Publications by authors named "Savaş Tay"

42 Publications

Masitinib is a broad coronavirus 3CL inhibitor that blocks replication of SARS-CoV-2.

Science 2021 Jul 20. Epub 2021 Jul 20.

Institut Pasteur, Viral Populations and Pathogenesis Unit, Centre National de la Recherche Scientifique UMR 3569, Paris, France.

There is an urgent need for antiviral agents that treat SARS-CoV-2 infection. We screened a library of 1,900 clinically safe drugs against OC43, a human beta-coronavirus that causes the common cold and evaluated the top hits against SARS-CoV-2. Twenty drugs significantly inhibited replication of both viruses in vitro. Eight of these drugs inhibited the activity of the SARS-CoV-2 main protease, 3CLpro, with the most potent being masitinib, an orally bioavailable tyrosine kinase inhibitor. X-ray crystallography and biochemistry show that masitinib acts as a competitive inhibitor of 3CLpro. Mice infected with SARS-CoV-2 and then treated with masitinib showed >200-fold reduction in viral titers in the lungs and nose, as well as reduced lung inflammation. Masitinib was also effective in vitro against all tested variants of concern (B.1.1.7, B.1.351 and P.1).
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http://dx.doi.org/10.1126/science.abg5827DOI Listing
July 2021

COVIDomic: A multi-modal cloud-based platform for identification of risk factors associated with COVID-19 severity.

PLoS Comput Biol 2021 07 14;17(7):e1009183. Epub 2021 Jul 14.

Insilico Medicine Hong Kong Ltd, Pak Shek Kok, New Territories, Hong Kong.

Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in December 2019 in Wuhan, China. It was quickly established that both the symptoms and the disease severity may vary from one case to another and several strains of SARS-CoV-2 have been identified. To gain a better understanding of the wide variety of SARS-CoV-2 strains and their associated symptoms, thousands of SARS-CoV-2 genomes have been sequenced in dozens of countries. In this article, we introduce COVIDomic, a multi-omics online platform designed to facilitate the analysis and interpretation of the large amount of health data collected from patients with COVID-19. The COVIDomic platform provides a comprehensive set of bioinformatic tools for the multi-modal metatranscriptomic data analysis of COVID-19 patients to determine the origin of the coronavirus strain and the expected severity of the disease. An integrative analytical workflow, which includes microbial pathogens community analysis, COVID-19 genetic epidemiology and patient stratification, allows to analyze the presence of the most common microbial organisms, their antibiotic resistance, the severity of the infection and the set of the most probable geographical locations from which the studied strain could have originated. The online platform integrates a user friendly interface which allows easy visualization of the results. We envision this tool will not only have immediate implications for management of the ongoing COVID-19 pandemic, but will also improve our readiness to respond to other infectious outbreaks.
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http://dx.doi.org/10.1371/journal.pcbi.1009183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8312936PMC
July 2021

NF-κB responds to absolute differences in cytokine concentrations.

Sci Signal 2021 Jan;14(666)

Pritzker School of Molecular Engineering, University of Chicago, Chicago, IL 60637, USA.

Cells receive a wide range of dynamic signaling inputs during immune regulation, but how gene regulatory networks measure such dynamic inputs is not well understood. Here, we used microfluidic single-cell analysis and mathematical modeling to study how the NF-κB pathway responds to immune inputs that vary over time such as increasing, decreasing, or fluctuating cytokine signals. We found that NF-κB activity responded to the absolute difference in cytokine concentration and not to the concentration itself. Our analyses revealed that negative feedback by the regulatory proteins A20 and IκBα enabled differential responses to changes in cytokine dose by providing a short-term memory of previous cytokine concentrations and by continuously resetting kinase cycling and receptor abundance. Investigation of NF-κB target gene expression showed that cells exhibited distinct transcriptional responses under different dynamic cytokine profiles. Our results demonstrate how cells use simple network motifs and transcription factor dynamics to efficiently extract information from complex signaling environments.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8244746PMC
January 2021

Sensitive detection and quantification of SARS-CoV-2 in saliva.

Sci Rep 2021 06 14;11(1):12425. Epub 2021 Jun 14.

Section of Hematology and Oncology, Pritzker School of Medicine, University of Chicago, Chicago, IL, USA.

Saliva has significant advantages as a test medium for detection of SARS-CoV-2 infection in patients, such as ease of collection, minimal requirement of supplies and trained personnel, and safety. Comprehensive validation in a large cohort of prospectively collected specimens with unknown SARS-CoV-2 status should be performed to evaluate the potential and limitations of saliva-based testing. We developed a saliva-based testing pipeline for detection of SARS-CoV-2 nucleic acids using real-time reverse transcription PCR (RT-PCR) and droplet digital PCR (ddPCR) readouts, and measured samples from 137 outpatients tested at a curbside testing facility and 29 inpatients hospitalized for COVID-19. These measurements were compared to the nasal swab results for each patient performed by a certified microbiology laboratory. We found that our saliva testing positively detects 100% (RT-PCR) and 93.75% (ddPCR) of curbside patients that were identified as SARS-CoV-2 positive by the Emergency Use Authorization (EUA) certified nasal swab testing assay. Quantification of viral loads by ddPCR revealed an extremely wide range, with 1 million-fold difference between individual patients. Our results demonstrate for both community screening and hospital settings that saliva testing reliability is on par with that of the nasal swabs in detecting infected cases, and has potential for higher sensitivity when combined with ddPCR in detecting low-abundance viral loads that evade traditional testing methods.
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http://dx.doi.org/10.1038/s41598-021-91835-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8203799PMC
June 2021

Discovery of SARS-CoV-2 main protease inhibitors using a synthesis-directed design model.

Chem Commun (Camb) 2021 Jun;57(48):5909-5912

PostEra Inc, 2 Embarcadero Centre, San Franciso, CA 94111, USA.

The SARS-CoV-2 main viral protease (Mpro) is an attractive target for antivirals given its distinctiveness from host proteases, essentiality in the viral life cycle and conservation across coronaviridae. We launched the COVID Moonshot initiative to rapidly develop patent-free antivirals with open science and open data. Here we report the use of machine learning for de novo design, coupled with synthesis route prediction, in our campaign. We discover novel chemical scaffolds active in biochemical and live virus assays, synthesized with model generated routes.
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http://dx.doi.org/10.1039/d1cc00050kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8204246PMC
June 2021

Cannabidiol Inhibits SARS-CoV-2 Replication and Promotes the Host Innate Immune Response.

bioRxiv 2021 Mar 10. Epub 2021 Mar 10.

The rapid spread of COVID-19 underscores the need for new treatments. Here we report that cannabidiol (CBD), a compound produced by the cannabis plant, inhibits SARS-CoV-2 infection. CBD and its metabolite, 7-OH-CBD, but not congeneric cannabinoids, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after cellular infection, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription. CBD induces interferon expression and up-regulates its antiviral signaling pathway. A cohort of human patients previously taking CBD had significantly lower SARS-CoV-2 infection incidence of up to an order of magnitude relative to matched pairs or the general population. This study highlights CBD, and its active metabolite, 7-OH-CBD, as potential preventative agents and therapeutic treatments for SARS-CoV-2 at early stages of infection.
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http://dx.doi.org/10.1101/2021.03.10.432967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987002PMC
March 2021

Single-Cell Proteomics.

Trends Biochem Sci 2021 Aug 27;46(8):661-672. Epub 2021 Feb 27.

Pritzker School of Molecular Engineering, University of Chicago, Chicago, IL 60637, USA; Institute for Genomics and Systems Biology, University of Chicago, Chicago, IL 60637, USA. Electronic address:

The inability to make broad, minimally biased measurements of a cell's proteome stands as a major bottleneck for understanding how gene expression translates into cellular phenotype. Unlike sequencing for nucleic acids, there is no dominant method for making single-cell proteomic measurements. Instead, methods typically focus on either absolute quantification of a small number of proteins or highly multiplexed protein measurements. Advances in microfluidics and output encoding have led to major improvements in both aspects. Here, we review the most recent progress that has enabled hundreds of protein measurements and ultrahigh-sensitivity quantification. We also highlight emerging technologies such as single-cell mass spectrometry that may enable unbiased measurement of cellular proteomes.
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http://dx.doi.org/10.1016/j.tibs.2021.01.013DOI Listing
August 2021

Sensitive detection and quantification of SARS-CoV-2 in saliva.

medRxiv 2020 Dec 7. Epub 2020 Dec 7.

Saliva has significant advantages as a test medium for detection of SARS-CoV-2 infection in patients, such as ease of collection, minimal requirement of supplies and trained personnel, and safety. Comprehensive validation in a large cohort of prospectively collected specimens with unknown SARS-CoV-2 status should be performed to evaluate the potential and limitations of saliva-based testing. We developed a saliva-based testing pipeline for detection of SARS-CoV-2 nucleic acids using real-time reverse transcription PCR (RT-PCR) and droplet digital PCR (ddPCR) readouts, and measured samples from 137 outpatients tested at a curbside testing facility and 29 inpatients hospitalized for COVID-19. These measurements were compared to the nasal swab results for each patient performed by a certified microbiology laboratory. We found that our saliva testing positively detects 100% (RT-PCR) and 93.75% (ddPCR) of curbside patients that were identified as SARS-CoV-2 positive by the Emergency Use Authorization (EUA) certified nasal swab testing assay. Quantification of viral loads by ddPCR revealed an extremely wide range, with 1 million-fold difference between individual patients. Our results demonstrate for both community screening and hospital settings that saliva testing reliability is on par with that of the nasal swabs in detecting infected cases, and has potential for higher sensitivity when combined with ddPCR in detecting low-abundance viral loads that evade traditional testing methods.
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http://dx.doi.org/10.1101/2020.12.04.20241059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7743089PMC
December 2020

Automated microfluidic platform for dynamic and combinatorial drug screening of tumor organoids.

Nat Commun 2020 10 19;11(1):5271. Epub 2020 Oct 19.

Pritzker School of Molecular Engineering, The University of Chicago, Chicago, IL, 60637, USA.

Three-dimensional (3D) cell culture technologies, such as organoids, are physiologically relevant models for basic and clinical applications. Automated microfluidics offers advantages in high-throughput and precision analysis of cells but is not yet compatible with organoids. Here, we present an automated, high-throughput, microfluidic 3D organoid culture and analysis system to facilitate preclinical research and personalized therapies. Our system provides combinatorial and dynamic drug treatments to hundreds of cultures and enables real-time analysis of organoids. We validate our system by performing individual, combinatorial, and sequential drug screens on human-derived pancreatic tumor organoids. We observe significant differences in the response of individual patient-based organoids to drug treatments and find that temporally-modified drug treatments can be more effective than constant-dose monotherapy or combination therapy in vitro. This integrated platform advances organoids models to screen and mirror real patient treatment courses with potential to facilitate treatment decisions for personalized therapy.
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http://dx.doi.org/10.1038/s41467-020-19058-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7573629PMC
October 2020

Fixed single-cell RNA sequencing for understanding virus infection and host response.

bioRxiv 2020 Sep 17. Epub 2020 Sep 17.

Pritzker School of Molecular Engineering, The University of Chicago, Chicago, IL, USA.

Single-cell RNA sequencing studies requiring intracellular protein staining, rare-cell sorting, or pathogen inactivation are severely limited because current high-throughput methods are incompatible with paraformaldehyde treatment, a very common and simple tissue/cell fixation and preservation technique. Here we present FD-seq, a high-throughput method for droplet-based RNA sequencing of paraformaldehyde-fixed, stained and sorted single-cells. We used FD-seq to address two important questions in virology. First, by analyzing a rare population of cells supporting lytic reactivation of the human tumor virus KSHV, we identified as a host factor that mediates reactivation. Second, we studied the transcriptome of lung cells infected with the coronavirus OC43, which causes the common cold and also serves as a safer model pathogen for SARS-CoV-2. We found that pro-inflammatory pathways are primarily upregulated in abortively-infected or uninfected bystander cells, which are exposed to the virus but fail to express high level of viral genes. FD-seq is suitable for characterizing rare cell populations of interest, for studying high-containment biological samples after inactivation, and for integrating intracellular phenotypic with transcriptomic information.
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http://dx.doi.org/10.1101/2020.09.17.302232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523121PMC
September 2020

Drug repurposing screen identifies masitinib as a 3CLpro inhibitor that blocks replication of SARS-CoV-2 .

bioRxiv 2020 Sep 1. Epub 2020 Sep 1.

There is an urgent need for anti-viral agents that treat SARS-CoV-2 infection. The shortest path to clinical use is repurposing of drugs that have an established safety profile in humans. Here, we first screened a library of 1,900 clinically safe drugs for inhibiting replication of OC43, a human beta-coronavirus that causes the common-cold and is a relative of SARS-CoV-2, and identified 108 effective drugs. We further evaluated the top 26 hits and determined their ability to inhibit SARS-CoV-2, as well as other pathogenic RNA viruses. 20 of the 26 drugs significantly inhibited SARS-CoV-2 replication in human lung cells (A549 epithelial cell line), with EC50 values ranging from 0.1 to 8 micromolar. We investigated the mechanism of action for these and found that masitinib, a drug originally developed as a tyrosine-kinase inhibitor for cancer treatment, strongly inhibited the activity of the SARS-CoV-2 main protease 3CLpro. X-ray crystallography revealed that masitinib directly binds to the active site of 3CLpro, thereby blocking its enzymatic activity. Mastinib also inhibited the related viral protease of picornaviruses and blocked picornaviruses replication. Thus, our results show that masitinib has broad anti-viral activity against two distinct beta-coronaviruses and multiple picornaviruses that cause human disease and is a strong candidate for clinical trials to treat SARS-CoV-2 infection.
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http://dx.doi.org/10.1101/2020.08.31.274639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7480023PMC
September 2020

How Can Microfluidic and Microfabrication Approaches Make Experiments More Physiologically Relevant?

Cell Syst 2020 09 3;11(3):209-211. Epub 2020 Sep 3.

Microfabricated and microfluidic devices enable standardized handling, precise spatiotemporal manipulation of cells and liquids, and recapitulation of cellular environments, tissues, and organ-level biology. We asked researchers how these devices can make in vitro experiments more physiologically relevant.
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http://dx.doi.org/10.1016/j.cels.2020.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7890516PMC
September 2020

Droplet-based high-throughput cultivation for accurate screening of antibiotic resistant gut microbes.

Elife 2020 06 17;9. Epub 2020 Jun 17.

Pritzker School of Molecular Engineering, The University of Chicago, Chicago, United States.

Traditional cultivation approaches in microbiology are labor-intensive, low-throughput, and yield biased sampling of environmental microbes due to ecological and evolutionary factors. New strategies are needed for ample representation of rare taxa and slow-growers that are often outcompeted by fast-growers in cultivation experiments. Here we describe a microfluidic platform that anaerobically isolates and cultivates microbial cells in millions of picoliter droplets and automatically sorts them based on colony density to enhance slow-growing organisms. We applied our strategy to a fecal microbiota transplant (FMT) donor stool using multiple growth media, and found significant increase in taxonomic richness and larger representation of rare and clinically relevant taxa among droplet-grown cells compared to conventional plates. Furthermore, screening the FMT donor stool for antibiotic resistance revealed 21 populations that evaded detection in plate-based assessment of antibiotic resistance. Our method improves cultivation-based surveys of diverse microbiomes to gain deeper insights into microbial functioning and lifestyles.
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http://dx.doi.org/10.7554/eLife.56998DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7351490PMC
June 2020

Ultrasensitive digital quantification of cytokines and bacteria predicts septic shock outcomes.

Nat Commun 2020 05 25;11(1):2607. Epub 2020 May 25.

Pritzker School of Molecular Engineering, The University of Chicago, Chicago, IL, 60637, USA.

Quantification of pathogen and host biomarkers is essential for the diagnosis, monitoring, and treatment of infectious diseases. Here, we demonstrate sensitive and rapid quantification of bacterial load and cytokines from human biological samples to generate actionable hypotheses. Our digital assay measures IL-6 and TNF-α proteins, gram-negative (GN) and gram-positive (GP) bacterial DNA, and the antibiotic-resistance gene bla with femtomolar sensitivity. We use our method to characterize bronchoalveolar lavage fluid from patients with asthma, and find elevated GN bacteria and IL-6 levels compared to healthy subjects. We then analyze plasma from patients with septic shock and find that increasing levels of IL-6 and bla are associated with mortality, while decreasing IL-6 levels are associated with recovery. Surprisingly, lower GN bacteria levels are associated with higher probability of death. Applying decision-tree analysis to our measurements, we are able to predict mortality and rate of recovery from septic shock with over 90% accuracy.
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http://dx.doi.org/10.1038/s41467-020-16124-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7248118PMC
May 2020

Ultra-sensitive digital quantification of proteins and mRNA in single cells.

Nat Commun 2019 08 7;10(1):3544. Epub 2019 Aug 7.

Pritzker School of Molecular Engineering, University of Chicago, Chicago, IL, 60637, USA.

Simultaneous measurement of proteins and mRNA in single cells enables quantitative understanding and modeling of cellular functions. Here, we present an automated microfluidic system for multi-parameter and ultra-sensitive protein/mRNA measurements in single cells. Our technology improves the sensitivity of digital proximity ligation assay by up to 55-fold, with a detection limit of 2277 proteins per cell and with detection efficiency of as few as 29 protein molecules. Our measurements using this system reveal higher mRNA/protein correlation in single mammalian cells than previous estimates. Furthermore, time-lapse imaging of herpes simplex virus 1 infected epithelial cells enabled by our device shows that expression of ICP4 -a major transcription factor regulating hundreds of viral genes- is only partially correlated with viral protein counts, suggesting that many cells go through abortive infection. These results highlight the importance of high-sensitivity protein/mRNA quantification for understanding fundamental molecular mechanisms in individual cells.
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http://dx.doi.org/10.1038/s41467-019-11531-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6685952PMC
August 2019

HSV-1 single-cell analysis reveals the activation of anti-viral and developmental programs in distinct sub-populations.

Elife 2019 05 15;8. Epub 2019 May 15.

Institute for Molecular Engineering, The University of Chicago, Chicago, United States.

Viral infection is usually studied at the population level by averaging over millions of cells. However, infection at the single-cell level is highly heterogeneous, with most infected cells giving rise to no or few viral progeny while some cells produce thousands. Analysis of Herpes Simplex virus 1 (HSV-1) infection by population-averaged measurements has taught us a lot about the course of viral infection, but has also produced contradictory results, such as the concurrent activation and inhibition of type I interferon signaling during infection. Here, we combine live-cell imaging and single-cell RNA sequencing to characterize viral and host transcriptional heterogeneity during HSV-1 infection of primary human cells. We find extreme variability in the level of viral gene expression among individually infected cells and show that these cells cluster into transcriptionally distinct sub-populations. We find that anti-viral signaling is initiated in a rare group of abortively infected cells, while highly infected cells undergo cellular reprogramming to an embryonic-like transcriptional state. This reprogramming involves the recruitment of β-catenin to the host nucleus and viral replication compartments, and is required for late viral gene expression and progeny production. These findings uncover the transcriptional differences in cells with variable infection outcomes and shed new light on the manipulation of host pathways by HSV-1.
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http://dx.doi.org/10.7554/eLife.46339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570482PMC
May 2019

Ultra-multiplexed analysis of single-cell dynamics reveals logic rules in differentiation.

Sci Adv 2019 04 3;5(4):eaav7959. Epub 2019 Apr 3.

Institute for Molecular Engineering, The University of Chicago, Chicago, IL 60637, USA.

Dynamical control of cellular microenvironments is highly desirable to study complex processes such as stem cell differentiation and immune signaling. We present an ultra-multiplexed microfluidic system for high-throughput single-cell analysis in precisely defined dynamic signaling environments. Our system delivers combinatorial and time-varying signals to 1500 independently programmable culture chambers in week-long live-cell experiments by performing nearly 10 pipetting steps, where single cells, two-dimensional (2D) populations, or 3D neurospheres are chemically stimulated and tracked. Using our system and statistical analysis, we investigated the signaling landscape of neural stem cell differentiation and discovered "cellular logic rules" that revealed the critical role of signal timing and sequence in cell fate decisions. We find synergistic and antagonistic signal interactions and show that differentiation pathways are highly redundant. Our system allows dissection of hidden aspects of cellular dynamics and enables accelerated biological discovery.
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http://dx.doi.org/10.1126/sciadv.aav7959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6447378PMC
April 2019

Viable cell culture in PDMS-based microfluidic devices.

Methods Cell Biol 2018 14;148:3-33. Epub 2018 Nov 14.

Institute of Molecular Engineering, University of Chicago, Chicago, IL, United States; Institute of Genomics and Systems Biology, University of Chicago, Chicago, IL, United States. Electronic address:

Microfluidics has played a vital role in developing novel methods to investigate biological phenomena at the molecular and cellular level during the last two decades. Microscale engineering of cellular systems is nevertheless a nascent field marked inherently by frequent disruptive advancements in technology such as PDMS-based soft lithography. Viable culture and manipulation of cells in microfluidic devices requires knowledge across multiple disciplines including molecular and cellular biology, chemistry, physics, and engineering. There has been numerous excellent reviews in the past 15 years on applications of microfluidics for molecular and cellular biology including microfluidic cell culture (Berthier et al., 2012; El-Ali, Sorger, & Jensen, 2006; Halldorsson et al., 2015; Kim et al., 2007; Mehling & Tay, 2014; Sackmann et al., 2014; Whitesides, 2006; Young & Beebe, 2010), cell culture models (Gupta et al., 2016; Inamdar & Borenstein, 2011; Meyvantsson & Beebe, 2008), cell secretion (Schrell et al., 2016), chemotaxis (Kim & Wu, 2012; Wu et al., 2013), neuron culture (Millet & Gillette, 2012a, 2012b), drug screening (Dittrich & Manz, 2006; Eribol, Uguz, & Ulgen, 2016; Wu, Huang, & Lee, 2010), cell sorting (Autebert et al., 2012; Bhagat et al., 2010; Gossett et al., 2010; Wyatt Shields Iv, Reyes, & López, 2015), single cell studies (Lecault et al., 2012; Reece et al., 2016; Yin & Marshall, 2012), stem cell biology (Burdick & Vunjak-Novakovic, 2009; Wu et al., 2011; Zhang & Austin, 2012), cell differentiation (Zhang et al., 2017a), systems biology (Breslauer, Lee, & Lee, 2006), 3D cell culture (Huh et al., 2011; Li et al., 2012; van Duinen et al., 2015), spheroids and organoids (Lee et al., 2016; Montanez-Sauri, Beebe, & Sung, 2015; Morimoto & Takeuchi, 2013; Skardal et al., 2016; Young, 2013), organ-on-chip (Bhatia & Ingber, 2014; Esch, Bahinski, & Huh, 2015; Huh et al., 2011; van der Meer & van den Berg, 2012), and tissue engineering (Andersson & Van Den Berg, 2004; Choi et al., 2007; Hasan et al., 2014). In this chapter, we provide an overview of PDMS-based microdevices for microfluidic cell culture. We discuss the advantages and challenges of using PDMS-based soft lithography for microfluidic cell culture and highlight recent progress and future directions in this area.
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http://dx.doi.org/10.1016/bs.mcb.2018.09.007DOI Listing
January 2019

Automated Microfluidic System for Dynamic Stimulation and Tracking of Single Cells.

Anal Chem 2018 09 30;90(18):10695-10700. Epub 2018 Jul 30.

Department of Biosystems Science and Engineering , ETH Zurich, Mattenstrasse 26 4058 Basel , Switzerland.

Dynamic environments determine cell fate decisions and function. Understanding the relationship between extrinsic signals on cellular responses and cell fate requires the ability to dynamically change environmental inputs in vitro, while continuously observing individual cells over extended periods of time. This is challenging for nonadherent cells, such as hematopoietic stem and progenitor cells, because media flow displaces and disturbs such cells, preventing culture and tracking of single cells. Here, we present a programmable microfluidic system designed for the long-term culture and time-lapse imaging of nonadherent cells in dynamically changing cell culture conditions without losing track of individual cells. The dynamic, valve-controlled design permits targeted seeding of cells in up to 48 independently controlled culture chambers, each providing sufficient space for long-term cell colony expansion. Diffusion-based media exchange occurs rapidly and minimizes displacement of cells and eliminates shear stress. The chip was successfully tested with long-term culture and tracking of primary hematopoietic stem and progenitor cells, and murine embryonic stem cells. This system will have important applications to analyze dynamic signaling inputs controlling fate choices.
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http://dx.doi.org/10.1021/acs.analchem.8b00312DOI Listing
September 2018

Integrated platform for cell culture and dynamic quantification of cell secretion.

Lab Chip 2017 11;17(23):4124-4133

Department of Biosystems Science and Engineering, ETH Zürich, Mattenstrasse 26, 4058 Basel, Switzerland.

We developed an automated microfluidic chip that can measure dynamic cytokine secretion and transcription factor activation from cells responding to time-varying stimuli. Our chip patterns antibodies, exposes cells to time-varying inputs, measures cell secretion dynamics, and quantifies secretion all in the same platform. Secretion dynamics are measured using micrometer-sized immunoassays patterned directly inside the chip. All processes are automated, so that no user input is needed for conducting a complete cycle of device preparation, cell experiments, and secretion quantification. Using this system, we simulated an immune response by exposing cells to stimuli indicative of chronic and increasing inflammation. Specifically, we quantified how macrophages respond to changing levels of the bacterial ligand LPS, in terms of transcription factor NF-κB activity and TNF cytokine secretion. The integration of assay preparation with experimental automation of our system simplifies protocols for measuring cell responses to dynamic and physiologically relevant conditions and enables simpler and more error free means of microfluidic cellular investigations.
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http://dx.doi.org/10.1039/c7lc00839bDOI Listing
November 2017

Universal signal generator for dynamic cell stimulation.

Lab Chip 2017 06;17(13):2218-2224

Department of Biosystems Science and Engineering, ETH Zürich, 4058, Mattenstrasse 26, 4058 Basel, Switzerland.

Dynamic cell stimulation is a powerful technique for probing gene networks and for applications in stem cell differentiation, immunomodulation and signaling. We developed a robust and flexible method and associated microfluidic devices to generate a wide-range of precisely formulated dynamic chemical signals to stimulate live cells and measure their dynamic response. This signal generator is capable of digital to analog conversion (DAC) through combinatoric selection of discrete input concentrations, and outperforms existing methods by both achievable resolution, dynamic range and simplicity in design. It requires no calibration, has minimal space requirements and can be easily integrated into microfluidic cell culture devices. The signal generator hardware and software we developed allows to choose the waveform, period and amplitude of chemical input signals and features addition of well-defined chemical noise to study the role of stochasticity in cellular information processing.
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http://dx.doi.org/10.1039/c7lc00531hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5767101PMC
June 2017

Cellular Decision Making by Non-Integrative Processing of TLR Inputs.

Cell Rep 2017 04;19(1):125-135

Department of Biosystems Science and Engineering, ETH Zürich 4058, Switzerland; Institute for Molecular Engineering, University of Chicago, Chicago, IL 60637, USA; Institute for Genomics and Systems Biology, University of Chicago, Chicago, IL 60637, USA. Electronic address:

Cells receive a multitude of signals from the environment, but how they process simultaneous signaling inputs is not well understood. Response to infection, for example, involves parallel activation of multiple Toll-like receptors (TLRs) that converge on the nuclear factor κB (NF-κB) pathway. Although we increasingly understand inflammatory responses for isolated signals, it is not clear how cells process multiple signals that co-occur in physiological settings. We therefore examined a bacterial infection scenario involving co-stimulation of TLR4 and TLR2. Independent stimulation of these receptors induced distinct NF-κB dynamic profiles, although surprisingly, under co-stimulation, single cells continued to show ligand-specific dynamic responses characteristic of TLR2 or TLR4 signaling rather than a mixed response, comprising a cellular decision that we term "non-integrative" processing. Iterating modeling and microfluidic experiments revealed that non-integrative processing occurred through interaction of switch-like NF-κB activation, receptor-specific processing timescales, cell-to-cell variability, and TLR cross-tolerance mediated by multilayer negative feedback.
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http://dx.doi.org/10.1016/j.celrep.2017.03.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766323PMC
April 2017

Noise Induces Hopping between NF-κB Entrainment Modes.

Cell Syst 2016 Dec;3(6):532-539.e3

Niels Bohr Institute, University of Copenhagen, 2100 Copenhagen, Denmark. Electronic address:

Oscillations and noise drive many processes in biology, but how both affect the activity of the transcription factor nuclear factor κB (NF-κB) is not understood. Here, we observe that when NF-κB oscillations are entrained by periodic tumor necrosis factor (TNF) inputs in experiments, NF-κB exhibits jumps between frequency modes, a phenomenon we call "cellular mode-hopping." By comparing stochastic simulations of NF-κB oscillations to deterministic simulations conducted inside and outside the chaotic regime of parameter space, we show that noise facilitates mode-hopping in all regimes. However, when the deterministic system is driven by chaotic dynamics, hops between modes are erratic and short-lived, whereas in experiments, the system spends several periods in one entrainment mode before hopping and rarely visits more than two modes. The experimental behavior matches our simulations of noise-induced mode-hopping outside the chaotic regime. We suggest that mode-hopping is a mechanism by which different NF-κB-dependent genes under frequency control can be expressed at different times.
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http://dx.doi.org/10.1016/j.cels.2016.11.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5783698PMC
December 2016

A microfluidic device for measuring cell migration towards substrate-bound and soluble chemokine gradients.

Sci Rep 2016 11 7;6:36440. Epub 2016 Nov 7.

Institute of Science and Technology Austria (IST Austria), 3400, Klosterneuburg, Austria.

Cellular locomotion is a central hallmark of eukaryotic life. It is governed by cell-extrinsic molecular factors, which can either emerge in the soluble phase or as immobilized, often adhesive ligands. To encode for direction, every cue must be present as a spatial or temporal gradient. Here, we developed a microfluidic chamber that allows measurement of cell migration in combined response to surface immobilized and soluble molecular gradients. As a proof of principle we study the response of dendritic cells to their major guidance cues, chemokines. The majority of data on chemokine gradient sensing is based on in vitro studies employing soluble gradients. Despite evidence suggesting that in vivo chemokines are often immobilized to sugar residues, limited information is available how cells respond to immobilized chemokines. We tracked migration of dendritic cells towards immobilized gradients of the chemokine CCL21 and varying superimposed soluble gradients of CCL19. Differential migratory patterns illustrate the potential of our setup to quantitatively study the competitive response to both types of gradients. Beyond chemokines our approach is broadly applicable to alternative systems of chemo- and haptotaxis such as cells migrating along gradients of adhesion receptor ligands vs. any soluble cue.
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http://dx.doi.org/10.1038/srep36440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5098208PMC
November 2016

High-Content Quantification of Single-Cell Immune Dynamics.

Cell Rep 2016 Apr 31;15(2):411-22. Epub 2016 Mar 31.

Department of Biosystems Science and Engineering, ETH Zürich, 4058 Basel, Switzerland; Institute for Molecular Engineering, University of Chicago, Chicago, IL 60637, USA; Institute for Genomics and Systems Biology, University of Chicago, Chicago, IL 60637, USA. Electronic address:

Cells receive time-varying signals from the environment and generate functional responses by secreting their own signaling molecules. Characterizing dynamic input-output relationships in single cells is crucial for understanding and modeling cellular systems. We developed an automated microfluidic system that delivers precisely defined dynamical inputs to individual living cells and simultaneously measures key immune parameters dynamically. Our system combines nanoliter immunoassays, microfluidic input generation, and time-lapse microscopy, enabling study of previously untestable aspects of immunity by measuring time-dependent cytokine secretion and transcription factor activity from single cells stimulated with dynamic inflammatory inputs. Employing this system to analyze macrophage signal processing under pathogen inputs, we found that the dynamics of TNF secretion are highly heterogeneous and surprisingly uncorrelated with the dynamics of NF-κB, the transcription factor controlling TNF production. Computational modeling of the LPS/TLR4 pathway shows that post-transcriptional regulation by TRIF is a key determinant of noisy and uncorrelated TNF secretion dynamics in single macrophages.
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http://dx.doi.org/10.1016/j.celrep.2016.03.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4835544PMC
April 2016

Digital Quantification of Proteins and mRNA in Single Mammalian Cells.

Mol Cell 2016 Mar;61(6):914-24

Department of Biosystems Science and Engineering, ETH Zürich, 4058 Basel, Switzerland; Institute for Molecular Engineering, University of Chicago, Chicago, IL 60637, USA. Electronic address:

Absolute quantification of macromolecules in single cells is critical for understanding and modeling biological systems that feature cellular heterogeneity. Here we show extremely sensitive and absolute quantification of both proteins and mRNA in single mammalian cells by a very practical workflow that combines proximity ligation assay (PLA) and digital PCR. This digital PLA method has femtomolar sensitivity, which enables the quantification of very small protein concentration changes over its entire 3-log dynamic range, a quality necessary for accounting for single-cell heterogeneity. We counted both endogenous (CD147) and exogenously expressed (GFP-p65) proteins from hundreds of single cells and determined the correlation between CD147 mRNA and the protein it encodes. Using our data, a stochastic two-state model of the central dogma was constructed and verified using joint mRNA/protein distributions, allowing us to estimate transcription burst sizes and extrinsic noise strength and calculate the transcription and translation rate constants in single mammalian cells.
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http://dx.doi.org/10.1016/j.molcel.2016.02.030DOI Listing
March 2016

The Immune-Metabolic Basis of Effector Memory CD4+ T Cell Function under Hypoxic Conditions.

J Immunol 2016 Jan 30;196(1):106-14. Epub 2015 Nov 30.

Immunobiology, Department of Biomedicine, University of Basel, 4031 Basel, Switzerland;

Effector memory (EM) CD4(+) T cells recirculate between normoxic blood and hypoxic tissues to screen for cognate Ag. How mitochondria of these cells, shuttling between normoxia and hypoxia, maintain bioenergetic efficiency and stably uphold antiapoptotic features is unknown. In this study, we found that human EM CD4(+) T cells had greater spare respiratory capacity (SRC) than did naive counterparts, which was immediately accessed under hypoxia. Consequently, hypoxic EM cells maintained ATP levels, survived and migrated better than did hypoxic naive cells, and hypoxia did not impair their capacity to produce IFN-γ. EM CD4(+) T cells also had more abundant cytosolic GAPDH and increased glycolytic reserve. In contrast to SRC, glycolytic reserve was not tapped under hypoxic conditions, and, under hypoxia, glucose metabolism contributed similarly to ATP production in naive and EM cells. However, both under normoxic and hypoxic conditions, glucose was critical for EM CD4(+) T cell survival. Mechanistically, in the absence of glycolysis, mitochondrial membrane potential (ΔΨm) of EM cells declined and intrinsic apoptosis was triggered. Restoring pyruvate levels, the end product of glycolysis, preserved ΔΨm and prevented apoptosis. Furthermore, reconstitution of reactive oxygen species (ROS), whose production depends on ΔΨm, also rescued viability, whereas scavenging mitochondrial ROS exacerbated apoptosis. Rapid access of SRC in hypoxia, linked with built-in, oxygen-resistant glycolytic reserve that functionally insulates ΔΨm and mitochondrial ROS production from oxygen tension changes, provides an immune-metabolic basis supporting survival, migration, and function of EM CD4(+) T cells in normoxic and hypoxic conditions.
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http://dx.doi.org/10.4049/jimmunol.1501766DOI Listing
January 2016

Digital signaling decouples activation probability and population heterogeneity.

Elife 2015 Oct 21;4:e08931. Epub 2015 Oct 21.

Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zürich, Basel, Switzerland.

Digital signaling enhances robustness of cellular decisions in noisy environments, but it is unclear how digital systems transmit temporal information about a stimulus. To understand how temporal input information is encoded and decoded by the NF-κB system, we studied transcription factor dynamics and gene regulation under dose- and duration-modulated inflammatory inputs. Mathematical modeling predicted and microfluidic single-cell experiments confirmed that integral of the stimulus (or area, concentration × duration) controls the fraction of cells that activate NF-κB in the population. However, stimulus temporal profile determined NF-κB dynamics, cell-to-cell variability, and gene expression phenotype. A sustained, weak stimulation lead to heterogeneous activation and delayed timing that is transmitted to gene expression. In contrast, a transient, strong stimulus with the same area caused rapid and uniform dynamics. These results show that digital NF-κB signaling enables multidimensional control of cellular phenotype via input profile, allowing parallel and independent control of single-cell activation probability and population heterogeneity.
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http://dx.doi.org/10.7554/eLife.08931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608393PMC
October 2015

Single-Cell Analysis: The Differences That Kill.

Authors:
Savaş Tay

Cell 2015 Sep;162(6):1208-10

Department of Biosystems Science and Engineering, ETH Zurich, Basel 4058, Switzerland; Institute for Molecular Engineering, University of Chicago, Chicago 60637, USA. Electronic address:

Using single-cell RNA sequencing, Avraham et al. investigate how variability in macrophage response to infection is controlled by variability within the pathogen population. They find that heterogeneous expression of the Salmonella virulence factor PhoP and subsequent cell-wall modifications lead to the bimodal induction of the interferon-response in infected macrophages.
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http://dx.doi.org/10.1016/j.cell.2015.08.053DOI Listing
September 2015

Automated co-culture system for spatiotemporal analysis of cell-to-cell communication.

Lab Chip 2015 May;15(10):2192-200

Department of Biosystems Science and Engineering, ETH Zürich, Mattenstrasse 26, 4058 Basel, Switzerland.

We present a microfluidic co-culture system that generates localized and precisely formulated immune signals among a population of cells, enabling spatiotemporal analysis of paracrine signal transmission between different cell types. The automated system allows us to create temporally modulated chemical inputs that can be delivered to single signal-transmitting and receiving cells in a highly controlled way. Using this system we stimulated a single macrophage with brief pulses of bacterial LPS and observed the macrophage transmitted TNF signal propagating in a population of fibroblasts via NF-κB activation. The signal receiving fibroblasts transformed the TNF signal into a spatiotemporally distributed NF-κB output, recapitulating the initiation of immune response to bacterial infection.
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http://dx.doi.org/10.1039/c5lc00182jDOI Listing
May 2015
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