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Cells can respond and adapt to complex forms of environmental change. Budding yeast is widely used as a model system for these stress response studies. In these studies, the precise control of the environment with high temporal resolution is most important. However, there is a lack of single-cell research platforms that enable precise control of the temperature and form of cell growth. This has hindered our understanding of cellular coping strategies in the face of diverse forms of temperature change. Here, we developed a novel temperature-controlled microfluidic platform that integrates a microheater (using liquid metal) and a thermocouple (liquid metal vs. conductive PDMS) on a chip. Three forms of temperature changes (step, gradient, and periodical oscillations) were realized by automated equipment. The platform has the advantages of low cost and a simple fabrication process. Moreover, we investigated the nuclear entry and exit behaviors of the transcription factor Msn2 in yeast in response to heat stress (37 °C) with different heating modes. The feasibility of this temperature-controlled platform for studying the protein dynamic behavior of yeast cells was demonstrated. [ABSTRACT FROM AUTHOR]

Aging is a major risk factor for chronic diseases and disability in humans. Nowadays, no effective anti-aging treatment is available clinically. In this study, oridonin was selected based on the drug screening strategy similar to Connectivity MAP (CMAP) but upon transcriptomes of 102 traditional Chinese medicines treated cell lines. Oridonin is a diterpenoid isolated from Rabdosia rubescens. As reported, Oridonin exhibits a variety of pharmacological activities, including antitumor, antibacterial and anti-inflammatory activities. Here, we found that oridonin inhibited cellular senescence in human diploid fibroblasts (2BS and WI-38), manifested by decreased senescence-associated β-galactosidase (SA-β-gal) staining. Compared with the elderly control group, the positive cell rate in the oridonin intervention group was reduced to 48.5%. Notably, oridonin prolonged the lifespan of yeast by 48.9%, and extended the average life span of naturally aged mice by 21.6%. Our mice behavior experiments exhibited that oridonin significantly improved the health status of naturally aged mice. In addition, oridonin also delayed doxorubicin-induced cellular senescence and mouse senescence. Compared with the model group, the percentage of SA-β-gal positive cells in the oridonin treatment group was reduced to 59.8%. It extended the average lifespan of mice by 53.8% and improved healthspan. Mechanistically, we showed that oridonin delayed aging through the AKT signaling pathway and reversed the genetic changes caused by doxorubicin-induced cell senescence. Therefore, oridonin is a potential candidate for the development of anti-aging drugs. [ABSTRACT FROM AUTHOR]

Yeast studies usually focus on exploring diversity in terms of a specific trait (such as growth rate, antibiotic resistance, or fertility) among extensive strains. Microfluidic chips improve these biological studies in a manner of high throughput and high efficiency. For a population study of yeast, it is of great significance to set a proper initial cell density for every strain under specific circumstances. Herein, we introduced a novel design of chip, which enables users to load cells in a gradient order (six alternatives) of initial cell density within one channel. We discussed several guidelines to choose the appropriate chamber to ensure successful data recording. With this chip, we successfully studied the growth rate of yeast strains under a mating response, which is crucial for yeasts to control growth behaviors for prosperous mating. We investigated the growth rate of eight different yeast strains under three different mating pheromone levels (0.3 μM, 1 μM, and 10 μM). Strains with, even, a six-fold in growth rate can be recorded, with the available data produced simultaneously. This work has provided an efficient and time-saving microfluidic platform, which enables loading cells in a pattern of multi-cell densities for a yeast population experiment, especially for a high-throughput study. Besides, a quantitatively analyzed growth rate of different yeast strains shall reveal inspiring perspectives for studies concerning yeast population behavior with a stimulated mating pheromone. [ABSTRACT FROM AUTHOR]

Current microfluidic methods for studying multicell strains (e.g. m-types) with multienvironments (e.g. n-types) require large numbers of inlets/outlets (m*n), a complicated procedure or expensive machinery. Here, we developed a novel two-layer-integrated method to combine different PDMS microchannel layers with different functions into one chip by a PDMS through-hole array, which improved the design of a PDMS-based microfluidic system. Using this method, we succeeded in converting 2 × m × n inlets/outlets into m + n inlets/outlets and reduced the time cost of loading processing (from m × n to m) of the device for studying multicell strains (e.g. m-types) in varied multitemporal environments (i.e. n-types). Using this device, the dynamic behavior of the cell-stress-response proteins was studied when the glucose concentration decreased from 2% to a series of lower concentrations. Our device could also be widely used in high-throughput studies of various stress responses, and the new concept of a multilayer-integrated fabrication method could greatly improve the design of PDMS-based microfluidic systems. [ABSTRACT FROM AUTHOR]

Among all the cell cycle synchronization technologies, the baby machine may be considered as the most artifact-free method. A baby machine incubates “mother cells” under normal conditions and collects their “babies”, producing cell cultures that are similar not only in cell cycle phase but also in age. Unlike many other synchronization methods, no cell-cycle-blocking agent or metabolic stress is introduced in this method. Several macroscale and microfluidic baby machines have been developed for producing synchronized cell colonies. However, for rod-shaped cells like fission yeast (Schizosaccharomyces pombe), it is still a challenge to immobilize only the mother cells in a microfluidic device. Here we presented a new baby machine suitable for fission yeast. The device is fixed one end of the cell and releases the free-end daughter cell every time the cell finishes cytokinesis. A variety of structures for cell immobilization were attempted to find the optimal design. For the convenience of collection and further assay, we integrated into our baby machine chip a cell screener, which exploited the deformation of polymer material to switch between opening and closing states. Synchronous populations of fission yeast cells were produced with this device, its working detail was analyzed and performance was evaluated. The device provides a new on-chip tool for cell biology studies. [ABSTRACT FROM AUTHOR]

Budding yeast has served as an important model organism for aging research, and previous genetic studies have led to the discovery of conserved genes/pathways that regulate lifespan across species. However, the molecular causes of aging and death remain elusive, because it is very difficult to directly observe the cellular and molecular events accompanying aging in single yeast cells by the traditional approach based on micromanipulation. We have developed a microfluidic system to track individual mother cells throughout their lifespan, allowing automated lifespan measurement and direct observation of cell cycle dynamics, cell/organelle morphologies, and various molecular markers. We found that aging of the wild-type cells is characterized by an increased general stress and a progressive lengthening of the cell cycle for the last few cell divisions; these features are much less apparent in the long-lived FOB1 deletion mutant. Following the fate of individual cells revealed that there are different forms of cell death that are characterized by different terminal cell morphologies, and associated with different levels of stress and lifespan. We have identified a molecular marker - the level of the expression of Hsp104, as a good predictor for the lifespan of individual cells. Our approach allows detailed molecular phenotyping of single cells in the process of aging and thus provides new insight into its mechanism. [ABSTRACT FROM AUTHOR]

Abstract: We propose a programmed syringe pumps system for diffusion-based concentration control in microcavities. Fluorescein is used to demonstrate the influence of different durations of unvarying injecting speed and different sizes of microcavities on the concentration control and distribution in the microcavities. With longer duration of unvarying injecting speed and smaller size of microcavities, the concentration control of the microcavities will be more consistent with the input signal. Using this system, we can precisely control sudden as well as gradual change of the microenvironment of cells within a period from several minutes to days by re-setting parameters of the program. As a demonstration, we apply this system to culture budding yeast cells in the microfluidic device and follow the growth status in precisely controlled nutritional environment. This system will be useful for high-throughput cell stimulation and differentiation studies. [Copyright &y& Elsevier]