Inactivating histone deacetylase HDA promotes longevity by mobilizing trehalose metabolism.

Histone acetylations are important epigenetic markers for transcriptional activation in response to metabolic changes and various stresses. Using the high-throughput SEquencing-Based Yeast replicative Lifespan screen method and the yeast knockout collection, we demonstrate that the HDA complex, a class-II histone deacetylase (HDAC), regulates aging through its target of acetylated H3K18 at storage carbohydrate genes. We find that, in addition to longer lifespan, disruption of HDA results in resistance to DNA damage and osmotic stresses. We show that these effects are due to increased promoter H3K18 acetylation and transcriptional activation in the trehalose metabolic pathway in the absence of HDA. Furthermore, we determine that the longevity effect of HDA is independent of the Cyc8-Tup1 repressor complex known to interact with HDA and coordinate transcriptional repression. Silencing the HDA homologs in C. elegans and Drosophila increases their lifespan and delays aging-associated physical declines in adult flies. Hence, we demonstrate that this HDAC controls an evolutionarily conserved longevity pathway.

Measuring the Replicative Lifespan of Saccharomyces cerevisiae Using the HYAA Microfluidic Platform.

The replicative aging of the budding yeast, Saccharomyces cerevisiae, has been a useful model for dissecting the molecular mechanisms of the aging process. Traditionally, the replicative lifespan (RLS) is measured by manually dissecting mother cells from daughter cells, which is a very tedious process. Since 2012, several microfluidic systems have been developed to automate the dissection process, significantly accelerating RLS determination. Here, we describe a detailed protocol of RLS measurement using a ommercially available microfluidic system based on the HYAA chip design, which enables data collection of up to 8000 cells in a single experiment.

Integrated Microfluidic System for Gene Silencing and Cell Migration.

Metastasis involves the phenotype transition of cancer cells to gain invasiveness, and the following migration at the tumor site. Here an integrated microfluidic chip to study this process is presented by combining on-chip delivery of siRNA for gene silencing and cell migration assay. The major advantage of the integrated chip is the simple input of cells and gene transfection materials, and the ultimate output of migration ability. The reverse-fishbone structure and 0.7× phosphate-buffered saline solution are the optimized parameters for improved delivery efficiency. Using the chip, it is validated that cofilin plays an essential role in regulating cancer cell migration. The integrated chip may provide a simple and effective platform for biologists to easily check the role of specific genes in metastasis.

Microfluidic Platforms for Yeast-Based Aging Studies.

The budding yeast Saccharomyces cerevisiae has been a powerful model for the study of aging and has enabled significant contributions to our understanding of basic mechanisms of aging in eukaryotic cells. However, the laborious low-throughput nature of conventional methods of performing aging assays limits the pace of discoveries in this field. Some of the technical challenges of conventional aging assay methods can be overcome by use of microfluidic systems coupled to time-lapse microscopy. One of the major advantages is the ability of a microfluidic system to perform long-term cell culture under well-defined environmental conditions while tracking individual yeast. Here, recent advancements in microfluidic platforms for various yeast-based studies including replicative lifespan assay, long-term culture and imaging, gene expression, and cell signaling are discussed. In addition, emerging problems and limitations of current microfluidic approaches are examined and perspectives on the future development of this dynamic field are presented.

CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation.

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) nuclease system represents an efficient tool for genome editing and gene function analysis. It consists of two components: single-guide RNA (sgRNA) and the enzyme Cas9. Typical sgRNA and Cas9 intracellular delivery techniques are limited by their reliance on cell type and exogenous materials as well as their toxic effects on cells (for example, electroporation). We introduce and optimize a microfluidic membrane deformation method to deliver sgRNA and Cas9 into different cell types and achieve successful genome editing. This approach uses rapid cell mechanical deformation to generate transient membrane holes to enable delivery of biomaterials in the medium. We achieved high delivery efficiency of different macromolecules into different cell types, including hard-to-transfect lymphoma cells and embryonic stem cells, while maintaining high cell viability. With the advantages of broad applicability across different cell types, particularly hard-to-transfect cells, and flexibility of application, this method could potentially enable new avenues of biomedical research and gene targeting therapy such as mutation correction of disease genes through combination of the CRISPR-Cas9-mediated knockin system.

Imaging of Cell-Cell Communication in a Vertical Orientation Reveals High-Resolution Structure of Immunological Synapse and Novel PD-1 Dynamics.

The immunological synapse (IS) is one of the most pivotal communication strategies in immune cells. Understanding the molecular basis of the IS provides critical information regarding how immune cells mount an effective immune response. Fluorescence microscopy provides a fundamental tool to study the IS. However, current imaging techniques for studying the IS cannot sufficiently achieve high resolution in real cell-cell conjugates. In this study, we present a new device that allows for high-resolution imaging of the IS with conventional confocal microscopy in a high-throughput manner. Combining micropits and single-cell trap arrays, we have developed a new microfluidic platform that allows visualization of the IS in vertically "stacked" cells. Using this vertical cell pairing (VCP) system, we investigated the dynamics of the inhibitory synapse mediated by an inhibitory receptor, programed death protein-1, and the cytotoxic synapse at the single-cell level. In addition to the technique innovation, we have demonstrated novel biological findings by this VCP device, including novel distribution of F-actin and cytolytic granules at the IS, programed death protein-1 microclusters at the NK IS, and kinetics of cytotoxicity. We propose that this high-throughput, cost-effective, easy-to-use VCP system, along with conventional imaging techniques, can be used to address a number of significant biological questions in a variety of disciplines.

High-throughput analysis of yeast replicative aging using a microfluidic system.

Saccharomyces cerevisiae has been an important model for studying the molecular mechanisms of aging in eukaryotic cells. However, the laborious and low-throughput methods of current yeast replicative lifespan assays limit their usefulness as a broad genetic screening platform for research on aging. We address this limitation by developing an efficient, high-throughput microfluidic single-cell analysis chip in combination with high-resolution time-lapse microscopy. This innovative design enables, to our knowledge for the first time, the determination of the yeast replicative lifespan in a high-throughput manner. Morphological and phenotypical changes during aging can also be monitored automatically with a much higher throughput than previous microfluidic designs. We demonstrate highly efficient trapping and retention of mother cells, determination of the replicative lifespan, and tracking of yeast cells throughout their entire lifespan. Using the high-resolution and large-scale data generated from the high-throughput yeast aging analysis (HYAA) chips, we investigated particular longevity-related changes in cell morphology and characteristics, including critical cell size, terminal morphology, and protein subcellular localization. In addition, because of the significantly improved retention rate of yeast mother cell, the HYAA-Chip was capable of demonstrating replicative lifespan extension by calorie restriction.

Sheathless size-based acoustic particle separation.

Particle separation is of great interest in many biological and biomedical applications. Flow-based methods have been used to sort particles and cells. However, the main challenge with flow based particle separation systems is the need for a sheath flow for successful operation. Existence of the sheath liquid dilutes the analyte, necessitates precise flow control between sample and sheath flow, requires a complicated design to create sheath flow and separation efficiency depends on the sheath liquid composition. In this paper, we present a microfluidic platform for sheathless particle separation using standing surface acoustic waves. In this platform, particles are first lined up at the center of the channel without introducing any external sheath flow. The particles are then entered into the second stage where particles are driven towards the off-center pressure nodes for size based separation. The larger particles are exposed to more lateral displacement in the channel due to the acoustic force differences. Consequently, different-size particles are separated into multiple collection outlets. The prominent feature of the present microfluidic platform is that the device does not require the use of the sheath flow for positioning and aligning of particles. Instead, the sheathless flow focusing and separation are integrated within a single microfluidic device and accomplished simultaneously. In this paper, we demonstrated two different particle size-resolution separations; (1) 3 µm and 10 µm and (2) 3 µm and 5 µm. Also, the effects of the input power, the flow rate, and particle concentration on the separation efficiency were investigated. These technologies have potential to impact broadly various areas including the essential microfluidic components for lab-on-a-chip system and integrated biological and biomedical applications.

A label-free cell separation using surface acoustic waves.

We present two-stage microfluidic platform for a continuous label-free cell separation using surface acoustic waves. In the proposed platform, cells are first lined up at the center of the channel by using standing surface acoustic waves without introducing any external sheath flow. After focused at the center of the channel, the cells are then entered to the actual cell separation stage where the larger cell are exposed to more lateral displacement in the channel towards the pressure node due to the acoustic force differences. Consequently, different size cells are separated into multiple collection outlets. The focusing and separation of the cells can be accomplished simultaneously in the present two-stage microfluidic device. The device doesn't require the use of the sheath flow for positioning or aligning of cells. In this study, we demonstrated the separation of two different size particle streams (3 µm and 10 µm) with this microfluidic platform without introducing any external sheath flow.