| 1. |
- Christakou, Athanasia. E., et al.
(författare)
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Aggregation and long-term positioning of cells by ultrasound in a multi-well microchip for high-resolution imaging of the natural killer cell immune synapse
- 2011
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Ingår i: 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011. - 9781618395955 ; , s. 329-331
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Konferensbidrag (refereegranskat)abstract
- 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.
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| 2. |
- Christakou, Athanasia E., et al.
(författare)
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Characterization of natural killer cells' cytotoxic heterogeneity using an array of sono-cages
- 2012
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Ingår i: Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012. - : Chemical and Biological Microsystems Society. - 9780979806452 ; , s. 1555-1557
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Konferensbidrag (refereegranskat)abstract
- 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).
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| 3. |
- Christakou, Athanasia E., et al.
(författare)
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Live cell imaging in a micro-array of acoustic traps facilitates quantification of natural killer cell heterogeneity
- 2013
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Ingår i: Integrative Biology. - : Oxford University Press (OUP). - 1757-9694 .- 1757-9708. ; 5:4, s. 712-719
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Christakou, Athanasia E., et al.
(författare)
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Solid tumor spheroid formation by temperature-controlled high voltage ultrasound in a multi-well microdevice
- 2014
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Ingår i: 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014. - : Chemical and Biological Microsystems SocietyChemical and Biological Microsystems Society. - 9780979806476 ; , s. 573-575
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Konferensbidrag (refereegranskat)abstract
- 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.
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| 5. |
- Forslund, E., et al.
(författare)
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Novel microchip-based tools facilitating live cell imaging and assessment of functional heterogeneity within NK cell populations
- 2012
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 3:OCT, s. 300-
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Frisk, Thomas, et al.
(författare)
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A silicon-glass microwell platform for high-resolution imaging and high-content screening with single cell resolution
- 2011
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Ingår i: Biomedical microdevices (Print). - : Springer Science and Business Media LLC. - 1387-2176 .- 1572-8781. ; 13:4, s. 683-693
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Frisk, T., et al.
(författare)
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Live-cell imaging of natural killer cell mediated tumor rejection in arrays of microwells
- 2010
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Ingår i: 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010. - 9781618390622 ; , s. 950-952
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Konferensbidrag (refereegranskat)abstract
- 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.
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| 8. |
- Guldevall, Karolin
(författare)
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Development of Microchip-based Assays to Study Immune Cell Interactions at the Single Cell Level
- 2011
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Immune cell populations are constantly divided into smaller and smaller subsets defined by newly emerging cellular markers. However, there is a growing awareness of the functional heterogeneities in between cells even within small populations, in addition to the heterogeneity over time. One may ask whether a population is correctly defined only by cellular markers or if the functionality should be regarded as well? Many of today’s techniques only measures at the population level, giving an average estimate of the behavior of that pool of cells, but failing to detect rare possibly important events. Thus, high-throughput experimental approaches to analyze single cells over time are required to address cellular heterogeneity. Progress in the fields of microfabrication, microscopy and computing have paved the way for increasingly efficient tools for studies on the single cell level, and a variety of devices have been described by others. However, few of them are suitable for long-term imaging of dynamic events such as cell-cell interactions or migration. In addition, for efficient recording of many individual events it is desirable to scale down the cells’ interaction volume; not only to shorten the time to interaction, but also to increase the number of individual events in a given area; thereby pushing a screening approach. To address these questions, a complete microwell array system for imaging of immune cell responses with single-cell resolution was designed. The platform consists of a range of silicon-glass microchips with arrays of miniature wells for incubation of cells and a custom made holder that fits conventional microscopes. The device has been designed to allow cells to be kept viable for several days in the wells, to be easy to use and to allow high-resolution imaging. Five different designs were fabricated; all with a specific type of assay in mind, and were evaluated regarding biocompatibility and functionality. One design is aimed towards screening applications, making an automatic cell counting protocol necessary in order to analyze the massive amount of data generated; this program is also described and evaluated. We here show that our silicon microwell platform allows long-term studies (up to several days), with the possibility of both time-lapse and high-resolution imaging of a variety of immune cell behavior. Using time-lapse imaging we confirmed immune cell heterogeneity in NK cell populations regarding both cytotoxicity and migrational behavior. The automatic counting program was tested and showed similar results compared to both manual counting and FACS. In addition, the large numbers of wells that can be simultaneously imaged, provide new statistical information that will lead to a better understanding of the function and regulation of the immune system at the single cell level. Altogether, our technique enables novel types of cellular imaging assays allowing data collection at a level of resolution not previously obtained – this was shown to be important for performing basic cell biological studies, but may also prove valuable in the proposed future medical applications.
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| 9. |
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| 10. |
- Guldevall, Karolin, et al.
(författare)
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Imaging Immune Surveillance of Individual Natural Killer Cells Confined in Microwell Arrays
- 2010
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 5:11, s. e15453-
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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