| 1. |
- Lindström, Sara, et al.
(författare)
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Nanoporous titania coating of microwell chips for stem cell culture and analysis
- 2010
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Ingår i: Journal of Biomechanical Science and Engineering. - : Japan Society of Mechanical Engineers. - 1880-9863. ; 5:3, s. 272-279
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Tidskriftsartikel (refereegranskat)abstract
- Stem cell research is today an active and promising field of research. To learn more about the biology of stem cells, technical improvements are needed such as tools to study stem cells in order to characterize them further and to gain insights to the molecular regulations of their maintenance, differentiation and identification. Common procedure when studying stem cells is to coat the surface where the stem cells are to be cultured with organic materials like matri-gel, poly-L-lysine and fibronectin. The resulting coating is usually relatively fragile and it is difficult to know if the coating is evenly distributed. In addition, these forms of coatings cannot be sterilized and re-used, but must be added as an initial, time-consuming step in the daily protocol. A microwell chip with hundreds of 500 nl wells has recently been shown to be a useful tool for stem cell culturing. This platform is here modified to facilitate and improve the coating conditions for adherent cell culture. A robust and highly porous film of TiO2 is deposited in the wells prior cell seeding. TiO2 is known to be biocompatible and provides a surface that is even and well characterized, simple to produce and re-usable. Furthermore it enables the microwell chips to be stored pre-coated for longer periods of time before use. We investigated the growth of rat mesenchymal stem cells on nanoporous titania films and found that they proliferated much faster than on conventional coatings. The combination of the robust TiO2 coating of the microwell chip enables thousands of individually separated single, or clones of, stem cells to be studied simultaneously and opens up the possibility for more user-friendly cell culturing.
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| 2. |
- Friedman, Mikaela, et al.
(författare)
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Engineering and characterization of a bispecific HER2 x EGFR-binding affibody molecule
- 2009
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Ingår i: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 54, s. 121-131
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Tidskriftsartikel (refereegranskat)abstract
- HER2 (human epidermal-growth-factor receptor-2; ErbB2) and EGFR (epidermal-growth-factor receptor) are overexpressed in various forms of cancer, and the co-expression of both HER2 and EGFR has been reported in a number of studies. The simultaneous targeting of HER2 and EGFR has been discussed as a strategy with which to potentially increase efficiency and selectivity in molecular imaging and therapy of certain cancers. In an effort to generate a molecule capable of bispecifically targeting HER2 and EGFR, a gene fragment encoding a bivalent HER2-binding affibody molecule was genetically fused in-frame with a bivalent EGFR-binding affibody molecule via a (G(4)S)(3) [(Gly(4)-Ser)(3)]-encoding gene fragment. The encoded 30 kDa affibody construct (Z(HER2))(2)-(G(4)S)(3)-(Z(EGFR))(2), with potential for bs (bispecific) binding to HER2 and EGFR, was expressed in Escherichia coli and characterized in terms of its binding capabilities. The retained ability to bind HER2 and EGFR separately was demonstrated using both biosensor technology and flow-cytometric analysis, the latter using HER2- and EGFR-overexpressing cells. Furthermore, simultaneous binding to HER2 and EGFR was demonstrated in: (i) a sandwich format employing real-time biospecific interaction analysis where the bs affibody molecule bound immobilized EGFR and soluble HER2; (ii) immunofluorescence microscopy, where the bs affibody molecule bound EGFR-overexpressing cells and soluble HER2; and (iii) a cell-cell interaction analysis where the bs affibody molecule bound HER2-overexpressing SKBR-3 cells and EGFR-overexpressing A-431 cells. This is, to our knowledge, the first reported bs affinity protein with potential ability for the simultaneous targeting of HER2 and EGFR. The potential future use of this and similar constructs, capable of bs targeting of receptors to increase the efficacy and selectivity in imaging and therapy, is discussed.
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| 3. |
- 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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| 4. |
- Khorshidi, Mohammad Ali, et al.
(författare)
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Analysis of transient migration behavior of natural killer cells imaged in situ and in vitro
- 2011
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Ingår i: Integrative Biology. - : Oxford University Press (OUP). - 1757-9694 .- 1757-9708. ; 3:7, s. 770-778
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Lindström, Sara, et al.
(författare)
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A microwell array device with integrated microfluidic components for enhanced single-cell analysis
- 2009
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 30:24, s. 4166-4171
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Tidskriftsartikel (refereegranskat)abstract
- Increasing awareness of the importance of cell heterogeneity in many biological and medical contexts is prompting increasing interest in systems that allow single-cell analysis rather than conventional bulk analysis (which provides average values for variables of interest from large numbers of cells). Recently, we presented a microwell chip for long-term, high-throughput single-cell analysis. The chip has proved to be useful for purposes such as screening individual cancer and stem cells for protein/gene markers. However, liquids in the wells can only be added or changed by manually rinsing the chip, or parts of it. This procedure has several well-known drawbacks - including risks of cross-contamination, large dead volumes and laboriousness - but there have been few previous attempts to integrate liquid rinsing/switching channels in "ready-to-use" systems for single-cell analysis. Here we present a microwell system designed (using flow simulations) for single-cell analysis with integrated microfluidic components (microchannels, magnetically driven micropumps and reservoirs) for supplying the cell culture wells with reagents, or rinsing, thus facilitating controlled, directed liquid handling. It can be used totally independently, since tubing is not essential. The practical utility of the integrated system has been demonstrated by culturing endothelial cells in the microwells, and successfully applying live-cell Calcein AM staining. Systems such as this combining high-density microwell chips with microfluidic components have great potential in numerous screening applications, such as exploring the important, but frequently undetected, heterogeneity in drug responses among individual cells.
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| 6. |
- Lindström, Sara, et al.
(författare)
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A microwell chip for parallel culture and analysis of stem cells
- 2009
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Ingår i: Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - : Chemical and Biological Microsystems Society. - 9780979806421 ; , s. 1309-1311
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Konferensbidrag (refereegranskat)abstract
- With recent findings on the role of reprogramming factors on stem cells, in vitro screening assays for studying (de)-differentiation is of great interest. We developed a miniaturized stem cell screening chip that is easily accessible and provides means of rapidly studying thousands of individual stem/progenitor cell samples, using low reagent volumes. Results presented here include weeklong culturing and differentiation assays of mouse embryonic stem cells, mouse adult neural stem cells, and human embryonic stem cells. The possibility to maintain the cells as stem/progenitor cells over time was shown, as was isolation and clonal analysis.
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| 7. |
- Lindström, Sara, et al.
(författare)
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High-Density Microwell Chip for Culture and Analysis of Stem Cells
- 2009
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Ingår i: PLos ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 4:9, s. e6997-
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Tidskriftsartikel (refereegranskat)abstract
- With recent findings on the role of reprogramming factors on stem cells, in vitro screening assays for studying (de)differentiation is of great interest. We developed a miniaturized stem cell screening chip that is easily accessible and provides means of rapidly studying thousands of individual stem/progenitor cell samples, using low reagent volumes. For example, screening of 700,000 substances would take less than two days, using this platform combined with a conventional bio-imaging system. The microwell chip has standard slide format and consists of 672 wells in total. Each well holds 500 nl, a volume small enough to drastically decrease reagent costs but large enough to allow utilization of standard laboratory equipment. Results presented here include weeklong culturing and differentiation assays of mouse embryonic stem cells, mouse adult neural stem cells, and human embryonic stem cells. The possibility to either maintain the cells as stem/progenitor cells or to study cell differentiation of stem/progenitor cells over time is demonstrated. Clonality is critical for stem cell research, and was accomplished in the microwell chips by isolation and clonal analysis of single mouse embryonic stem cells using flow cytometric cell-sorting. Protocols for practical handling of the microwell chips are presented, describing a rapid and user-friendly method for the simultaneous study of thousands of stem cell cultures in small microwells. This microwell chip has high potential for a wide range of applications, for example directed differentiation assays and screening of reprogramming factors, opening up considerable opportunities in the stem cell field.
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| 8. |
- Lindström, Sara, 1980-
(författare)
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Microwell devices for single-cell analyses
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Powerful tools for detailed cellular studies are emerging, increasing the knowledge ofthe ultimate target of all drugs: the living cell. Today, cells are commonly analyzed inensembles, i.e. thousands of cells per sample, yielding results on the average responseof the cells. However, cellular heterogeneity implies the importance of studying howindividual cells respond, one by one, in order to learn more about drug targeting andcellular behavior. In vitro assays offering low volume sampling and rapid analysis in ahigh-throughput manner are of great interest in a wide range of single-cellapplications. This work presents a microwell device in silicon and glass, developed using standardmicrofabrication techniques. The chip was designed to allow flow-cytometric cellsorting, a controlled way of analyzing and sorting individual cells for dynamic cultureand clone formation, previously shown in larger multiwell plates only. Dependent onthe application, minor modifications to the original device were made resulting in agroup of microwell devices suitable for various applications. Leukemic cancer cellswere analyzed with regard to their clonogenic properties and a method forinvestigation of drug response of critical importance to predict long-term clinicaloutcome, is presented. Stem cells from human and mouse were maintainedpluripotent in a screening assay, also shown useful in studies on neural differentiation.For integrated liquid handling, a fluidic system was integrated onto the chip fordirected and controlled addition of reagents in various cell-based assays. The chip wasproduced in a slide format and used as an imaging tool for low-volume sampling withthe ability to run many samples in parallel, demonstrated in a protein-binding assay fora novel bispecific affinity protein. Moving from cells and proteins into geneticanalysis, a method for screening genes from clones in a rapid manner was shown bygene amplification and mutation analysis in individual wells. In summary, a microwelldevice with associated methods were developed and applied in a range of biologicalinvestigations, particularly interesting from a cell-heterogeneity perspective.
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| 9. |
- Lindström, Sara, et al.
(författare)
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Overview of single-cell analyses : microdevices and applications
- 2010
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 10:24, s. 3363-3372
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Forskningsöversikt (refereegranskat)abstract
- Numerous microdevices developed for single-cell analyses have been presented in the last decades. Practical usefulness in biological and clinical settings has become an important focus during the development and implementation of new structures and assays. Single-cell analysis has been applied in intracellular research, gene-and protein content and expression, PCR, cell culture and division, clone formation, differentiation, morphology, lysis, separation, sorting, cytotoxicity and fluorescence screens, antibody secretion, etc. as discussed here along with brief descriptions of the technical devices used for the studies, e. g. well-, trap-, pattern-, and droplet-based structures. This review aims to serve as an overview of available techniques for single-cell analysis by describing the different biological single-cell assays that have been performed to date and how each individual application requires a particular device design.
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| 10. |
- Lindström, Sara, et al.
(författare)
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PCR amplification and genetic analysis in a microwell cell cultivation chip
- 2008
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Ingår i: 12th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The Proceedings of MicroTAS 2008 Conference. - : Chemical and Biological Microsystems Society. ; , s. 576-578
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Konferensbidrag (refereegranskat)abstract
- We present a method for long-term single cell/clone cultivation followed by cell lysis, DNA amplification and detection of PCR product in a chip containing 672 individual microwells. By performing all steps on-chip in microwells, the proliferation and cell morphology of every single cell or clone can be linked to its genetic information. In this study two mammalian cell lines (mutated A431 vs. wild type U-2 OS) were used as a model system for mutation screening in the p53 gene. The presented method could improve the sensitivity in mutation frequency analysis of heterogeneous tumor samples.
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