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  • 1.
    Christakou, Athanasia. E.
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Ohlin, Mathias
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Khorshidi, Mohammad Ali
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Frisk, Thomas
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Vanherberghen, Bruno
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Aggregation and long-term positioning of cells by ultrasound in a multi-well microchip for high-resolution imaging of the natural killer cell immune synapse2011Inngår i: 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011, 2011, s. 329-331Konferansepaper (Fagfellevurdert)
    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.

  • 2.
    Christakou, Athanasia E.
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Ohlin, Mathias
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Vanherberghen, Bruno
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Khorshidi, Mohammad Ali
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Kadri, Nadir
    Frisk, Thomas
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Live cell imaging in a micro-array of acoustic traps facilitates quantification of natural killer cell heterogeneity2013Inngår i: Integrative Biology, ISSN 1757-9694, E-ISSN 1757-9708, Vol. 5, nr 4, s. 712-719Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 3.
    Frisk, Thomas
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Khorshidi, Mohammad Ali
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Guldevall, Karolin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Vanherberghen, Bruno
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    A silicon-glass microwell platform for high-resolution imaging and high-content screening with single cell resolution2011Inngår i: Biomedical microdevices (Print), ISSN 1387-2176, E-ISSN 1572-8781, Vol. 13, nr 4, s. 683-693Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 4.
    Guldevall, Karolin
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Frisk, Thomas
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Vanherberghen, Bruno
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Khorsidi, Mohammed Ali
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Manneberg, Otto
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Christakou, Athanasia
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Wiklund, Martin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Biomedicinsk fysik och röntgenfysik.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Imaging immune surveillance by individual Natural Killer cells isolated in arrays of nanoliter wells2010Konferansepaper (Fagfellevurdert)
  • 5.
    Khorshidi, Mohammad A.
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Vanherberghen, Bruno
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Natural Killer Cell-Mediated Tumor Surveillance: Correlation Between Killing Efficiency, Transient Migration Behavior and Morphology2012Inngår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 102, nr 3, s. 706A-706AArtikkel i tidsskrift (Annet vitenskapelig)
  • 6.
    Khorshidi, Mohammad Ali
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Live Single Cell Imaging and Analysis Using Microfluidic Devices2013Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Today many cell biological techniques study large cell populations where an average estimate of individual cells’ behavior is observed. On the other hand, single cell analysis is required for studying functional heterogeneities between cells within populations. This thesis presents work that combines the use of microfluidic devices, optical microscopy and automated image analysis to design various cell biological assays with single cell resolution including cell proliferation, clonal expansion, cell migration, cell-cell interaction and cell viability tracking. In fact, automated high throughput single cell techniques enable new studies in cell biology which are not possible with conventional techniques.

    In order to automatically track dynamic behavior of single cells, we developed a microwell based device as well as a droplet microfluidic platform. These high throughput microfluidic assays allow automated time-lapse imaging of encapsulated single cells in micro droplets or confined cells inside microwells. Algorithms for automatic quantification of cells in individual microwells and micro droplets are developed and used for the analysis of cell viability and clonal expansion. The automatic counting protocols include several image analysis steps, e.g. segmentation, feature extraction and classification. The automatic quantification results were evaluated by comparing with manual counting and revealed a high success rate. In combination these automatic cell counting protocols and our microfluidic platforms can provide statistical information to better understand behavior of cells at the individual level under various conditions or treatments in vitro exemplified by the analysis of function and regulation of immune cells. Thus, together these tools can be used for developing new cellular imaging assays with resolution at the single cell level.

    To automatically characterize transient migration behavior of natural killer (NK) cells compartmentalized in microwells, we developed a method for single cell tracking. Time-lapse imaging showed that the NK cells often exhibited periods of high motility, interrupted with periods of slow migration or complete arrest. These transient migration arrest periods (TMAPs) often overlapped with periods of conjugations between NK cells and target cells. Such conjugation periods sometimes led to cell-mediated killing of target cells. Analysis of cytotoxic response of NK cells revealed that a small sub-class of NK cells called serial killers was able to kill several target cells. In order to determine a starting time point for cell-cell interaction, a novel technique based on ultrasound was developed to aggregate NK and target cells into the center of the microwells. Therefore, these assays can be used to automatically and rapidly assess functional and migration behavior of cells to detect differences between health and disease or the influence of drugs.

    The work presented in this thesis gives good examples of how microfluidic devices combined with automated imaging and image analysis can be helpful to address cell biological questions where single cell resolution is necessary. 

  • 7.
    Khorshidi, Mohammad Ali
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Periyannan Rajeswari, Prem Kumar
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Wahlby, C.
    Jönsson, Håkan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Andersson Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Dynamic behavior analysis of single cells using droplet microfluidics2013Inngår i: 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013, 2013, Vol. 3, s. 1674-1676Konferansepaper (Fagfellevurdert)
    Abstract [en]

    We present a droplet microfluidic platform to automatically track and characterize the behavior of single cells over time. Time series analysis of single cells was enabled by encapsulating the cells in microdroplets followed by trapping microdroplets in static array of microwells that were fabricated on chip to make the droplets addressable by their position and imaging them over time. In this paper, we demonstrate the potential of automated time-lapse imaging and image analysis approach in droplet microfluidics by studying the viability of large number of single cells over time.

  • 8.
    Khorshidi, Mohammad Ali
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Periyannan Rajeswari, Prem Kumar
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Wählby, Carolina
    Jönsson, Håkan N.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Andersson Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Automated analysis of dynamic behavior of single cells in picoliter droplets2014Inngår i: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 14, nr 5, s. 931-937Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We present a droplet-based microfluidic platform to automatically track and characterize the behavior of single cells over time. This high-throughput assay allows encapsulation of single cells in micro-droplets and traps intact droplets in arrays of miniature wells on a PDMS-glass chip. Automated time-lapse fluorescence imaging and image analysis of the incubated droplets on the chip allows the determination of the viability of individual cells over time. In order to automatically track the droplets containing cells, we developed a simple method based on circular Hough transform to identify droplets in images and quantify the number of live and dead cells in each droplet. Here, we studied the viability of several hundred single isolated HEK293T cells over time and demonstrated a high survival rate of the encapsulated cells for up to 11 hours. The presented platform has a wide range of potential applications for single cell analysis, e.g. monitoring heterogeneity of drug action over time and rapidly assessing the transient behavior of single cells under various conditions and treatments in vitro.

  • 9.
    Khorshidi, Mohammad Ali
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Vanherberghen, Bruno
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Kowalewski, Jacob M.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Garrod, Kym R.
    Lindström, Sara
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi.
    Andersson-Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi.
    Brismar, Hjalmar
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Cahalan, Michael D.
    Önfelt, Björn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Analysis of transient migration behavior of natural killer cells imaged in situ and in vitro2011Inngår i: Integrative Biology, ISSN 1757-9694, E-ISSN 1757-9708, Vol. 3, nr 7, s. 770-778Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 10.
    Vanherberghen, Bruno
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Olofsson, Per E.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Forslund, Elin
    Sternberg-Simon, Michal
    Khorshidi, Mohammad Ali
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Pacouret, Simon
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik.
    Guldevall, Karolin
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Enqvist, Monika
    Malmberg, Karl-Johan
    Mehr, Ramit
    Önfelt, Bjorn
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Classification of human natural killer cells based on migration behavior and cytotoxic response2013Inngår i: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 121, nr 8, s. 1326-1334Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Despite intense scrutiny of the molecular interactions between natural killer (NK) and target cells, few studies have been devoted to dissection of the basic functional heterogeneity in individual NK cell behavior. Using a microchip-based, time-lapse imaging approach allowing the entire contact history of each NK cell to be recorded, in the present study, we were able to quantify how the cytotoxic response varied between individual NK cells. Strikingly, approximately half of the NK cells did not kill any target cells at all, whereas a minority of NK cells was responsible for a majority of the target cell deaths. These dynamic cytotoxicity data allowed categorization of NK cells into 5 distinct classes. A small but particularly active subclass of NK cells killed several target cells in a consecutive fashion. These "serial killers" delivered their lytic hits faster and induced faster target cell death than other NK cells. Fast, necrotic target cell death was correlated with the amount of perforin released by the NK cells. Our data are consistent with a model in which a small fraction of NK cells drives tumor elimination and inflammation.

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