| 1. |
- Ohlin, Mathias, et al.
(författare)
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Influence of acoustic streaming on ultrasonic particle manipulation in a 100-well ring-transducer microplate
- 2013
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Ingår i: Journal of Micromechanics and Microengineering. - : IOP Publishing. - 0960-1317 .- 1361-6439. ; 23:3, s. 035008-
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
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| 3. |
- 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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| 4. |
- 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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| 5. |
- 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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| 6. |
- Wiklund, Martin, et al.
(författare)
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Ultrasound-Induced Cell-Cell Interaction Studies in a Multi-Well Microplate
- 2014
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Ingår i: Micromachines. - : MDPI AG. - 2072-666X. ; 5:1, s. 27-49
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Forskningsöversikt (refereegranskat)abstract
- This review describes the use of ultrasound for inducing and retaining cell-cell contact in multi-well microplates combined with live-cell fluorescence microscopy. This platform has been used for studying the interaction between natural killer (NK) cells and cancer cells at the level of individual cells. The review includes basic principles of ultrasonic particle manipulation, design criteria when building a multi-well microplate device for this purpose, biocompatibility aspects, and finally, two examples of biological applications: Dynamic imaging of the inhibitory immune synapse, and studies of the heterogeneity in killing dynamics of NK cells interacting with cancer cells.
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| 7. |
- Wilkinson, Kai, et al.
(författare)
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Visualization of custom-tailored iron oxide nanoparticles chemistry, uptake, and toxicity
- 2012
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Ingår i: Nanoscale. - 2040-3364. ; 4:23, s. 7383-7393
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Tidskriftsartikel (refereegranskat)abstract
- Nanoparticles of iron oxide generated by wearing of vehicles have been modelled with a tailored solution of size-uniform engineered magnetite particles produced by the Bradley reaction, a solvothermal metal–organic approach rendering hydrophilic particles. The latter does not bear any pronounced surface charge in analogy with that originating from anthropogenic sources in the environment. Physicochemical properties of the nanoparticles were thoroughly characterized by a wide range of methods, including XPD, TEM, SEM, DLS and spectroscopic techniques. The magnetite nanoparticles were found to be sensitive for transformation into maghemite under ambient conditions. This process was clearly revealed by Raman spectroscopy for high surface energy magnetite particles containing minor impurities of the hydromaghemite phase and was followed by quantitative measurements with EXAFS spectroscopy. In order to assess the toxicological effects of the produced nanoparticles in humans, with and without surface modification with ATP (a model of bio-corona formed in alveolar liquid), a pathway of potential uptake and clearance was modelled with a sequence of in vitro studies using A549 lung epithelial cells, lymphocyte 221-B cells, and 293T embryonal kidney cells, respectively. Raman microscopy unambiguously showed that magnetite nanoparticles are internalized within the A549 cells after 24 h co-incubation, and that the ATP ligand is retained on the nanoparticles throughout the uptake process. The toxicity of the nanoparticles was estimated using confocal fluorescence microscopy and indicated no principal difference for unmodified and modified particles, but revealed considerably different biochemical responses. The IL-8 cytokine response was found to be significantly lower for the magnetite nanoparticles compared to TiO2, while an enhancement of ROS was observed, which was further increased for the ATP-modified nanoparticles, implicating involvement of the ATP signalling pathway in the epithelium.
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| 8. |
- Hurtig, Johan, et al.
(författare)
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Intercellular nanotubes : insights from imaging studies and beyond
- 2010
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Ingår i: WILEY INTERDISCIPLINARY REVIEWS-NANOMEDICINE AND NANOBIOTECHNOLOGY. - : Wiley. - 1939-5116 .- 1939-0041. ; 2:3, s. 260-276
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Forskningsöversikt (refereegranskat)abstract
- 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
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| 9. |
- Olofsson, Karl, et al.
(författare)
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Single cell resolution analysis of ultrasound-produced multi-cellular tumor spheroids
- 2020
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Ingår i: 21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017. - : Chemical and Biological Microsystems Society. ; , s. 955-956
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Konferensbidrag (refereegranskat)abstract
- We have previously presented an ultrasonic standing wave (USW) 3D culture platform enabling parallel production, staining and processing of 100 uniformly sized multi-cellular tumor spheroids (MCTS) [1]. Here, we use the system for single cell resolution analysis of A498 renal carcinoma MCTS by off-chip fluorescence-activated cell sorting (FACS) and on-chip automatic image analysis methods based on 3D confocal microscopy images.
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