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  • 1.
    Friedman, Mikaela
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    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.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Engineering and characterization of a bispecific HER2 × EGFR-binding affibody molecule2009In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 54, p. 121-131Article in journal (Refereed)
    Abstract [en]

    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.

  • 2.
    Grönwall, Caroline
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Jonsson, Andreas
    Affibody AB, Bromma.
    Lindström, Sara
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Gunneriusson, Elin
    Affibody AB, Bromma.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Herne, Nina
    Affibody AB, Bromma.
    Selection and characterization of Affibody ligands binding to Alzheimer amyloid beta peptides2007In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 128, no 1, p. 162-183Article in journal (Refereed)
    Abstract [en]

    Affibody (Affibody) ligands specific for human amyloid beta (Abeta) peptides (40 or 42 amino acid residues in size), involved in the progress of Alzheimer's disease, were selected by phage display technology from a combinatorial protein library based on the 58-amino acid residue staphylococcal protein A-derived Z domain. Post-selection screening of 384 randomly picked clones, out of which 192 clones were subjected to DNA sequencing and clustering, resulted in the identification of 16 Affibody variants that were produced and affinity purified for ranking of their binding properties. The two most promising Affibody variants were shown to selectively and efficiently bind to Abeta peptides, but not to the control proteins. These two Affibody ligands were in dimeric form (to gain avidity effects) coupled to affinity resins for evaluation as affinity devices for capture of Abeta peptides from human plasma and serum. It was found that both ligands could efficiently capture Abeta that were spiked (100 microgml(-1)) to plasma and serum samples. A ligand multimerization problem that would yield suboptimal affinity resins, caused by a cysteine residue present at the binding surface of the Affibody ligands, could be circumvented by the generation of second-generation Affibody ligands (having cysteine to serine substitutions). In an epitope mapping effort, the preferred binding site of selected Affibody ligands was mapped to amino acids 30-36 of Abeta, which fortunately would indicate that the Affibody molecules should not bind the amyloid precursor protein (APP). In addition, a significant effort was made to analyze which form of Abeta (monomer, dimer or higher aggregates) that was most efficiently captured by the selected Affibody ligand. By using Western blotting and a dot blot assay in combination with size exclusion chromatography, it could be concluded that selected Affibody ligands predominantly bound a non-aggregated form of analyzed Abeta peptide, which we speculate to be dimeric Abeta. In conclusion, we have successfully selected Affibody ligands that efficiently capture Abeta peptides from human plasma and serum. The potential therapeutic use of these optimized ligands for extracorporeal capture of Abeta peptides in order to slow down or reduce amyloid plaque formation, is discussed.

  • 3.
    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.

  • 4.
    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.

  • 5.
    Lindstrom, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Proteomics.
    Miniaturization of biological assays: Overview on microwell devices for single-cell analyses2011In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1810, no 3, p. 308-316Article, review/survey (Refereed)
    Abstract [en]

    Background: Today, cells are commonly analyzed in ensembles, i.e. thousands of cells per sample, yielding results on the average response of the cells. However, cellular heterogeneity implies the importance of studying how individual cells respond, one by one, in order to learn more about drug targeting and cellular behavior. Scope of review: This review discusses general aspects on miniaturization of biological assays and in particular summarizes single-cell assays in microwell formats. A range of microwell-based chips are discussed with regard to their well characteristics, cell handling, choice of material etc. along with available detection systems for single-cell studies. History and trends in microsystem technology, various commonly used materials for device fabrication, and conventional methods for single-cell analysis are also discussed, before a closing section with a detailed example from our research in the field. Major conclusions:A range of miniaturized and microwell devices have shown useful for studying individual cells. General significance: In vitro assays offering low volume sampling and rapid analysis in a high-throughput manner are of great interest in a wide range of single-cell applications. Size compatibility between a cell and micron-sized tools has encouraged the field of micro- and nanotechnologies to move into areas such as life sciences and molecular biology. To test as many compounds as possible against a given amount of patient sample requires miniaturized tools where low volume sampling is sufficient for accurate results and on which a high number of experiments per cm(2) can be performed. This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine. (C) 2010 Elsevier B.V. All rights reserved.

  • 6.
    Lindström, Sara
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Microwell devices for single-cell analyses2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    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.

  • 7.
    Lindström, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    A microwell array device with integrated microfluidic components for enhanced single-cell analysis2009In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 30, no 24, p. 4166-4171Article in journal (Refereed)
    Abstract [en]

    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.

  • 8.
    Lindström, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Eriksson, M.
    Vazin, Tandis
    KTH, School of Biotechnology (BIO), Gene Technology. National Institute of Health, United States .
    Sandberg, Julia
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Frisén, J.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    A microwell chip for parallel culture and analysis of stem cells2009In: Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Chemical and Biological Microsystems Society , 2009, p. 1309-1311Conference paper (Refereed)
    Abstract [en]

    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.

  • 9.
    Lindström, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Eriksson, Malin
    Vazin, Tandis
    KTH, School of Biotechnology (BIO), Gene Technology.
    Sandberg, Julia
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Frisén, Jonas
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO).
    High-Density Microwell Chip for Culture and Analysis of Stem Cells2009In: PLos ONE, ISSN 1932-6203, Vol. 4, no 9, p. e6997-Article in journal (Refereed)
    Abstract [en]

    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.

  • 10.
    Lindström, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Hammond, Maria
    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.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    PCR amplification and genetic analysis in a microwell cell culturing chip2009In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, p. 3465-3471Article in journal (Refereed)
    Abstract [en]

    We have previously described a microwell chip designed for high throughput, long-term single-cell culturing and clonal analysis in individual wells providing a controlled way of studying high numbers of individual adherent or non-adherent cells. Here we present a method for the genetic analysis of cells cultured on-chip by PCR and minisequencing, demonstrated using two human adherent cell lines: one wild type and one with a single-base mutation in the p53 gene. Five wild type or mutated cells were seeded per well (in a defined set of wells, each holding 500 nL of culture medium) in a 672-microwell chip. The cell chip was incubated overnight, or cultured for up to five days, depending on the desired colony size, after which the cells were lysed and subjected to PCR directly in the wells. PCR products were detected, in the wells, using a biotinylated primer and a fluorescently labelled primer, allowing the products to be captured on streptavidin-coated magnetic beads and detected by a fluorescence microscope. In addition, to enable genetic analysis by minisequencing, the double-stranded PCR products were denatured and the immobilized strands were kept in the wells by applying a magnetic field from the bottom of the wells while the wells were washed, a minisequencing reaction mixture was added, and after incubation in appropriate conditions the expected genotypes were detected in the investigated microwells, simultaneously, by an array scanner. We anticipate that the technique could be used in mutation frequency screening, providing the ability to correlate cells' proliferative heterogeneity to their genetic heterogeneity, in hundreds of samples simultaneously. The presented method of single-cell culture and DNA amplification thus offers a potentially powerful alternative to single-cell PCR, with advantageous robustness and sensitivity

  • 11.
    Lindström, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Hammond, Maria
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Gantelius, Jesper
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    PCR amplification and genetic analysis in a microwell cell cultivation chip2008In: 12th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The Proceedings of MicroTAS 2008 Conference, Chemical and Biological Microsystems Society , 2008, p. 576-578Conference paper (Refereed)
    Abstract [en]

    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.

  • 12.
    Lindström, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Iles, A.
    Persson, Johanna
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Hosseinkhani, H.
    Hosseinkhani, M.
    Khademhosseini, A.
    Lindström, H.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Nanoporous titania coating of microwell chips for stem cell culture and analysis2010In: Journal of Biomechanical Science and Engineering, ISSN 1880-9863, Vol. 5, no 3, p. 272-279Article in journal (Refereed)
    Abstract [en]

    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.

  • 13.
    Lindström, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Larsson, R.
    Andersson, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    High throughput single cell clone analysis2006In: Micro Total Analysis Systems - Proceedings of MicroTAS 2006 Conference: 10th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Japan Academic Association Inc , 2006, p. 410-412Conference paper (Refereed)
    Abstract [en]

    A novel microplate has been developed for high throughput single cell/clone analysis. Rapid single cell seeding using a conventional FACS into micro wells allows several thousands of single cells to be cultivated, short-term (72 h) or long-term (10-14 days), and analyzed individually. The platform requires a remarkably low number of cells, a major advantage when screening limited amounts of patient cell samples. Analysis of single cell heterogeneity and colony formation related to drug sensitivity can be accomplished in a high throughput manner.

  • 14.
    Lindström, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Larsson, Rolf
    Univ Uppsala Hosp, Dept Med Sci, Uppsala, Sweden.
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Towards high-throughput single cell/clone cultivation and analysis2008In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 29, p. 1219-1227Article in journal (Refereed)
    Abstract [en]

    In order to better understand cellular processes and behavior, a controlled way of studying high numbers of single cells and their clone formation is greatly needed. Numerous ways of ordering single cells into arrays have previously been described, but platforms in which each cell/clone can be addressed to an exact position in the microplate, cultivated for weeks and treated separately in a high-throughput manner have until now been missing. Here, a novel microplate developed for high-throughput single cell/clone cultivation and analysis is presented. Rapid single cell seeding into microwells, using conventional flow cytometry, allows several thousands of single cells to be cultivated, short-term (72 h) or long-term (10-14 days), and analyzed individually. By controlled sorting of individual cells to predefined locations in the microplate, analysis of single cell heterogeneity and clonogenic properties related to drug sensitivity can be accomplished. Additionally, the platform requires remarkably low number of cells, a major advantage when screening limited amounts of patient cell samples. By seeding single cells into the microplate it is possible to analyze the cells for over 14 generations, ending up with more than 10 000 cells in each well. Described here is a proof-of-concept on compartmentalization and cultivation of thousands of individual cells enabling heterogeneity analysis of various cells/clones and their response to different drugs.

  • 15.
    Vanherberghen, Bruno
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Kowalewski, Jacob
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Garrod, Kym
    Lindström, Sara
    KTH, School of Biotechnology (BIO).
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO).
    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.
    Single Cell Tracking of Natural Killer CellMigration in vivo and in vitro reveals Transient Migration Arrest PeriodsManuscript (preprint) (Other (popular science, discussion, etc.))
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