| 1. |
- Welkenhuysen, Niek, 1988, et al.
(författare)
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Single-cell study links metabolism with nutrient signaling and reveals sources of variability
- 2017
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Ingår i: Bmc Systems Biology. - : Springer Science and Business Media LLC. - 1752-0509. ; 11:59
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Tidskriftsartikel (refereegranskat)abstract
- Background: The yeast AMPK/SNF1 pathway is best known for its role in glucose de/repression. When glucose becomes limited, the Snf1 kinase is activated and phosphorylates the transcriptional repressor Mig1, which is then exported from the nucleus. The exact mechanism how the Snf1-Mig1 pathway is regulated is not entirely elucidated. Results: Glucose uptake through the low affinity transporter Hxt1 results in nuclear accumulation of Mig1 in response to all glucose concentrations upshift, however with increasing glucose concentration the nuclear localization of Mig1 is more intense. Strains expressing Hxt7 display a constant response to all glucose concentration upshifts. We show that differences in amount of hexose transporter molecules in the cell could cause cell-to-cell variability in the Mig1-Snf1 system. We further apply mathematical modelling to our data, both general deterministic and a nonlinear mixed effect model. Our model suggests a presently unrecognized regulatory step of the Snf1-Mig1 pathway at the level of Mig1 dephosphorylation. Model predictions point to parameters involved in the transport of Mig1 in and out of the nucleus as a majorsource of cell to cell variability. Conclusions: With this modelling approach we have been able to suggest steps that contribute to the cell-to-cell variability. Our data indicate a close link between the glucose uptake rate, which determines the glycolytic rate, and the activity of the Snf1/Mig1 system. This study hence establishes a close relation between metabolism and signalling.
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| 2. |
- Abbaszadehbanaeiyan, Amin, et al.
(författare)
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Liver-lobule-on-a-chip microfluidic device for long-term maintenance of human hepatocytes
- 2017
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Ingår i: Presented at EMBEC’17 & NBC’17 (conference), 11-15 juni 2017, Tampere, Finland.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The pressing need for in vitro micro-physiological platforms for drug discovery and development has given rise to the emergence of organs-on-a-chip (OOC) microfluidic devices. The possibility of reproducing the native niche of each organ in a dynamic microenvironment offers advantages over current static 2D and 3D cell culture techniques. Constant removal of waste products and metabolites from the culture while providing a continuous flow of growth media is one of the major benefits of dynamic OOC systems. Additionally, physiological flow conditions can be introduced to the system allowing for reproduction of the vasculature parameters of organs in vitro. The liver is the main organ in the body for drug clearance and detoxification. The key role of the liver in the metabolism system of the human body makes it an interesting target organ to mimic in the dynamic OOC systems. Here we present a PDMS-based liver-lobule-on-a-chip microfluidic device designed to reproduce the geometrical as well as convection-diffusion mass transport aspects of the classic liver lobule. We cultured human induced pluripotent stem cell (hiPSC)-derived hepatocytes (CDI) in honeycomb cell culture chambers with involvement of two different extra-cellular matrix (ECM) materials. In the first approach, microfluidic devices were pre-treated with rat-tail collagen I and cell suspension was seeded in the devices afterwards. Cells were seeded in the devices with the supplemented plating medium (RPMI) and culture for 5 days. The medium was changed to the supplemented mainte-nance media (RPMI) thereafter and replaced every other day. In the second approach, we mixed the cell suspension with 20% diluted GeltrexTM (15 mg/ml) in a 1:1 ratio. Cells were seeded in the supplemented plating media (RPMI) and were kept under conditions identical to approach one during the hepatocyte maturation period. After day 5, however, the formulation of maintenance media was changed to supplemented DMEM/F12. Cultures were kept viable and functional for at least three weeks. In both scenarios cells formed 3D tissue-like structures and formation of bile canaliculi network was observed in the devices versus 2D static cultures. The compatibility of the device for drug toxicity applications and multi-cellular in vitro organotype construction is currently under exploration.
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| 3. |
- Almquist, Joachim, 1980, et al.
(författare)
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A nonlinear mixed effects approach for modeling the cell-to-cell variability of Mig1 dynamics in yeast.
- 2015
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:4
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Tidskriftsartikel (refereegranskat)abstract
- The last decade has seen a rapid development of experimental techniques that allow data collection from individual cells. These techniques have enabled the discovery and characterization of variability within a population of genetically identical cells. Nonlinear mixed effects (NLME) modeling is an established framework for studying variability between individuals in a population, frequently used in pharmacokinetics and pharmacodynamics, but its potential for studies of cell-to-cell variability in molecular cell biology is yet to be exploited. Here we take advantage of this novel application of NLME modeling to study cell-to-cell variability in the dynamic behavior of the yeast transcription repressor Mig1. In particular, we investigate a recently discovered phenomenon where Mig1 during a short and transient period exits the nucleus when cells experience a shift from high to intermediate levels of extracellular glucose. A phenomenological model based on ordinary differential equations describing the transient dynamics of nuclear Mig1 is introduced, and according to the NLME methodology the parameters of this model are in turn modeled by a multivariate probability distribution. Using time-lapse microscopy data from nearly 200 cells, we estimate this parameter distribution according to the approach of maximizing the population likelihood. Based on the estimated distribution, parameter values for individual cells are furthermore characterized and the resulting Mig1 dynamics are compared to the single cell times-series data. The proposed NLME framework is also compared to the intuitive but limited standard two-stage (STS) approach. We demonstrate that the latter may overestimate variabilities by up to almost five fold. Finally, Monte Carlo simulations of the inferred population model are used to predict the distribution of key characteristics of the Mig1 transient response. We find that with decreasing levels of post-shift glucose, the transient response of Mig1 tend to be faster, more extended, and displays an increased cell-to-cell variability.
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| 4. |
- Babazadeh, Roja, et al.
(författare)
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The yeast osmostress response is carbon source dependent
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Adaptation to altered osmotic conditions is a fundamental property of living cells and has been studied in detail in the yeast Saccharomyces cerevisiae. Yeast cells accumulate glycerol as compatible solute, controlled at different levels by the High Osmolarity Glycerol (HOG) response pathway. Up to now, essentially all osmostress studies in yeast have been performed with glucose as carbon and energy source, which is metabolised by glycolysis with glycerol as a by-product. Here we investigated the response of yeast to osmotic stress when yeast is respiring ethanol as carbon and energy source. Remarkably, yeast cells do not accumulate glycerol under these conditions and it appears that trehalose may partly take over the role as compatible solute. The HOG pathway is activated in very much the same way as during growth on glucose and is also required for osmotic adaptation. Slower volume recovery was observed in ethanol-grown cells as compared to glucose-grown cells. Dependence on key regulators as well as the global gene expression profile were similar in many ways to those previously observed in glucose-grown cells. However, there are indications that cells re-arrange redox-metabolism when respiration is hampered under osmostress, a feature that could not be observed in glucose-grown cells. © 2017 The Author(s).
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| 5. |
- Banaeiyan, Amin A, et al.
(författare)
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Design and fabrication of a scalable liver-lobule-on-a-chip microphysiological platform
- 2017
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Ingår i: Biofabrication. - : IOP Publishing. - 1758-5082 .- 1758-5090. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- The design and fabrication of a very large-scale liver-lobule (VLSLL)-on-a-chip device, providing a microphysiological niche for hepatocytes, is described. The device consists of an integrated network of liver-lobule-like hexagonal tissue-culture chambers constructed in a hybrid layout with a separate seed-feed network. As a key feature, each chamber contains a central outlet mimicking the central vein of a liver lobule. Separating chamber walls located between the culture area and feed network protects cells from the shear force of the convective flow. Arrays of designated passages convey nutrients to the cells by diffusion-dominated mass transport. We simulated the flow velocity, shear stress and diffusion of glucose molecules inside and outside the culture chambers under a continuous flow rate of 1 μl min-1. As proof of concept, human hepatocellular carcinoma cells (HepG2) were cultured for periods of 5 and 14 days and human-induced pluripotent stem cell (hiPSC)-derived hepatocytes for 21 days. Stabilized albumin secretion and urea synthesis were observed in the microfluidic devices and cells maintained morphology and functionality during the culture period. Furthermore, we observed 3D tissue-like structure and bile-canaliculi network formation in the chips. Future applications of the described platform include drug development and toxicity studies, as well as the modeling of patient-specific liver diseases, and integration in multi-organ human-on-a-chip systems. © 2017 IOP Publishing Ltd.
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| 6. |
- Dalsbecker, Philip, 1991, et al.
(författare)
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ENABLING INTRACELLULAR ASSAYS DURING ON-CHIP DIFFERENTIATION OF INDUCED PLURIPOTENT STEMCELLS INTO HEPATOCYTES
- 2017
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Ingår i: 17th International Multidisciplinary Scientific GeoConference SGEM 27 - 29 November, 2017, Vienna, Austria. SGEM2017 Conference Proceedings, vol. 17, issue 63, s. 225-234. - : STEF92 Technology. - 1314-2704. - 9786197408294
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Konferensbidrag (refereegranskat)abstract
- The oxygen plasma-based bonding of a liver-on-a-chip platform has been optimized for immunostaining and RNA extraction during differentiation of induced pluripotent stem cells into hepatocytes on-chip. The device is a structural and fluidic mimicry of the liver’s lobules, consisting of a double layer of polydimethylsiloxane (PDMS) bonded to glass. This bonding has been adjusted in terms of power, exposure time and post-exposure baking to make the device separable from its glass slide, making cells cultured inside the device readily accessible. A panel of functionality and maturity assays have been optimized to be used with this device. Using these assays, the differentiation and maturation of the hepatocytes can be monitored over time as the cells develop, and emerging differences between device culture and conventional well plate culture can be studied. Two proof-of-concept studies were carried out using the new bonding, showcasing that the cells can be cultured in the same way as previously reported and still be easily accessed at the end of the culture period. Together, these methods show promise as a means of studying induced pluripotent stem cell-derived hepatocytes in a realistic, liver-mimicking environment, allowing for future drug screening and personalized medicine applications.
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| 7. |
- Dalsbecker, Philip, 1991, et al.
(författare)
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On-chip maturation of induced pluripotent stem-cell derived hepatocytes
- 2017
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Ingår i: SelectBio Organ-on-a-Chip Europe (conference), 10-11 May 2017, Munich, Germany..
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The increasing interest in induced pluripotent stem cells (iPSCs) for research towards personalized medicine fits together well with recent advances in organ-on-chip development. Here, results of on-chip maturation of iPS-derived hepatocytes in a liver-on-a-chip platform are presented. The device itself, previously described by Banaeiyan et al., is designed to mimic the lobular structure of the liver and has successfully been used for 3D culture of both hepatocellular carcinoma cells (HepG2) and iPSC-derived hepatocytes. As the next step of device validation, iPS-derived hepatic cells have been matured on-chip into functional, mature hepatocytes, expressing important markers such as albumin and forming bile canalicular structures not seen in corresponding 2D cultures. Future plans for the platform include on-chip differentiation of iPSC-derived definitive endoderm cells into mature hepatocytes. To this end, a combination of immunofluorescent and metabolic assays have been tested on said definitive endoderm cells during directed differentiation into hepatocytes in a conventional 2D culture. These assays will be used to verify the corresponding differentiation in the on-chip 3D culture.
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| 8. |
- Gustavsson, Anna-Karin, 1986, et al.
(författare)
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Entrainment of heterogeneous glycolytic oscillations in single cells
- 2015
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- Cell signaling, gene expression, and metabolism are affected by cell-cell heterogeneity and random changes in the environment. The effects of such fluctuations on cell signaling and gene expression have recently been studied intensively using single-cell experiments. In metabolism heterogeneity may be particularly important because it may affect synchronisation of metabolic oscillations, an important example of cell-cell communication. This synchronisation is notoriously difficult to describe theoretically as the example of glycolytic oscillations shows: neither is the mechanism of glycolytic synchronisation understood nor the role of cell-cell heterogeneity. To pin down the mechanism and to assess its robustness and universality we have experimentally investigated the entrainment of glycolytic oscillations in individual yeast cells by periodic external perturbations. We find that oscillatory cells synchronise through phase shifts and that the mechanism is insensitive to cell heterogeneity (robustness) and similar for different types of external perturbations (universality).
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| 9. |
- Gustavsson, Anna-Karin, 1986, et al.
(författare)
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Studying Glycolytic Oscillations in Individual Yeast Cells by Combining Fluorescence Microscopy with Microfluidics and Optical Tweezers.
- 2019
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Ingår i: Current protocols in cell biology. - : Wiley. - 1934-2616 .- 1934-2500. ; 82:1
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Tidskriftsartikel (refereegranskat)abstract
- In this unit, we provide a clear exposition of the methodology employed to study dynamic responses in individual cells, using microfluidics for controlling and adjusting the cell environment, optical tweezers for precise cell positioning, and fluorescence microscopy for detecting intracellular responses. This unit focuses on the induction and study of glycolytic oscillations in single yeast cells, but the methodology can easily be adjusted to examine other biological questions and cell types. We present a step-by-step guide for fabrication of the microfluidic device, for alignment of the optical tweezers, for cell preparation, and for time-lapse imaging of glycolytic oscillations in single cells, including a discussion of common pitfalls. A user who follows the protocols should be able to detect clear metabolite time traces over the course of up to an hour that are indicative of dynamics on the second scale in individual cells during fast and reversible environmental adjustments. © 2018 by John Wiley & Sons, Inc.
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| 10. |
- Mojica Benavides, Martin, 1983, et al.
(författare)
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An Optical Tweezers, Epi-Fluorescence/Spinning disk confocal- and microfluidic-setup for synchronization studies of glycolytic oscillations in living yeast cells
- 2016
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Ingår i: Proceedings SPIE 9922, Optical Trapping and Optical Micromanipulation XIII. San Diego; USA. 28 August -1 September 2016. - : SPIE. - 9781510602359
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Konferensbidrag (refereegranskat)abstract
- Due to the significant importance of glycolytic oscillations studies and the recent breakthroughs on single cell analysis, a further interest arrives with intracellular and intercellular responses. Understanding cell-cell communication can give insight to oscillatory behaviors in biological systems, such as insulin secretion from pancreatic beta-cells. The aim of this work consists on the manipulation of living yeast cells to study propagation and synchronization of induced glycolytic oscillations. A setup, consisting of an optical tweezers system and microfluidic devices coupled with fluorescence imaging was designed to perform a time dependent observation during artificially induced glycolytic oscillations. Multi-channel flow devices and diffusion chambers were fabricated using soft lithography. Automatized pumps controlled specific flow rates of infused glucose and cyanide solutions, used to induce the oscillations. Flow and diffusion in the microfluidic devices were simulated to assure experimentally the desired coverage of the solutions across the yeast cells, a requirement for time dependent measurements. Using near infrared optical tweezers, yeast cells were trapped and positioned in array configurations, ranging from a single cell to clusters of various symmetries, in order to obtain information about cell-cell communications during the metabolic cycles. Confocal illumination of an entire focal plane using a spinning disk, will allow acquirement of NADH periodic fluorescence signals during glycolytic oscillations. This method permits an improvement of the 2D projection images obtained with wide field microscopy to a tomographic description of the subcellular propagation of the oscillations.
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| 11. |
- van Niekerk, David, et al.
(författare)
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Phosphofructokinase controls the acetaldehyde induced phase shift in isolated yeast glycolytic oscillators.
- 2019
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Ingår i: The Biochemical journal. - 1470-8728. ; 476:2, s. 353-363
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Tidskriftsartikel (refereegranskat)abstract
- The response of oscillatory systems to external perturbations is crucial for emergent properties such as synchronization and phase locking, and can be quantified in a phase response curve. In individual, oscillating yeast cells, we characterized experimentally the phase response of glycolytic oscillations for external acetaldehyde pulses, and followed the transduction of the perturbation through the system. Subsequently, we analyzed the control of the relevant system components in a detailed mechanistic model. The observed responses are interpreted in terms of the functional coupling and regulation in the reaction network. We find that our model quantitatively predicts the phase dependent phase shift observed in the experimental data. The phase shift is in agreement with an adaptation leading to synchronization with an external signal. Our model analysis establishes that phosphofructokinase plays a key role in the phase shift dynamics as shown in the phase response curve, and adaptation time to external perturbations. Specific mechanism-based interventions, made possible through such analyses of detailed models, can improve upon standard trial and error methods, e.g. melatonin supplementation to overcome jet-lag, which are error prone, specifically, since the effects are phase and dose dependent.
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| 12. |
- Welkenhuysen, Niek, 1988, et al.
(författare)
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Applying microfluidic systems to study effects of glucose at single-cell level
- 2018
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Ingår i: Glucose Transport. Methods in Molecular Biology. - New York : Humana Press. - 9781493975075 ; 1713, s. 109-121
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Microfluidic systems in combination with microscopy (e.g., fluorescence) can be a powerful tool to study, at single-cell level, the behavior and morphology of biological cells after uptake of glucose. Here, we briefly discuss the advantages of using microfluidic systems. We further describe how microfluidic systems are fabricated and how they are utilized. Finally, we discuss how the large amount of data can be analyzed in a “semi-automatic” manner using custom-made software. In summary, we provide a guide to how to use microfluidic systems in single-cell studies. © Springer Science+Business Media LLC 2018.
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| 13. |
- Wolfson, D., et al.
(författare)
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Rapid 3D fluorescence imaging of individual optically trapped living immune cells
- 2015
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Ingår i: Journal of Biophotonics. - : Wiley. - 1864-0648 .- 1864-063X. ; 8:3, s. 208-216
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate an approach to rapidly characterize living suspension cells in 4 dimensions while they are immobilized and manipulated within optical traps. A single, high numerical aperture objective lens is used to separate the imaging plane from the trapping plane. This facilitates full control over the position and orientation of multiple trapped cells using a spatial light modulator, including directed motion and object rotation, while also allowing rapid 4D imaging. This system is particularly useful in the handling and investigation of the behavior of non-adherent immune cells. We demonstrate these capabilities by imaging and manipulating living, fluorescently stained Jurkat T cells.
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