Understanding the Link between Inflammasome and Apoptosis through the Response of THP-1 Cells against Drugs Using Droplet-Based Microfluidics.

Droplet-based microfluidic devices are used to investigate monocytic THP-1 cells in response to drug administration. Consistent and reproducible droplets are created, each of which acts as a bioreactor to carry out single cell experiments with minimized contamination and live cell tracking under an inverted fluorescence microscope for more than 2 days. Here, the effects of three different drugs (temsirolimus, rifabutin, and BAY 11-7082) on THP-1 are examined and the results are analyzed in the context of the inflammasome and apoptosis relationship. The ASC adaptor gene tagged with GFP is monitored as the inflammasome reporter. Thus, a systematic way is presented for deciphering cell-to-cell heterogeneity, which is an important issue in cancer treatment. The drug temsirolimus, which has effects of disrupting the mTOR pathway and triggering apoptosis in tumor cells, causes THP-1 cells to express ASC and to be involved in apoptosis. Treatment with rifabutin, which inhibits proliferation and initiates apoptosis in cells, affects ASC expression by first increasing and then decreasing it. CASP-3, which has a role in apoptosis and is directly related to ASC, has an increasing level in inflammasome conditioning. Thus, the cell under the effect of rifabutin might be faced with programmed cell death faster. The drug BAY 11-7082, which is responsible for NFκB inhibition, shows similar results to temsirolimus with more than 60% of cells having high fluorescence intensity (ASC expression). The microfluidic platform presented here offers strong potential for studying newly developed small-molecule inhibitors for personalized/precision medicine.

Fabrication Protocol for Thermoplastic Microfluidic Devices: Nanoliter Volume Bioreactors for Cell Culturing.

Microfluidic devices consist of microchannels etched or embossed into substrates made of polymer, glass or silicon. Intricate connections of the microchannels to reactors with some smart mechanical structures such as traps or curvatures fulfil the desired functionalities such as mixing, separation, flow control or setting the environment for biochemical reactions. Here, we describe the fabrication methods of a thermoplastic microbioreactor step by step. First, material selection is made, then, production methods are determined with the equipment that can be easily procured in a laboratory. COP with its outstanding characteristics among many polymers was chosen. Two types of microbioreactors, with and without electrodes, are designed with AutoCAD and L-edit softwares. Photolithography and electrochemical wet etching are used for master mold preparation. Thermal evaporator is employed for pure chromium and gold deposition on COP substrate and etchants are used to form the interdigitated electrodes. Once the master mold produced, hot embossing is used to obtain the designed shape on drilled and planarized COP. Cover COP, with or without electrodes, is bonded to the hot embossed COP via thermo-compression and thermoplastic microfluidic device is realized. Tubings are connected to the device and a bridge between the macro and micro world is established. Yeast or mammalian cells labeled or tagged with GFP/RFP on specific gene products are loaded into the microfluidic device, and real time data on cell dimensions and fluorescence intensity are collected using inverted fluorescence microscope, and finally image processing is used to analyze the acquired data.

Real-Time Single-Cell Monitoring of Drug Effects Using Droplet-Based Microfluidic Technology: A Proof-of-Concept Study.

Drugs that act on ribosome biogenesis and cell proliferation play important roles in treatment of human diseases. Moreover, measurement of drug effects at a single-cell level would create vast opportunities for pharmaceutical innovation. We present in this study an original proof-of-concept study of single-cell measurement of drug effects with a focus on inhibition of ribosome biogenesis and cell proliferation, and using yeast (Saccharomyces cerevisiae) as a model eukaryotic organism. We employed a droplet-based microfluidic technology and nucleolar protein-tagged strain of the yeast for real-time monitoring of the cells. We report a comprehensive account of the ways in which interrelated pathways are impacted by drug treatment in a single-cell level. Self-organizing maps, transcription factor, and Gene Ontology enrichment analyses were utilized to these ends. This article makes a contribution to advance single-cell measurement of drug effects. We anticipate the microfluidic technology platform presented herein is well poised for future applications in personalized/precision medicine research as well as in industrial settings for drug discovery and clinical development. In addition, the study offers new insights on ribosome biogenesis and cell proliferation that should prove useful in cancer research and other complex human diseases impacted by these key cellular processes.

Thermoplastic microfluidic bioreactors with integrated electrodes to study tumor treating fields on yeast cells.

Tumor-treating fields (TTFields) are alternating electrical fields of intermediate frequency and low intensity that can slow or inhibit tumor growth by disrupting mitosis division of cancerous cells through cell cycle proteins. In this work, for the first time, an in-house fabricated cyclo-olefin polymer made microfluidic bioreactors are integrated with Cr/Au interdigitated electrodes to test TTFields on yeast cells with fluorescent protein:Nop56 gene. A small gap between electrodes (50 µm) allows small voltages (<150 mV) to be applied on the cells; hence, uninsulated gold electrodes are used in the non-faradaic region without causing any electrochemical reaction at the electrode-medium interface. Electrochemical modeling as well as impedance characterization and analysis of the electrodes are done using four different cell nutrient media. The experiments with yeast cells are done with 150 mV, 150 kHz and 30 mV, 200 kHz sinusoidal signals to generate electrical field magnitudes of 6.58 V/cm and 1.33 V/cm, respectively. In the high electrical field experiment, the cells go through electroporation. In the experiment with the low electrical field magnitude for TTFields, the cells have prolonged mitosis from typical 80-90 min to 200-300 min. Our results confirm the validity of the electrochemical model and the importance of applying a correct magnitude of the electrical field. Compared to the so far reported alternatives with insulated electrodes, the here developed thermoplastic microfluidic bioreactors with uninsulated electrodes provide a new, versatile, and durable platform for in vitro cell studies toward the improvement of anti-cancer therapies including personalized treatment.

Cell trapping microfluidic chip made of Cyclo olefin polymer enabling two concurrent cell biology experiments with long term durability.

Cyclo Olefin Polymer (COP) based microbioreactors on a microfluidic chip were produced in house by hot-embossing and thermo-compression bonding methods. The chip allows two different experiments to be performed on trapped cells at the same time. On one side of the chip, red fluorescent protein (RFP) tagged nucleolar Nop56 protein was used to track changes in cell cycle as well as protein synthesis within the yeast cells under the application of the anti-tumor agent hydroxyurea (HU). Simultaneously, on the other side of the chip, the response of yeast cells to the drug metformin, mTOR inhibitor, was investigated to reveal the role of TOR signaling in ribosome biogenesis and cell proliferation. The results of 20 h long experiments are captured by taking brightfield and fluorescent microscopy images of the trapped cells every 9 min. The expression of Nop56 protein of ribosome assembly and synthesis was densely observed during G1 phase of cell cycle, and later towards the end of cell cycle the ribosomal protein expression slowed down. Under HU treatment, the morphology of yeast cells changed, but after cessation of HU, the biomass synthesis rate was sustained as monitored by the cell perimeter. Under metformin treatment, the perimeters of single cells were observed to decrease, implying a decrease in biomass growth; however these cells continued their proliferation during and after the drug application. The relation between ribosome biogenesis and cell cycle was successfully investigated on single cell basis, capturing cell-to-cell variations, which cannot be tracked by regular macroscale bioreactors.

A Drug Repurposing and Protein-Protein Interaction Network Study of Ribosomopathies Using Yeast as a Model System.

Ribosomopathies result in various cancers, neurodegenerative and viral diseases, and other pathologies such as Diamond-Blackfan anemia and Shwachman-Diamond syndrome. Their pathophysiology at a proteome and functional level remains to be determined. Protein networks and highly connected hub proteins for ribosome biogenesis in Saccharomyces cerevisiae offer a potential as a model system to inform future therapeutic innovation in ribosomopathies. In this context, we report a ribosome biogenesis protein-protein interaction network in S. cerevisiae, created with 1772 proteins and 22,185 physical interactions connecting them. Moreover, by network decomposition analysis, we determined the linear pathways between the transcription factors and target proteins with a view to drug repurposing. While considering only the paths containing the three C/D box proteins (Nop56, Nop58, and Nop1), the most frequently encountered proteins were Aft1, Htz1, Ssa1, Ssb1, Ssb2, Gcn5, Cka1, Tef1, Nop1, Cdc28, Act1, Krr1, Rpl8B, and Tor1, which were then identified as potential drug targets. For drug repurposing, these candidate proteins were further searched in the DrugBank to find other diseases associated with them, as well as the drugs used to treat these diseases. To support the computational results, an experimental study was conducted using in-house manufactured microfluidic bioreactor platform, while the effect of the drug temsirolimus, Tor1 inhibitor, on yeast cells was investigated by following Nop56 protein expression. In conclusion, these results inform the ways in which ribosomopathies and associated common complex human diseases materialize and how drug repurposing might accelerate therapeutic innovation through bioinformatic studies of yeast.

A low cost PS based microfluidic platform to investigate cell cycle towards developing a therapeutic strategy for cancer.

Inhibition of DNA damage response pathway in combination with DNA alkylating agents may enhance the selective killing of cancer cells leading to better therapeutic effects. MDM2 binding protein (MTBP) in human has a role in G1 phase (interphase of cell cycle) and its overexpression leads to breast and ovarian cancers. Sld7 is an uncharacterized protein in budding yeast and a potential functional homologue of MTBP. To investigate the role of Sld7 as a therapeutic target, the behavior of the wild-type cells and sld7∆ mutants were monitored in 0.5 nL microbioreactors. The brightfield microscopy images were used to analyze the change in the cell size and to determine the durations of G1 and S/G2/M phases of wild type cells and mutants. With the administration of the alkylating agent, the cell size decreased and the duration of cell cycle increased. The replacement of the medium with the fresh one enabled the cells to repair their DNA. The application of calorie restriction together with DNA alkylating agent to mutant cells resulted in smaller cell size and longer G1 phase compared to those in control environment. For therapeutic purposes, the potential of MTBP in humans or Sld7 in yeast as a drug target deserves further exploration. The fabrication simplicity, robustness and low-cost of this microfluidic bioreactor made of polystyrene allowed us to perform yeast culturing experiments and show a potential for further cell culturing studies. The device can successfully be used for therapeutic applications including the discovery of new anti-microbial, anti-inflammatory, anti-cancer drugs.

Advances in microfluidic devices made from thermoplastics used in cell biology and analyses.

Silicon and glass were the main fabrication materials of microfluidic devices, however, plastics are on the rise in the past few years. Thermoplastic materials have recently been used to fabricate microfluidic platforms to perform experiments on cellular studies or environmental monitoring, with low cost disposable devices. This review describes the present state of the development and applications of microfluidic systems used in cell biology and analyses since the year 2000. Cultivation, separation/isolation, detection and analysis, and reaction studies are extensively discussed, considering only microorganisms (bacteria, yeast, fungi, zebra fish, etc.) and mammalian cell related studies in the microfluidic platforms. The advantages/disadvantages, fabrication methods, dimensions, and the purpose of creating the desired system are explained in detail. An important conclusion of this review is that these microfluidic platforms are still open for research and development, and solutions need to be found for each case separately.

Fabrication of cyclo olefin polymer microfluidic devices for trapping and culturing of yeast cells.

A microfluidic platform is designed and fabricated to investigate the role of uncharacterized YOR060C (Sld7) protein in aging in yeast cells for the first time. Saccharomyces cerevisiae yeast cells are trapped in the series of C-shaped regions (0.5 nL) of COP (cyclo olefin polymer), PMMA (poly methylmethacrylate), or PS (polystyrene) microbioreactors. The devices are fabricated using hot embossing and thermo-compression bonding methods. Photolithography and electrochemical etching are used to form the steel mold needed for hot embossing. The cell cycle processes are investigated by monitoring green fluorescent protein (GFP) tagged Sld7 expressions under normal as well as calorie restricted conditions. The cells are loaded at 1 µL/min flowrate and trapped successfully within each chamber. The medium is continuously fed at 0.1 µL/min throughout the experiments. Fluorescent signals of the low abundant Sld7 proteins could be distinguished only on COP devices. The background fluorescence of COP is found 1.22 and 7.24 times lower than that of PMMA, and PS, respectively. Hence, experiments are continued with COP, and lasted for more than 40 h without any contamination. The doubling time of the yeast cells are found as 72 min and 150 min, and the growth rates as 9.63 × 10-3 min-1 and 4.62 × 10-3 min-1, in 2% glucose containing YPD and YNB medium, respectively. The product concentration (Sld7p:GFP) increased in accordance with cell growth. The dual role of Sld7 protein in both cell cycle and chronological aging needs to be further investigated following the preliminary experimental results.