Abstract：
BACKGROUND: Budding yeast, Saccharomyces cerevisiae, has been extensively favored as a model organism in aging and age-related studies, thanks to versatile microfluidic chips for cell dynamics assay and replicative lifespan (RLS) determination at single-cell resolution. However, previous microfluidic structures aiming to immobilize haploid yeast may impose excessive spatial constraint and mechanical stress on cells, especially for larger diploid cells that sprout in a bipolar pattern.
RESULTS: We developed a high-throughput microfluidic chip for diploid yeast long-term culturing (DYLC), optical inspection and cell-aging analysis. The DYLC chip features 1100 \"leaky bowl\"-shaped traps formatted in an array to dock single cells under laminar-perfused medium and effectively remove daughter cells by hydraulic shear forces. The delicate microstructures of cell traps enable hydrodynamic rotation of newborn buds, so as to ensure bud reorientation towards downstream and concerted daughter dissection thereafter. The traps provide sufficient space for cell-volume enlargement during aging, and thus properly alleviate structural compression and external stress on budding yeast. Trapping efficiency and long-term maintenance of single cells were optimized according to computational fluid dynamics simulations and experimental characterization in terms of critical parameters of the trap and array geometries. Owing to the self-filling of daughter cells dissected from traps upstream, an initial trapping efficiency of about 70% can rapidly reach a high value of over 92% after 4-hour cell culturing. During yeast proliferation and aging, cellular processes of growth, budding and daughter dissection were continuously tracked for over 60 h by time-lapse imaging. Yeast RLS and budding time interval (BTI) were directly calculated by the sequential two-digit codes indicating the budding status in images. With the employed diploid yeast strain, we obtained an RLS of 24.29 ± 3.65 generations, and verified the extension of BTI in the first couple of generations after birth and the last several generations approaching death, as well as cell de-synchronization along diploid yeast aging.
CONCLUSIONS: The DYLC chip offers a promising platform for reliable capture and culturing of diploid yeast cells and for life-long tracking of cell dynamics and replicative aging processes so that grasping comprehensive insights of aging mechanism in complex eukaryotic cells.
RESULTS: We developed a high-throughput microfluidic chip for diploid yeast long-term culturing (DYLC), optical inspection and cell-aging analysis. The DYLC chip features 1100 \"leaky bowl\"-shaped traps formatted in an array to dock single cells under laminar-perfused medium and effectively remove daughter cells by hydraulic shear forces. The delicate microstructures of cell traps enable hydrodynamic rotation of newborn buds, so as to ensure bud reorientation towards downstream and concerted daughter dissection thereafter. The traps provide sufficient space for cell-volume enlargement during aging, and thus properly alleviate structural compression and external stress on budding yeast. Trapping efficiency and long-term maintenance of single cells were optimized according to computational fluid dynamics simulations and experimental characterization in terms of critical parameters of the trap and array geometries. Owing to the self-filling of daughter cells dissected from traps upstream, an initial trapping efficiency of about 70% can rapidly reach a high value of over 92% after 4-hour cell culturing. During yeast proliferation and aging, cellular processes of growth, budding and daughter dissection were continuously tracked for over 60 h by time-lapse imaging. Yeast RLS and budding time interval (BTI) were directly calculated by the sequential two-digit codes indicating the budding status in images. With the employed diploid yeast strain, we obtained an RLS of 24.29 ± 3.65 generations, and verified the extension of BTI in the first couple of generations after birth and the last several generations approaching death, as well as cell de-synchronization along diploid yeast aging.
CONCLUSIONS: The DYLC chip offers a promising platform for reliable capture and culturing of diploid yeast cells and for life-long tracking of cell dynamics and replicative aging processes so that grasping comprehensive insights of aging mechanism in complex eukaryotic cells.
摘要：
背景：萌芽酵母，酿酒酵母，在衰老和与年龄相关的研究中被广泛用作模型生物，由于多功能微流控芯片的细胞动力学分析和复制寿命（RLS）在单细胞分辨率测定。然而,先前旨在固定单倍体酵母的微流体结构可能会对细胞施加过度的空间约束和机械应力，特别是对于以双极模式发芽的较大的二倍体细胞。
结果：我们开发了一种用于二倍体酵母长期培养（DYLC）的高通量微流控芯片，光学检查和细胞老化分析。DYLC芯片具有1100“漏碗”形陷阱，该陷阱以阵列形式格式化，可将单细胞停靠在层状灌注培养基下，并通过液压剪切力有效地去除子细胞。细胞陷阱的微妙微观结构使新生芽的流体动力旋转，以确保芽向下游重新定向，并在此后进行协调的女儿解剖。陷阱为老化过程中的细胞体积扩大提供了足够的空间，从而适当地减轻出芽酵母的结构压缩和外部应力。根据计算流体动力学模拟和实验表征，根据陷阱和阵列几何形状的关键参数，优化了单个细胞的捕获效率和长期维护。由于从上游陷阱解剖的子细胞的自我填充，约70%的初始捕获效率在4小时细胞培养后可迅速达到超过92%的高值。在酵母增殖和老化过程中，细胞生长过程，通过延时成像连续跟踪出芽和女儿夹层超过60小时。酵母RLS和出芽时间间隔（BTI）是通过连续的两位数代码直接计算的，这些代码指示了图像中的出芽状态。使用二倍体酵母菌株，我们获得了24.29±3.65代的RLS，并验证了BTI在出生后的前几代和接近死亡的最后几代中的延伸，以及二倍体酵母老化过程中的细胞去同步。
结论：DYLC芯片提供了一个有前途的平台，可以可靠地捕获和培养二倍体酵母细胞，并可以终身跟踪细胞动力学和复制性衰老过程，从而掌握复杂真核细胞衰老机制的全面见解。
结果：我们开发了一种用于二倍体酵母长期培养（DYLC）的高通量微流控芯片，光学检查和细胞老化分析。DYLC芯片具有1100“漏碗”形陷阱，该陷阱以阵列形式格式化，可将单细胞停靠在层状灌注培养基下，并通过液压剪切力有效地去除子细胞。细胞陷阱的微妙微观结构使新生芽的流体动力旋转，以确保芽向下游重新定向，并在此后进行协调的女儿解剖。陷阱为老化过程中的细胞体积扩大提供了足够的空间，从而适当地减轻出芽酵母的结构压缩和外部应力。根据计算流体动力学模拟和实验表征，根据陷阱和阵列几何形状的关键参数，优化了单个细胞的捕获效率和长期维护。由于从上游陷阱解剖的子细胞的自我填充，约70%的初始捕获效率在4小时细胞培养后可迅速达到超过92%的高值。在酵母增殖和老化过程中，细胞生长过程，通过延时成像连续跟踪出芽和女儿夹层超过60小时。酵母RLS和出芽时间间隔（BTI）是通过连续的两位数代码直接计算的，这些代码指示了图像中的出芽状态。使用二倍体酵母菌株，我们获得了24.29±3.65代的RLS，并验证了BTI在出生后的前几代和接近死亡的最后几代中的延伸，以及二倍体酵母老化过程中的细胞去同步。
结论：DYLC芯片提供了一个有前途的平台，可以可靠地捕获和培养二倍体酵母细胞，并可以终身跟踪细胞动力学和复制性衰老过程，从而掌握复杂真核细胞衰老机制的全面见解。
