8):2181-2193. doi: 10.1007/s00216-021-03186-x. Epub 2021 Jan 31.
Microfluidic methodologies allow for automatic and high-throughput replicative lifespan (RLS) determination of single budding yeast cells. However, the resulted RLS is highly impacted by the robustness of experimental conditions, especially the microfluidic yeast-trapping structures, which are designed for cell retention, growth, budding, and daughter cell dissection. In this work, four microfluidic yeast-trapping structures, which were commonly used to immobilize mother cells and remove daughter cells for entire lifespan of budding yeast, were systematically investigated by means of finite element modeling (FEM). The results from this analysis led us to propose an optimized design, the yeast rotation (YRot) trap, which is a "leaky bowl"-shaped structure composed of two mirrored microcolumns facing each other. The YRot trap enables stable retention of mother cells in its "bowl" and hydrodynamic rotation of buds into its "leaky orifice" such that matured progenies can be dissected in a coincident direction. We validated the functions of the YRot trap in terms of cell rotation and daughter dissection by both FEM simulations and experiments. With the integration of denser YRot traps in microchannels, the microfluidic platform with stable single-yeast immobilization, long-term cell culturing, and coincident daughter dissection could potentially improve the robustness of experimental conditions for precise RLS determination in yeast aging studies.
微流控方法允许自动且高通量地测定单个出芽酵母细胞的复制寿命 (RLS)。然而，得到的 RLS 高度受到实验条件的稳健性的影响，特别是用于细胞保留、生长、出芽和子细胞分离的微流控酵母捕获结构。在这项工作中，通过有限元建模 (FEM) 系统地研究了用于固定母细胞并去除子细胞以完成整个出芽酵母寿命的四种常用微流控酵母捕获结构。该分析的结果使我们提出了一种优化设计，即酵母旋转 (YRot) 阱，它是一种由两个彼此相对的镜像微柱组成的“漏碗”形结构。YRot 阱能够在其“碗”中稳定地保留母细胞，并使芽体在其“漏口”中进行流体动力旋转，从而可以在一致的方向上分离成熟的后代。我们通过有限元模拟和实验验证了 YRot 阱在细胞旋转和子细胞分离方面的功能。通过在微通道中集成更密集的 YRot 阱，具有稳定的单个酵母固定、长期细胞培养和一致的子细胞分离的微流控平台有可能提高实验条件的稳健性，从而在酵母衰老研究中更精确地确定 RLS。
