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  • 1. Benninger, R. K. P.
    et al.
    Önfelt, Björn
    Neil, M. A. A.
    Davis, D. M.
    French, P. M. W.
    Fluorescence imaging of two-photon linear dichroism: Cholesterol depletion disrupts molecular orientation in cell membranes2005In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 88, no 1, p. 609-622Article in journal (Refereed)
    Abstract [en]

    The plasma membrane of cells is an ordered environment, giving rise to anisotropic orientation and restricted motion of molecules and proteins residing in the membrane. At the same time as being an organized matrix of defined structure, the cell membrane is heterogeneous and dynamic. Here we present a method where we use fluorescence imaging of linear dichroism to measure the orientation of molecules relative to the cell membrane. By detecting linear dichroism as well as fluorescence anisotropy, the orientation parameters are separated from dynamic properties such as rotational diffusion and homo energy transfer ( energy migration). The sensitivity of the technique is enhanced by using two-photon excitation for higher photo-selection compared to single photon excitation. We show here that we can accurately image lipid organization in whole cell membranes and in delicate structures such as membrane nanotubes connecting two cells. The speed of our wide-field imaging system makes it possible to image changes in orientation and anisotropy occurring on a subsecond timescale. This is demonstrated by time-lapse studies showing that cholesterol depletion rapidly disrupts the orientation of a fluorophore located within the hydrophobic region of the cell membrane but not of a surface bound probe. This is consistent with cholesterol having an important role in stabilizing and ordering the lipid tails within the plasma membrane.

  • 2. Benninger, Richard K. P.
    et al.
    Hofmann, Oliver
    Önfelt, Björn
    Munro, Ian
    Dunsby, Chris
    Davis, Daniel M.
    Neil, Mark A. A.
    French, Paul M. W.
    de Mello, Andrew J.
    Fluorescence-lifetime imaging of DNA-dye interactions within continuous-flow microfluidic systems2007In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 46, no 13, p. 2228-2231Article in journal (Refereed)
  • 3. Benninger, Richard K. P.
    et al.
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Young, Stephen
    Taner, Sabrina B.
    Culley, Fiona I.
    Schnyder, Tim
    Neil, Mark A. A.
    Wuestner, Daniel
    French, Paul M. W.
    Davis, Daniel M.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Live Cell Linear Dichroism Imaging Reveals Extensive Membrane Ruffling within the Docking Structure of Natural Killer Cell Immune Synapses2009In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 96, no 2, p. L13-L15Article in journal (Refereed)
    Abstract [en]

    We have applied fluorescence imaging of two-photon linear dichroism to measure the subresolution organization of the cell membrane during formation of the activating (cytolytic) natural killer (NK) cell immune synapse (IS). This approach revealed that the NK cell plasma membrane is convoluted into ruffles at the periphery, but not in the center of a mature cytolytic NK cell IS. Time-lapse imaging showed that the membrane ruffles formed at the initial point of contact between NK cells and target cells and then spread radialy across the intercellular contact as the size of the IS increased, becoming absent from the center of the mature synapse. Understanding the role of such extensive membrane ruff ling in the assembly of cytolytic synapses is an intriguing new goal.

  • 4.
    Brandt, Ludwig
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics.
    Pfefferle, Aline
    Goodridge, Jodie
    Malmberg, Karl-Johan
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Cytotoxicity and killing kinetics of KIR educated NK cells2017In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 86, no 4, p. 301-301Article in journal (Other academic)
  • 5. Carannante, Valentina
    et al.
    Olofsson, Karl
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Van Oojen, Hanna
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Edwards, Steven
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Lundqvist, Andreas
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Novel platform for studying infiltration, migration and cytotoxicity of human Natural Killer cells in solid tumors2017In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 86, no 4, p. 315-315Article in journal (Other academic)
  • 6.
    Christakou, Athanasia E.
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Ohlin, Mathias
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Kadri, N.
    Frisk, Thomas
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics. Karolinska Institute, Sweden.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Characterization of natural killer cells' cytotoxic heterogeneity using an array of sono-cages2012In: Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, Chemical and Biological Microsystems Society , 2012, p. 1555-1557Conference paper (Refereed)
    Abstract [en]

    Using a multi-well device as an array of sono-cages for single cell analysis, we quantify for the first time the heterogeneity of natural killer (NK) cells' cytotoxic response against cancer cells. We report a fraction of inactive NK cells within the tested population (36%), as well as the existence of few 'serial killers' that eliminate up to six targets during 4 hours. We also characterize the multi-well acoustic device in terms of trapping efficiency at different actuation voltages, using adherent and non-adherent cell lines. We show that the acoustic forces applied on the cells can be compared to forces of biological processes (i.e. cell adherence).

  • 7.
    Christakou, Athanasia. E.
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Ohlin, Mathias
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Khorshidi, Mohammad Ali
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Frisk, Thomas
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Aggregation and long-term positioning of cells by ultrasound in a multi-well microchip for high-resolution imaging of the natural killer cell immune synapse2011In: 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011, 2011, p. 329-331Conference paper (Refereed)
    Abstract [en]

    In this study we investigate the ability of Natural Killer (NK) cells to form ultrasound-mediated intercellular contacts with target cells in a multi-well microdevice by high-resolution confocal-microscopy imaging of inhibitory immune synapses. Furthermore, we compare the NK-Target cell cluster migration with and without ultrasound actuation. Experiments indicate that clusters of cells are positioned and maintained centered in the wells for 17 hours when they are exposed continuously to ultrasound. Our system can be used for both screening high numbers of events in low resolution and also for high resolution imaging of long term cell-cell interactions.

  • 8.
    Christakou, Athanasia E.
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Ohlin, Mathias
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Khorshidi, Mohammad Ali
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Kadri, Nadir
    Frisk, Thomas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Live cell imaging in a micro-array of acoustic traps facilitates quantification of natural killer cell heterogeneity2013In: Integrative Biology, ISSN 1757-9694, E-ISSN 1757-9708, Vol. 5, no 4, p. 712-719Article in journal (Refereed)
    Abstract [en]

    Natural killer (NK) cells kill virus-infected or cancer cells through the release of cytotoxic granules into a tight intercellular contact. NK cell populations comprise individual cells with varying sensitivity to distinct input signals, leading to disparate responses. To resolve this NK cell heterogeneity, we have designed a novel assay based on ultrasound-assisted cell-cell aggregation in a multiwell chip allowing high-resolution time-lapse imaging of one hundred NK-target cell interactions in parallel. Studying human NK cells' ability to kill MHC class I deficient tumor cells, we show that approximately two thirds of the NK cells display cytotoxicity, with some NK cells being particularly active, killing up to six target cells during the assay. We also report that simultaneous interaction with several susceptible target cells increases the cytotoxic responsiveness of NK cells, which could be coupled to a previously unknown regulatory mechanism with implications for NK-mediated tumor elimination.

  • 9.
    Christakou, Athanasia E.
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Ohlin, Mathias
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Solid tumor spheroid formation by temperature-controlled high voltage ultrasound in a multi-well microdevice2014In: 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014, Chemical and Biological Microsystems SocietyChemical and Biological Microsystems Society , 2014, p. 573-575Conference paper (Refereed)
    Abstract [en]

    In the present work we demonstrate effective 3D growth of human hepatocellular carcinoma (HCC) HepG2 cell spheroids in parallel in a multi-well microdevice actuated with high voltage ultrasound in a temperature-controlled system. We compare the spheroid formation during continuous ultrasound exposure for one week where we formed spheroids in 59% of the wells, with the spheroid formation without ultrasound actuation, where we obtained 0% spheroids. Furthermore, we present an application of the tumor spheroids for investigating natural killer (NK) cells behavior against solid tumors.

  • 10.
    Christakou, Athanasia E.
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Ohlin, Mathias
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Ultrasonic three-dimensional cell culture on chip for dynamic studies of tumor immune surveillance by natural killer cellsManuscript (preprint) (Other academic)
  • 11.
    Christakou, Athanasia E.
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Ohlin, Mathias
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Ultrasound-assisted three-dimensional tumor formation in a multi well microplate for monitoring natural killer cell functional behaviorManuscript (preprint) (Other academic)
  • 12.
    Christakou, Athanasia
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Ohlin, Mathias
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Characterization of natural killer cell immune surveillance against solid liver tumors2015In: MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Chemical and Biological Microsystems Society , 2015, p. 915-917Conference paper (Refereed)
    Abstract [en]

    We demonstrate a method for investigating natural killer (NK) cell aggression against ultrasound-assisted human hepatocellular carcinoma (HCC) HepG2 solid tumors in a multi-well microdevice. We quantify the activity of human primary IL-2 activated NK cells against HepG2 tumors for up to five days and we present the correlation between NK cell numbers versus average tumor volume and final tumor outcome (growth or defeat). We suggest future applications on formation of tumors originated from primary tumors cells and other tumor components as well as primary NK originating from the patient for use in personalized immunotherapy.

  • 13.
    Christakou, Athanasia
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Ohlin, Mathias
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Ultrasonic three-dimensional on-chip cell culture for dynamic studies of tumor immune surveillance by natural killer cells2015In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 15, no 15, p. 3222-31Article in journal (Refereed)
    Abstract [en]

    We demonstrate a simple method for three-dimensional (3D) cell culture controlled by ultrasonic standing waves in a multi-well microplate. The method gently arranges cells in a suspension into a single aggregate in each well of the microplate and, by this, nucleates 3D tissue-like cell growth for culture times between two and seven days. The microplate device is compatible with both high-resolution optical microscopy and maintenance in a standard cell incubator. The result is a scaffold- and coating-free method for 3D cell culture that can be used for controlling the cellular architecture, as well as the cellular and molecular composition of the microenvironment in and around the formed cell structures. We demonstrate the parallel production of one hundred synthetic 3D solid tumors comprising up to thousands of human hepatocellular carcinoma (HCC) HepG2 cells, we characterize the tumor structure by high-resolution optical microscopy, and we monitor the functional behavior of natural killer (NK) cells migrating, docking and interacting with the tumor model during culture. Our results show that the method can be used for determining the collective ability of a given number of NK cells to defeat a solid tumor having a certain size, shape and composition. The ultrasound-based method itself is generic and can meet any demand from applications where it is advantageous to monitor cell culture from production to analysis of 3D tissue or tumor models using microscopy in one single microplate device.

  • 14. Coates, C. G.
    et al.
    Olofsson, J.
    Coletti, M.
    McGarvey, J. J.
    Önfelt, Björn
    Lincoln, P.
    Norden, B.
    Tuite, E.
    Matousek, P.
    Parker, A. W.
    Picosecond time-resolved resonance Raman probing of the light-switch states of Ru(Phen)(2)dppz (2+)2001In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 105, no 50, p. 12653-12664Article in journal (Refereed)
    Abstract [en]

    Picosecond time-resolved resonance Raman (picosecond-TR3) spectroscopy has been used to conduct an extensive photophysical characterization of the light- switch complex [Ru(phen)(2)dppz](2+) as a function of environment, in which studies have been carried out in aqueous and nonaqueous media and in DNA. The results are considered in rotation to a previous report describing environment-sensitive lowest triplet MLCT states. Vibrational marker features and enhancement patterns were used to determine the rapid progression (< 20 ps) between two triplet MLCT states in aqueous environment, followed by subnanosecond, nonradiative deactivation to the ground state. In nonaqueous environment, the long-lived, emissive triplet MLCT state is spectrally identified as the short-lived first triplet MLCT state observed in water, in agreement with the earlier proposed mechanism. The present data are shown to correlate well with previous nanosecond RR findings for the complex in each environment. Interestingly, a precursor state has been identified upon excitation in both nonaqueous solvent and in DNA, which precedes the triplet MLCT state, and the lifetime of which appears to be environment dependent. Observation of this state is discussed in relation to other recent femtosecond spectroscopic studies on this complex.

  • 15. Dunsby, C.
    et al.
    Lanigan, P. M. P.
    McGinty, J.
    Elson, D. S.
    Requejo-Isidro, J.
    Munro, I.
    Galletly, N.
    McCann, F.
    Treanor, B.
    Önfelt, Björn
    Davis, D. M.
    Neil, M. A. A.
    French, P. M. W.
    An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy2004In: Journal of Physics D: Applied Physics, ISSN 0022-3727, E-ISSN 1361-6463, Vol. 37, no 23, p. 3296-3303Article in journal (Refereed)
    Abstract [en]

    Fluorescence imaging is used widely in microscopy and macroscopic imaging applications for fields ranging from biomedicine to materials science. A critical component for any fluorescence imaging system is the excitation source. Traditionally, wide-field systems use filtered thermal or arc-generated white light sources, while point scanning confocal microscope systems require spatially coherent (point-like) laser sources. Unfortunately, the limited range of visible wavelengths available from conventional laser sources constrains the design and usefulness of fluorescent probes in confocal microscopy. A 'hands-off' laser-like source, electronically tunable across the visible spectrum, would be invaluable for fluorescence imaging and provide new opportunities, e.g. automated excitation fingerprinting and in situ measurement of excitation cross-sections. Yet more information can be obtained using fluorescence lifetime imaging (FLIM), which requires that the light source be pulsed or rapidly modulated. We show how a white light continuum, generated by injecting femtosecond optical radiation into a micro-structured optical fibre, coupled with a simple prism-based tunable filter arrangement, can fulfil all these roles as a continuously electronically tunable (435-1150 nm) visible ultrafast light source in confocal, wide-field and FLIM systems.

  • 16. Eleme, K.
    et al.
    Taner, S. B.
    Önfelt, Björn
    Collinson, L. M.
    McCann, F. E.
    Chalupny, N. J.
    Cosman, D.
    Hopkins, C.
    Magee, A. I.
    Davis, D. M.
    Cell surface organization of stress-inducible proteins ULBP and MICA that stimulate human NK cells and T cells via NKG2D2004In: Journal of Experimental Medicine, ISSN 0022-1007, E-ISSN 1540-9538, Vol. 199, no 7, p. 1005-1010Article in journal (Refereed)
    Abstract [en]

    Cell surface proteins major histocompatibility complex (MHC) class I-related chain A (MICA) and UL16-binding proteins (ULBP) 1, 2, and 3 are up-regulated upon infection or tumor transformation and can activate human natural killer (NK) cells. Patches of cross-linked raft resident ganglioside GM1 colocalized with ULBP1, 2, 3, or MICA, but not CD45. Thus, ULBPs and MICA are expressed in lipid rafts at the cell surface. Western blotting revealed that glycosylphosphatidylinositol (GPI)-anchored ULBP3 but not transmembrane MICA, MHC class I protein, or transferrin receptor, accumulated in detergent-resistant membranes containing GM1. Thus, MICA may have a weaker association with lipid rafts than ULBP3, yet both proteins accumulate at an activating human N-K cell immune synapse. Target cell lipid rafts marked by green fluorescent protein-tagged GPI also accumulate with ULBP3 at some synapses. Electron microscopy reveals constitutive clusters of ULBP at the cell surface. Regarding a specific molecular basis for the organization of these proteins, ULBP1, 2, and 3 and MICA are lipid modified. ULBP1, 2, and 3 are GPI anchored, and we demonstrate here that MICA is S-acylated. Finally, expression of a truncated form of MICA that lacks the putative site for S-acylation and the cytoplasmic tall can be expressed at the cell surface, but is unable to activate NK cells.

  • 17. Enqvist, M.
    et al.
    Ask, E. H.
    Forslund, E.
    Carlsten, M.
    Abrahamsen, G.
    Béziat, V.
    Andersson, S.
    Schaffer, M.
    Spurkland, A.
    Bryceson, Y.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Malmberg, K. -J
    Coordinated expression of DNAM-1 and LFA-1 in educated NK cells2015In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 194, no 9, p. 4518-4527Article in journal (Refereed)
    Abstract [en]

    The functional capacity of NK cells is dynamically tuned by integrated signals from inhibitory and activating cell surface receptors in a process termed NK cell education. However, the understanding of the cellular and molecular mechanisms behind this functional tuning is limited. In this study, we show that the expression of the adhesion molecule and activation receptor DNAX accessory molecule 1 (DNAM-1) correlates with the quantity and quality of the inhibitory input by HLA class I-specific killer cell Ig-like receptors and CD94/NKG2A as well as with the magnitude of functional responses. Upon target cell recognition, the conformational state of LFA-1 changed in educated NK cells, associated with rapid colocalization of both active LFA-1 and DNAM-1 at the immune synapse. Thus, the coordinated expression of LFA-1 and DNAM-1 is a central component of NK cell education and provides a potential mechanism for controlling cytotoxicity by functionally mature NK cells.

  • 18.
    Fogelqvist, Emelie
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Kördel, Mikael
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Carannante, Valentina
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Hertz, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics. Karolinska Institutet, Sweden.
    Laboratory cryo x-ray microscopy for 3D cell imaging2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 13433Article in journal (Refereed)
    Abstract [en]

    Water-window x-ray microscopy allows two-and three-dimensional (2D and 3D) imaging of intact unstained cells in their cryofixed near-native state with unique contrast and high resolution. Present operational biological water-window microscopes are based at synchrotron facilities, which limits their accessibility and integration with complementary methods. Laboratory-source microscopes have had difficulty addressing relevant biological tasks with proper resolution and contrast due to long exposure times and limited up-time. Here we report on laboratory cryo x-ray microscopy with the exposure time, contrast, and reliability to allow for routine high-spatial resolution 3D imaging of intact cells and cell-cell interactions. Stabilization of the laser-plasma source combined with new optics and sample preparation provide high-resolution cell imaging, both in 2D with ten-second exposures and in 3D with twenty-minute tomography. Examples include monitoring of the distribution of carbon-dense vesicles in starving HEK293T cells and imaging the interaction between natural killer cells and target cells.

  • 19.
    Fogelqvist, Emelie
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Kördel, Mikael
    Carannante, Valentina
    Önfelt, Björn
    Hertz, Hans
    Laboratory cryo x-ray microscopy for 3D cell imagingManuscript (preprint) (Other academic)
  • 20. Forslund, E.
    et al.
    Guldevall, Karolin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Olofsson, Per E.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Frisk, Thomas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Christakou, Athanasia E.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Novel microchip-based tools facilitating live cell imaging and assessment of functional heterogeneity within NK cell populations2012In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 3, no OCT, p. 300-Article in journal (Refereed)
    Abstract [en]

    Each individual has a heterogeneous pool of NK cells consisting of cells that may be specialized towards specific functional responses such as secretion of cytokines or killing of tumor cells. Many conventional methods are not fit to characterize heterogeneous populations as they measure the average response of all cells. Thus, there is a need for experimental platforms that provide single cell resolution. In addition, there are transient and stochastic variations in functional responses at the single cell level, calling for methods that allow studies of many events over extended periods of time. This paper presents a versatile microchip platform enabling long-term microscopic studies of individual NK cells interacting with target cells. Each microchip contains an array of microwells, optimized for medium or high-resolution time-lapse imaging of single or multiple NK and target cells, or for screening of thousands of isolated NK-target cell interactions. Individual NK cells confined with target cells in small microwells is a suitable setup for high-content screening and rapid assessment of heterogeneity within populations, while microwells of larger dimensions are appropriate for studies of NK cell migration and sequential interactions with multiple target cells. By combining the chip technology with ultrasonic manipulation, NK and target cells can be forced to interact and positioned with high spatial accuracy within individual microwells.This setup effectively and synchronously creates NK-target conjugates at hundreds of parallel positions in the microchip. Thus, this facilitates assessment of temporal aspects of NK-target cell interactions, e.g., conjugation, immune synapse formation, and cytotoxic events.The microchip platform presented here can be used to effectively address questions related to fundamental functions of NK cells that can lead to better understanding of how the behavior of individual cells add up to give a functional response at the population level.

  • 21. Forslund, Elin
    et al.
    Sohlberg, Ebba
    Enqvist, Monika
    Olofsson, Per E.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Malmberg, Karl-Johan
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Inst, Sci Life Lab, Dept Microbiol Tumor & Cell Biol, S-17165 Stockholm, Sweden.
    Microchip-Based Single-Cell Imaging Reveals That CD56(dim) CD57(-)KIR(-)NKG2A(+) NK Cells Have More Dynamic Migration Associated with Increased Target Cell Conjugation and Probability of Killing Compared to CD56(dim)CD57(-)KIR(-)NKG2A(-) NK Cells2015In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 195, no 7, p. 3374-3381Article in journal (Refereed)
    Abstract [en]

    NK cells are functionally educated by self-MHC specific receptors, including the inhibitory killer cell Ig-like receptors (KIRs) and the lectin-like CD94/NKG2A heterodimer. Little is known about how NK cell education influences qualitative aspects of cytotoxicity such as migration behavior and efficacy of activation and killing at the single-cell level. In this study, we have compared the behavior of FACS-sorted CD56(dim)CD57(-)KIR(-)NKG2A(+) (NKG2A(+)) and CD56(dim)CD57(-)KIR(-)NKG2A(+) (lacking inhibitory receptors; IR-) human NK cells by quantifying migration, cytotoxicity, and contact dynamics using microchip-based live cell imaging. NKG2A(+) NK cells displayed a more dynamic migration behavior and made more contacts with target cells than IR-NK cells. NKG2A(+) NK cells also more frequently killed the target cells once a conjugate had been formed. NK cells with serial killing capacity were primarily found among NKG2A(+) NK cells. Conjugates involving IR- NK cells were generally more short-lived and IR- NK cells did not become activated to the same extent as NKG2A(+) NK cells when in contact with target cells, as evident by their reduced spreading response. In contrast, NKG2A(+) and IR- NK cells showed similar dynamics in terms of duration of conjugation periods and NK cell spreading response in conjugates that led to killing. Taken together, these observations suggest that the high killing capacity of NKG2A(+) NK cells is linked to processes regulating events in the recognition phase of NK-target cell contact rather than events after cytotoxicity has been triggered.

  • 22. Frisk, T.
    et al.
    Guldevall, Karolin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Vanherbergen, B.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Live-cell imaging of natural killer cell mediated tumor rejection in arrays of microwells2010In: 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010: Volume 2, 2010, p. 950-952Conference paper (Refereed)
    Abstract [en]

    Due to the inherent heterogeneity of immune cell populations a comprehensive view of immune functions can only be achieved by collecting data from many individual cells. We here demonstrate a novel multi-well microchip platform for cell analysis, which we use to study the interaction between natural killer (NK) cells and their target cells.

  • 23.
    Frisk, Thomas
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Khorshidi, Mohammad Ali
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Guldevall, Karolin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    A silicon-glass microwell platform for high-resolution imaging and high-content screening with single cell resolution2011In: Biomedical microdevices (Print), ISSN 1387-2176, E-ISSN 1572-8781, Vol. 13, no 4, p. 683-693Article in journal (Refereed)
    Abstract [en]

    We present a novel microwell array platform suited for various cell-imaging assays where single cell resolution is important. The platform consists of an exchangeable silicon-glass microchip for cell biological applications and a custom made holder that fits in conventional microscopes. The microchips presented here contain arrays of miniature wells, where the well sizes and layout have been designed for different applications, including single cell imaging, studies of cell-cell interactions or ultrasonic manipulation of cells. The device has been designed to be easy to use, to allow long-term assays (spanning several days) with read-outs based on high-resolution imaging or high-content screening. This study is focused on screening applications and an automatic cell counting protocol is described and evaluated. Finally, we have tested the device and automatic counting by studying the selective survival and clonal expansion of 721.221 B cells transfected to express HLA Cw6-GFP compared to untransfected 721.221 B cells when grown under antibiotic selection for 3 days. The device and automated analysis protocol make up the foundation for development of several novel cellular imaging assays.

  • 24.
    Girnyk, Maksym A.
    et al.
    KTH, School of Electrical Engineering (EES), Communication Theory.
    Vehkapera, Mikko
    Rasmussen, Lars Kildehoj
    Christakou, Athanasia
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Onfelt, Bjorn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Orange, Jordan
    Lytic granule convergence is essential for NK cells to promote targeted killing while preventing collateral damage2016In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 196Article in journal (Other academic)
  • 25. Goodridge, Jodie P.
    et al.
    Onfelt, Bjorn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. Karolinska Inst, Microbiol Tumor & Cell Biol.
    Malmberg, Karl-Johan
    Newtonian cell interactions shape natural killer cell education2015In: Immunological Reviews, ISSN 0105-2896, E-ISSN 1600-065X, Vol. 267, no 1, p. 197-213Article, review/survey (Refereed)
    Abstract [en]

    Newton's third law of motion states that for every action on a physical object there is an equal and opposite reaction. The dynamic change in functional potential of natural killer (NK) cells during education bears many features of such classical mechanics. Cumulative physical interactions between cells, under a constant influence of homeostatic drivers of differentiation, lead to a reactive spectrum that ultimately shapes the functionality of each NK cell. Inhibitory signaling from an array of self-specific receptors appear not only to suppress self-reactivity but also aid in the persistence of effector functions over time, thereby allowing the cell to gradually build up a functional potential. Conversely, the frequent non-cytolytic interactions between normal cells in the absence of such inhibitory signaling result in continuous stimulation of the cells and attenuation of effector function. Although an innate cell, the degree to which the fate of the NK cell is predetermined versus its ability to adapt to its own environment can be revealed through a Newtonian view of NK cell education, one which is both chronological and dynamic. As such, the development of NK cell functional diversity is the product of qualitatively different physical interactions with host cells, rather than simply the sum of their signals or an imprint based on intrinsically different transcriptional programs.

  • 26.
    Guldevall, Karolin
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brandt, Ludwig
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Forslund, Elin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Sweden.
    Olofsson, Karl
    Frisk, Thomas W.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Olofsson, Per E.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gustafsson, Karin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Manneberg, Otto
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Karre, Klas
    Uhlin, Michael
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Microchip screening Platform for single cell assessment of NK cell cytotoxicity2016In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 7, article id 119Article in journal (Refereed)
    Abstract [en]

    Here, we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon-glass microchip containing 32,400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis, the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (approximate to 75%) were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (>= 3) target cells within the 12-h long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors, it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g., in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy.

  • 27.
    Guldevall, Karolin
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Frisk, Thomas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Khorsidi, Mohammed Ali
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Manneberg, Otto
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Christakou, Athanasia
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Imaging immune surveillance by individual Natural Killer cells isolated in arrays of nanoliter wells2010Conference paper (Refereed)
  • 28.
    Guldevall, Karolin
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gustafsson, Karin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Forslund, Elin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Frisk, Thomas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Manneberg, Otto
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Olofsson, Per E.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Tauriainen, Johanna
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Stikvoort, Arwen
    Karolinska Institute.
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Mattsson, Jonas
    Karolinska Institute.
    Kärre, Klas
    Karolinska Institute.
    Uhlin, Michael
    Karolinska Institute.
    Önfelt, Bjorn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Microchip screening platform for assessment of cytotoxic effector cellsManuscript (preprint) (Other academic)
    Abstract [en]

    Here we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) or T cells within larger populations. Human primary NK cells or human Epstein-Barr virus (EBV)- specific T cells were distributed across a silicon-glass microchip containing 32 400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis the numbers of effector and live or dead target cells in each well could be assessed at different time-points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic to the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12 hours long experiment. We demonstrate that this assay can be used to enumerate and characterize cytotoxic cells, something that could find clinical applications, e.g. in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy.

  • 29.
    Guldevall, Karolin
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Frisk, Thomas
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet.
    Hurtig, Johan
    Department of Chemsitry, University of Washington, Seattle, USA.
    Christakou, Athanasia
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Manneberg, Otto
    Department of Environmental Health, Harvard School of Public Health, Boston, USA.
    Lindström, Sara
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Imaging Immune Surveillance of Individual Natural Killer Cells Confined in Microwell Arrays2010In: PLOS ONE, ISSN 1932-6203, Vol. 5, no 11, p. e15453-Article in journal (Refereed)
    Abstract [en]

    New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level.

  • 30. Hsu, Hsiang-Ting
    et al.
    Mace, Emily M.
    Carisey, Alexandre F.
    Viswanath, Dixita I.
    Christakou, Athanasia
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Önfelt, Bjorn
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Orange, Jordan S.
    NK cells converge lytic granules to promote cytotoxicity and prevent bystander killing2016In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 215, no 6, p. 875-889Article in journal (Refereed)
    Abstract [en]

    Natural killer (NK) cell activation triggers sequential cellular events leading to destruction of diseased cells. We previously identified lytic granule convergence, a dynein-and integrin signal-dependent movement of lysosome-related organelles to the microtubule-organizing center, as an early step in the cell biological process underlying NK cell cytotoxicity. Why lytic granules converge during NK cell cytotoxicity, however, remains unclear. We experimentally controlled the availability of human ligands to regulate NK cell signaling and promote granule convergence with either directed or nondirected degranulation. By the use of acoustic trap microscopy, we generated specific effector-target cell arrangements to define the impact of the two modes of degranulation. NK cells with converged granules had greater targeted and less nonspecific "bystander" killing. Additionally, NK cells in which dynein was inhibited or integrin blocked under physiological conditions demonstrated increased nondirected degranulation and bystander killing. Thus, NK cells converge lytic granules and thereby improve the efficiency of targeted killing and prevent collateral damage to neighboring healthy cells.

  • 31. Hurtig, Johan
    et al.
    Chiu, Daniel T.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Intercellular nanotubes: insights from imaging studies and beyond2010In: WILEY INTERDISCIPLINARY REVIEWS-NANOMEDICINE AND NANOBIOTECHNOLOGY, ISSN 1939-5116, Vol. 2, no 3, p. 260-276Article, review/survey (Refereed)
    Abstract [en]

    Cell-cell communication is critical to the development, maintenance, and function of multicellular organisms. Classical mechanisms for intercellular communication include secretion of molecules into the extracellular space and transport of small molecules through gap junctions. Recent reports suggest that cells also can communicate over long distances via a network of transient intercellular nanotubes. Such nanotubes have been shown to mediate intercellular transfer of organelles as well as membrane components and cytoplasmic molecules. Moreover, intercellular nanotubes have been observed in vivo and have been shown to enhance the transmission of pathogens such as human immunodeficiency virus (HIV)-1 and prions in vitro. These studies indicate that intercellular nanotubes may play a role both in normal physiology and in disease. (C) 2010 John Wiley & Sons, Inc. WIREs Nanomed Nanobiotechnol 2010 2 260-276

  • 32.
    Kamali-Zare, Padideh
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Kowalewski, Jacob M
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Zelenina, Marina
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Aperia, Anita
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Role of diffusion limited space on water and salt homeostasisManuscript (preprint) (Other academic)
  • 33.
    Kamali-Zare, Padideh
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Kowalewski, Jacob
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Zelenina, Marina
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Aperia, Anita
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Diffusion limited space contributes to K+ siphoning by regulation of K+ and water homeostasis in astrocytesManuscript (preprint) (Other academic)
    Abstract [en]

    Diffusion Limited Space (DLS) is defined as a region where diffusion is limited by the geometry. Two examples of DLS in the brain are the neuronal synapse, and the narrow region between astrocyte endfeet and blood capillaries. In a series of geometrical models we show that DLS plays a role in regulation of water and K+ homeostasis in the brain by an indirect functional coupling of aquaporins (AQPs) and inward rectifying K+ (Kir) channels in a membrane microdomain.

    1. Simulations in geometrical models of a synapse region show that following a step increase in synaptic [K+], both K+ and water are taken up by astrocytes via AQPs and Kir channels lining the synapse.  This uptake creates a transient depletion of water in the synapse region that, enhanced by the DLS, facilitates K+ uptake and an efficient clearance of excess K+ from the synapse.

    2. Simulations in a geometrical model of astrocytes show that the DLS formed between astrocyte endfeet and blood capillaries, facilitate the siphoning of accumulated K+ into the extracellular space facing the blood capillaries. The DLS geometry creates an efficient coupling between AQPs and Kir channels.

    3. Furthermore, the models show that a local coupling between water and K+ transport is important for the maintenance of membrane potential and the net K+ spatial buffering capacity in the astrocytes.

    4. In the full geometrical model of K+ spatial buffering we show that the geometry of the extracellular space both in the synapse region and in the endfeet is an essential component for the cell volume regulation.

    Our results suggest that for regulation of K+ and water homeostasis in astrocytes, not only the classical aspects of functional couplings between proteins, but also the geometry of the cell and the microdomains are important. Further, our results suggest a central role for AQPs in the astrocyte endfeet and identify their contribution to K+ siphoning.

  • 34.
    Khorshidi, Mohammad A.
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Natural Killer Cell-Mediated Tumor Surveillance: Correlation Between Killing Efficiency, Transient Migration Behavior and Morphology2012In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 102, no 3, p. 706A-706AArticle in journal (Other academic)
  • 35.
    Khorshidi, Mohammad Ali
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Kowalewski, Jacob M.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Garrod, Kym R.
    Lindström, Sara
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Cahalan, Michael D.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Analysis of transient migration behavior of natural killer cells imaged in situ and in vitro2011In: Integrative Biology, ISSN 1757-9694, E-ISSN 1757-9708, Vol. 3, no 7, p. 770-778Article in journal (Refereed)
    Abstract [en]

    We present a simple method for rapid and automatic characterization of lymphocyte migration from time-lapse fluorescence microscopy data. Time-lapse imaging of natural killer (NK) cells in vitro and in situ, both showed that individual cells transiently alter their migration behavior. Typically, NK cells showed periods of high motility, interrupted by transient periods of slow migration or almost complete arrests. Analysis of in vitro data showed that these periods frequently coincided with contacts with target cells, sometimes leading to target cell lysis. However, NK cells were also commonly observed to stop independently of contact with other cells. In order to objectively characterize the migration of NK cells, we implemented a simple method to discriminate when NK cells stop or have low motilities, have periods of directed migration or undergo random movement. This was achieved using a sliding window approach and evaluating the mean squared displacement (MSD) to assess the migration coefficient and MSD curvature along trajectories from individual NK cells over time. The method presented here can be used to quickly and quantitatively assess the dynamics of individual cells as well as heterogeneity within ensembles. Furthermore, it may also be used as a tool to automatically detect transient stops due to the formation of immune synapses, cell division or cell death. We show that this could be particularly useful for analysis of in situ time-lapse fluorescence imaging data where most cells, as well as the extracellular matrix, are usually unlabelled and thus invisible.

  • 36.
    Manneberg, Otto
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Svennebring, Jessica
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Hertz, Hans M.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    A three-dimensional ultrasonic cage for characterization of individual cells2008In: Applied Physics Letters, ISSN 0003-6951, E-ISSN 1077-3118, Vol. 93, p. 063901-Article in journal (Refereed)
    Abstract [en]

    We demonstrate enrichment, controlled aggregation, and manipulation of microparticles and cells by an ultrasonic cage integrated in a microfluidic chip compatible with high-resolution optical microscopy. The cage is designed as a dual-frequency resonant filleted square box integrated in the fluid channel. Individual particles may be trapped three dimensionally, and the dimensionality of one-dimensional to three-dimensional aggregates can be controlled. We investigate the dependence of the shape and position of a microparticle aggregate on the actuation voltages and aggregate size, and demonstrate optical monitoring of individually trapped live cells with submicrometer resolution.

  • 37.
    Manneberg, Otto
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Svennebring, Jessica
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Hertz, Hans M.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Ultrasonic microcages for high-resolution characterization of individual cells2008Conference paper (Refereed)
  • 38.
    Manneberg, Otto
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Flow-free transport of cells in microchannels by frequency-modulated ultrasound2009In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 9, p. 833-837Article in journal (Refereed)
    Abstract [en]

    We demonstrate flow-free transport of cells and particles by the use of frequency-modulated ultrasonic actuation of a microfluidic chip. Two different modulation schemes are combined: A rapid (1 kHz) linear frequency sweep around similar to 6.9 MHz is used for two-dimensional spatial stabilization of the force field over a 5 mm long inlet channel of constant cross section, and a slow (0.2-0.7 Hz) linear frequency sweep around similar to 2.6 MHz is used for flow-free ultrasonic transport and positioning of cells or particles. The method is used for controlling the motion and position of cells monitored with high-resolution optical microscopy, but can also be used more generally for improving the robustness and performance of ultrasonic manipulation micro-devices.

  • 39. McCann, Fiona E.
    et al.
    Eissmann, Philipp
    Önfelt, Björn
    Leung, Rufina
    Davis, Daniel M.
    The activating NKG2D ligand MHC class I-related chain a transfers from target cells to NK cells in a manner that allows functional consequences2007In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 178, no 6, p. 3418-3426Article in journal (Refereed)
    Abstract [en]

    Recently, it has become apparent that surface proteins commonly transfer between immune cells in contact. Inhibitory receptors and ligands exchange between cells during NK cell surveillance and we report here that NK cells also acquire activating ligands from target cells. Specifically, the stress-inducible activating ligand for NKG2D, MHC class I-related chain A (MICA), transferred to NK cells upon conjugation with MICA-expressing target cells. Acquisition of MICA from target cells was dependent on cell contact and occurred after accumulation of MICA at the immunological synapse. Moreover, transfer of MICA was facilitated by specific molecular recognition via NKG2D and augmented by Src kinase signaling. Importantly, MICA associated with its new host NK cell membrane in an orientation that allowed engagement with NKG2D in trans and indeed could down-regulate NKG2D in subsequent homotypic interactions with other NK cells. MICA captured from target cells could subsequently transfer between NK cells and, more importantly, NK cell degranulation was triggered in such NK cell-NK cell interactions. Thus, NK cells can influence other NK cells with proteins acquired from target cells and our data specifically suggest that NK cells could lyse other NK cells upon recognition of activating ligands acquired from target cells. This mechanism could constitute an important function for immunoregulation of NK cell activity.

  • 40. Meinke, Stephan
    et al.
    Brandt, Ludwig
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics.
    Sandgren, Per
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hoglund, Petter
    Platelets become NK cell targets in the presence of anti-platelet antibodies2017In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 86, no 4, p. 257-258Article in journal (Other academic)
  • 41. Nilsson, Jesper R.
    et al.
    Li, Shiming
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Andreasson, Joakim
    Light-induced cytotoxicity of a photochromic spiropyran2011In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 47, no 39, p. 11020-11022Article in journal (Refereed)
    Abstract [en]

    In this work we present a novel water soluble spiropyran photoswitch that can be photonically activated inside live cells from a form that has no significant effect on the cellular survival to a form that induces a dramatic toxic response.

  • 42.
    Oei, Vincent Yi Sheng
    et al.
    Radiumhospitalet, Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway.;Univ Oslo, Inst Clin Med, KG Jebsen Ctr Canc Immunotherapy, Oslo, Norway..
    Siernicka, Marta
    Med Univ Warsaw, Dept Immunol, Ctr Biostruct Res, Warsaw, Poland.;Med Univ Warsaw, Postgrad Sch Mol Med, Warsaw, Poland..
    Graczyk-Jarzynka, Agnieszka
    Med Univ Warsaw, Dept Immunol, Ctr Biostruct Res, Warsaw, Poland..
    Hoel, Hanna Julie
    Radiumhospitalet, Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway..
    Yang, Weiwen
    Radiumhospitalet, Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway..
    Palacios, Daniel
    Radiumhospitalet, Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway.;Univ Oslo, Inst Clin Med, KG Jebsen Ctr Canc Immunotherapy, Oslo, Norway..
    Almasbak, Hilde
    Radiumhospitalet, Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway..
    Bajor, Malgorzata
    Med Univ Warsaw, Dept Immunol, Ctr Biostruct Res, Warsaw, Poland..
    Clement, Dennis
    Radiumhospitalet, Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway.;Univ Oslo, Inst Clin Med, KG Jebsen Ctr Canc Immunotherapy, Oslo, Norway..
    Brandt, Ludwig
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Inst.
    Goodridge, Jodie
    Radiumhospitalet, Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway.;Univ Oslo, Inst Clin Med, KG Jebsen Ctr Canc Immunotherapy, Oslo, Norway..
    Winiarska, Magdalena
    Med Univ Warsaw, Dept Immunol, Ctr Biostruct Res, Warsaw, Poland..
    Zagozdzon, Radoslaw
    Med Univ Warsaw, Dept Immunol, Ctr Biostruct Res, Warsaw, Poland.;Med Univ Warsaw, Inst Transplantat, Dept Clin Immunol, Warsaw, Poland..
    Olweus, Johanna
    Radiumhospitalet, Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway.;Univ Oslo, Inst Clin Med, KG Jebsen Ctr Canc Immunotherapy, Oslo, Norway..
    Kyte, Jon-Amund
    Radiumhospitalet, Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway.;Oslo Univ Hosp, Dept Oncol, Oslo, Norway..
    Malmberg, Karl-Johan
    Radiumhospitalet, Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway.;Univ Oslo, Inst Clin Med, KG Jebsen Ctr Canc Immunotherapy, Oslo, Norway.;Karolinska Inst, Dept Med Huddinge, Ctr Infect Med, Stockholm, Sweden..
    Intrinsic Functional Potential of NK-Cell Subsets Constrains Retargeting Driven by Chimeric Antigen Receptors2018In: CANCER IMMUNOLOGY RESEARCH, ISSN 2326-6066, Vol. 6, no 4, p. 467-480Article in journal (Refereed)
    Abstract [en]

    Natural killer (NK) cells hold potential as a source of allogeneic cytotoxic effector cells for chimeric antigen receptor (CAR)-mediated therapies. Here, we explored the feasibility of transfecting CAR-encoding mRNA into primary NK cells and investigated how the intrinsic potential of discrete NK-cell subsets affects retargeting efficiency. After screening five second- and third-generation anti-CD19 CAR constructs with different signaling domains and spacer regions, a third-generation CAR with the CH2-domain removed was selected based on its expression and functional profiles. Kinetics experiments revealed that CAR expression was optimal after 3 days of IL15 stimulation prior to transfection, consistently achieving over 80% expression. CAR-engineered NK cells acquired increased degranulation toward CD19(+) targets, and maintained their intrinsic degranulation response toward CD19(-) K562 cells. The response of redirected NK-cell subsets against CD19(+) targets was dependent on their intrinsic thresholds for activation determined through both differentiation and education by killer cell immunoglobulin-like receptors (KIR) and/or CD94/NKG2A binding to self HLA class I and HLA-E, respectively. Redirected primary NK cells were insensitive to inhibition through NKG2A/HLA-E interactions but remained sensitive to inhibition through KIR depending on the amount of HLA class I expressed on target cells. Adaptive NK cells, expressing NKG2C, CD57, and self-HLA-specific KIR(s), displayed superior ability to kill CD19(+), HLA low, or mismatched tumor cells. These findings support the feasibility of primary allogeneic NK cells for CAR engineering and highlight a need to consider NK-cell diversity when optimizing efficacy of cancer immunotherapies based on CAR-expressing NK cells.

  • 43.
    Ohlin, Mathias
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Christakou, Athanasia E.
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Frisk, Thomas
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Analysis of trapping and streaming in an ultrasoundactuated multi-well microplate for single-cell studies2012In: Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, Chemical and Biological Microsystems Society , 2012, p. 497-499Conference paper (Refereed)
    Abstract [en]

    The dynamics of the acoustic streaming and the acoustic positioning performance in an ultrasound-actuated multiwell microplate are investigated by two different ultrasonic frequency actuation schemes: Frequency-modulation and single- frequency actuation. Our results show a significant decrease in size of the field of view when using frequencymodulation compared to single-frequency actuation, which can be used for improving the scanning time for 3D highresolution confocal microscopy by almost one order of magnitude. Furthermore, in the ultrasound-actuated multi-well microplate the high-voltage acoustic streaming show a complex time and temperature dependence and could gain stability by the use of temperature control.

  • 44.
    Ohlin, Mathias
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Christakou, Athanasia E.
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Frisk, Thomas
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Controlling acoustic streaming in a multi-well microplate for improving live cell assays2011In: 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011, 2011, p. 1612-1614Conference paper (Refereed)
    Abstract [en]

    Acoustic streaming in a multi-well microplate is investigated using two different ultrasonic actuation frequency-schemes: Single-frequency and frequency-modulation. The streaming is tracked by the use of 1 μm fluorescent polymer beads and micro-particle image velocimetry. The suspension also contained human B cells for studying the acoustic trapping and aggregation performance simultaneously with the acoustic streaming. Our results show a significant difference in the acoustic streaming between the two ultrasonic actuation schemes. A rotational fluid flow speed decreased a factor of 30 when frequency-modulation was applied compared to single-frequency actuation without apparently interfering with the acoustic cell trapping function.

  • 45.
    Ohlin, Mathias
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Christakou, Athanasia E.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Frisk, Thomas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Influence of acoustic streaming on ultrasonic particle manipulation in a 100-well ring-transducer microplate2013In: Journal of Micromechanics and Microengineering, ISSN 0960-1317, E-ISSN 1361-6439, Vol. 23, no 3, p. 035008-Article in journal (Refereed)
    Abstract [en]

    We characterize and quantify the performance of ultrasonic particle aggregation and positioning in a 100-well microplate. We analyze the result when operating a planar ultrasonic ring transducer at different single actuation frequencies in the range 2.20-2.40 MHz, and compare with the result obtained from different schemes of frequency-modulated actuation. Compared to our previously used wedge transducer design, the ring transducer has a larger contact area facing the microplate, resulting in lower temperature increase for a given actuation voltage. Furthermore, we analyze the dynamics of acoustic streaming occurring simultaneously with the particle trapping in the wells of the microplate, and we define an adaptive ultrasonic actuation scheme for optimizing both efficiency and robustness of the method. The device is designed as a tool for ultrasound-mediated cell aggregation and positioning. This is a method for high-resolution optical characterization of time-dependent cellular processes at the level of single cells. In this paper, we demonstrate how to operate our device in order to optimize the scanning time of 3D confocal microscopy with the aim to perform high-resolution time-lapse imaging of cells or cell-cell interactions in a highly parallel manner.

  • 46. Olofsson, J.
    et al.
    Önfelt, Björn
    Lincoln, P.
    Three-state light switch of Ru(phen)(2)dppz (2+): Distinct excited-state species with two, one, or no hydrogen bonds from solvent2004In: Journal of Physical Chemistry A, ISSN 1089-5639, E-ISSN 1520-5215, Vol. 108, no 20, p. 4391-4398Article in journal (Refereed)
    Abstract [en]

    The ruthenium complexes of dppz (dipyrido[3,2-a:2',3'-c]phenazine) have found wide interest due to their environment-sensitive luminescence and are used, for example, as spectroscopic probes for DNA. The deactivation process for the excited state of the light-switch complex [Ru(phen)(2)dppz](2+) (phen = 1,10-phenanthroline) has been studied in water, glycerol, ethylene glycol, and 1,2- and 1,3-propandiol by using fluorescence spectroscopy and single photon counting. In all solvents anomalous temperature dependence is found (increasing quantum yield and excited-state lifetime with increasing temperature). Model-independent analysis shows that only two emissive species, with solvent- and temperature-invariant emission spectral profiles, are sufficient to account for all the data in the polyol solvents. Van't Hoff plots of the ratio of the two species are linear at higher temperatures in all solvents, indicating rapid thermal equilibration of the two species, except for lower temperatures in the most viscous solvent glycerol. Kinetic modeling of the system with microscopic rate constants with positive Arrhenius activation energies requires a third nonemissive species, which is assigned to an excited state with two hydrogen bonds from the solvent, whereas the first two species are assigned to the mono-hydrogen-bonded and non-hydrogen-bonded excited-state species. This assignment is supported by the observation of a growing luminescence intensity as temperature is increased, but no wavelength shift, of high-purity [Ru(phen)(2)dppz](2+) in water solution.

  • 47. Olofsson, J.
    et al.
    Önfelt, Björn
    Lincoln, P.
    Norden, B.
    Matousek, P.
    Parker, A. W.
    Tuite, E.
    Picosecond Kerr-gated time-resolved resonance Raman spectroscopy of the Ru(phen)(2)dppz (2+) interaction with DNA2002In: Journal of Inorganic Biochemistry, ISSN 0162-0134, E-ISSN 1873-3344, Vol. 91, no 1, p. 286-297Article in journal (Refereed)
    Abstract [en]

    To investigate the basis of the 'light-switch' effect, the solvent dependence of the Kerr-gated picosecond-time resolved resonance Raman (TR3) spectra of [Ru(bpy),dppz](2+), [Ru(phen)(2)dppz](2+), and the modified complex [Ru(phen)(2)cpdppzOMe](2+) and a dimer [mu-C4(cpdppz)(2)-(phen)(4)Ru-2](4+) were studied. The investigation focussed on comparing the behaviour of [Ru(phen)(2)dppz](2+) in acetonitrile, ethanol, H2O, D2O, and DNA. The data are consistent with a model wherein excitation induces metal-to-ligand charge transfer (MLCT) to any of the ligands (termed the 'precursor' state) which, by interligand electron transfer (ILET), produces an excited state localised on the dppz ligand, MLCT1. In water this state relaxes with a characteristic time of similar to6 ps to a non-emissive state (MLCT2). The TR3 spectra in water, acetonitrile and DNA are all distinctly different. However. the early (4 ps) water spectrum resembles the spectrum in DNA. This interesting observation suggests that the DNA-bound excited state of the complex can be thought of as a model for the initial, poorly solvated state in water.

  • 48.
    Olofsson, Karl
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Carannante, V.
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden..
    Ohlin, Mathias
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Frisk, Thomas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Kushiro, K.
    Univ Tokyo, Dept Bioengn, Tokyo, Japan..
    Takai, M.
    Univ Tokyo, Dept Bioengn, Tokyo, Japan..
    Lundqvist, A.
    Karolinska Inst, Dept Oncol Pathol, Stockholm, Sweden..
    Önfelt, Björn
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Acoustic formation of multicellular tumor spheroids enabling on-chip functional and structural imaging2018In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 18, no 16, p. 2466-2476Article in journal (Refereed)
    Abstract [en]

    Understanding the complex 3D tumor microenvironment is important in cancer research. This microenvironment can be modelled in vitro by culturing multicellular tumor spheroids (MCTS). Key challenges when using MCTS in applications such as high-throughput drug screening are overcoming imaging and analytical issues encountered during functional and structural investigations. To address these challenges, we use an ultrasonic standing wave (USW) based MCTS culture platform for parallel formation, staining and imaging of 100 whole MCTS. A protein repellent amphiphilic polymer coating enables flexible production of high quality and unanchored MCTS. This enables high-content multimode analysis based on flow cytometry and in situ optical microscopy. We use HepG2 hepatocellular carcinoma, A498 and ACHN renal carcinoma, and LUTC-2 thyroid carcinoma cell lines to demonstrate (i) the importance of the ultrasound-coating combination, (ii) bright field image based automatic characterization of MTCS, (iii) detailed deep tissue confocal imaging of whole MCTS mounted in a refractive index matching solution, and (iv) single cell functional analysis through flow cytometry of single cell suspensions of disintegrated MTCS. The USW MCTS culture platform is customizable and holds great potential for detailed multimode MCTS analysis in a high-content manner.

  • 49.
    Olofsson, Per E
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Forslund, Elin
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Chechet, Ksenia
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Mickelin, Oscar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Rivera Ahlin, Alexander
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Everhorn, Tobias
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Distinct Migration and Contact Dynamics of Resting and IL-2-Activated Human Natural Killer Cells.2014In: Frontiers in immunology, ISSN 1664-3224, Vol. 5, p. 80-Article in journal (Refereed)
    Abstract [en]

    Natural killer (NK) cells serve as one of the first lines of defense against viral infections and transformed cells. NK cell cytotoxicity is not dependent on antigen presentation by target cells, but is dependent on integration of activating and inhibitory signals triggered by receptor-ligand interactions formed at a tight intercellular contact between the NK and target cell, i.e., the immune synapse. We have studied the single-cell migration behavior and target-cell contact dynamics of resting and interleukin (IL)-2-activated human peripheral blood NK cells. Small populations of NK cells and target cells were confined in microwells and imaged by fluorescence microscopy for >8 h. Only the IL-2-activated population of NK cells showed efficient cytotoxicity against the human embryonic kidney 293T target cells. We found that although the average migration speeds were comparable, activated NK cells showed significantly more dynamic migration behavior, with more frequent transitions between periods of low and high motility. Resting NK cells formed fewer and weaker contacts with target cells, which manifested as shorter conjugation times and in many cases a complete lack of post-conjugation attachment to target cells. Activated NK cells were approximately twice as big as the resting cells, displayed a more migratory phenotype, and were more likely to employ "motile scanning" of the target-cell surface during conjugation. Taken together, our experiments quantify, at the single-cell level, how activation by IL-2 leads to altered NK cell cytotoxicity, migration behavior, and contact dynamics.

  • 50.
    Olofsson, Per
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Magnusson, Klas E. G.
    KTH, School of Electrical Engineering (EES), Signal Processing.
    Frisk, Thomas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Jaldén, Joakim
    KTH, School of Electrical Engineering (EES), Signal Processing.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    A collagen-based microwell migration assay to study NK—target cell interactionsManuscript (preprint) (Other academic)
    Abstract [en]

    Natural killer (NK) cell cytotoxicity is highly dependent on the ability of NK cells to migrate through the extracellular matrix (ECM) microenvironment. Traditional imaging studies of NK cell migration and cytotoxicity have utilized 2-D surfaces, which do not properly reproduce the structural and mechanical cues that shape the migratory response of NK cells in vivo. In addition, current in vivo imaging does not allow for the accurate long-term single-cell imaging required to dissect the functional heterogeneity of NK cell populations, and importantly, it does not allow studies of human cells. Therefore, it is desirable to implement in vitro migration and killing assays that better mimic in vivo conditions.

    We have combined a microwell assay that allows long-term imaging and tracking of small, well-defined populations of NK cells with an interstitial ECM-like matrix to more closely approximate in vivo conditions. The microwells, which are loaded with a gel mixture containing NK and target cells, allows for long-term imaging of NK–target cell interactions within a confined 3-D volume. The microwells were optically sectioned by confocal fluorescence microscopy once every 2 min for 12 h. NK cells were tracked by the Baxter Algorithms to assess motility parameters and interactions with target cells were manually scored for duration and outcome.

    We found marked differences in motility between individual cells with a significant fraction of the cells moving slowly and being confined to a small area within the matrix, while other cells moved more freely, probably reflecting local variations in the matrix structure and inherent difference in motility between individual cells. A majority of NK cells also exhibited transient variation in their mobility alternating between periods of migration arrest and random movement. NK cells that alternated between different modes of migration switched on average once every 3 h.

    NK cells made fewer and shorter contacts with target cells than in comparable 2-D assays. The difference was particularly pronounced for the process of post-conjugation attachment when NK and target cells separate. The timing of this process is likely influenced by a biomechanical component only present in 3-D environments where the cells are offered multiple anchor points with the matrix that can be used to generate the forces needed to pull apart.

    The developed microwell-based assay is suitable for 3-D time-lapse imaging of NK cells migration and cytotoxicity. As it allows for experiments with human cells, it could be used as a complement to in vivo imaging to study the influence of e.g. education and cytokine activation on NK cell heterogeneity in migration and cytotoxicity.

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