8 results on '"Braeckmans, Kevin"'
Search Results
2. Cas9 RNP transfection by vapor nanobubble photoporation for ex vivo cell engineering
- Author
-
Raes, Laurens, Pille, Melissa, Harizaj, Aranit, Goetgeluk, Glenn, van Hoeck, Jelter, Stremersch, Stephan, Fraire, Juan C, Brans, Toon, de Jong, Olivier G, Maas - Bakker, Roel, Mastrobattista, Enrico, Vader, Pieter, de Smedt, Stefaan C, Vandekerckhove, Bart, Raemdonck, Koen, Braeckmans, Kevin, Afd Pharmaceutics, and Pharmaceutics
- Subjects
Cell type ,intracellular delivery ,T cells ,RM1-950 ,photoporation ,MESENCHYMAL STEM-CELLS ,Genome engineering ,ACTIVATION ,Cell therapy ,DELIVERY ,stem cells ,Drug Discovery ,Medicine and Health Sciences ,Primary cell ,gene editing ,Chemistry ,Mesenchymal stem cell ,Biology and Life Sciences ,Transfection ,CAR ,Cell biology ,T-CELLS ,Molecular Medicine ,Therapeutics. Pharmacology ,CRISPR-Cas9 ,Stem cell ,SYSTEM ,Intracellular - Abstract
The CRISPR-Cas9 technology represents a powerful tool for genome engineering in eukaryotic cells, advancing both fundamental research and therapeutic strategies. Despite the enormous potential of the technology, efficient and direct intracellular delivery of Cas9 ribonucleoprotein (RNP) complexes in target cells poses a significant hurdle, especially in refractive primary cells. In the present work, vapor nanobubble (VNB) photoporation was explored for Cas9 RNP transfection in a variety of cell types. Proof of concept was first demonstrated in H1299-EGFP cells, before proceeding to hard-to-transfect stem cells and T cells. Gene knock-out levels over 80% and up to 60% were obtained for H1299 cells and mesenchymal stem cells, respectively. In these cell types, the unique possibility of VNB photoporation to knock out genes according to user-defined spatial patterns was demonstrated as well. Next, effective targeting of the programmed cell death 1 receptor and Wiskott-Aldrich syndrome gene in primary human T cells was demonstrated, reaching gene knock-out levels of 25% and 34%, respectively. With a throughput of >200,000 T cells per second, VNB photoporation is a scalable and versatile intracellular delivery method that holds great promise for CRISPR-Cas9-mediated ex vivo engineering of cell therapy products.
- Published
- 2021
3. Hydrogel-Induced Cell Membrane Disruptions Enable Direct Cytosolic Delivery of Membrane-Impermeable Cargo
- Author
-
Van Hoeck, Jelter, Van de Vyver, Thijs, Harizaj, Aranit, Goetgeluk, Glenn, Merckx, Pieterjan, Liu, Jing, Wels, Mike, Sauvage, Félix, De Keersmaecker, Herlinde, Vanhove, Christian, Vader, Pieter, de Jong, Olivier G., Dewitte, Heleen, Vandekerckhove, Bart, Braeckmans, Kevin, de Smedt, S.C., Raemdonck, Koen, Afd Pharmaceutics, and Pharmaceutics
- Subjects
protein delivery ,Materials science ,Cell Membrane Permeability ,contrast-enhanced MRI ,intracellular delivery ,membrane disruption ,Cell ,Contrast Media ,02 engineering and technology ,010402 general chemistry ,01 natural sciences ,Cell therapy ,Cell membrane ,chemistry.chemical_compound ,nanogels ,Cytosol ,Materials Science(all) ,Nanocapsules ,medicine ,Organometallic Compounds ,Animals ,Humans ,General Materials Science ,Fluorescein isothiocyanate ,Fluorescent Dyes ,Electroporation ,Mechanical Engineering ,Cell Membrane ,Epithelium, Corneal ,Proteins ,Dextrans ,Hydrogels ,021001 nanoscience & nanotechnology ,0104 chemical sciences ,Cell biology ,medicine.anatomical_structure ,Cross-Linking Reagents ,chemistry ,Cell culture ,Mechanics of Materials ,Self-healing hydrogels ,Cattle ,cell therapy ,0210 nano-technology ,Intracellular ,Fluorescein-5-isothiocyanate ,HeLa Cells - Abstract
Intracellular delivery of membrane-impermeable cargo offers unique opportunities for biological research and the development of cell-based therapies. Despite the breadth of available intracellular delivery tools, existing protocols are often suboptimal and alternative approaches that merge delivery efficiency with both biocompatibility, as well as applicability, remain highly sought after. Here, a comprehensive platform is presented that exploits the unique property of cationic hydrogel nanoparticles to transiently disrupt the plasma membrane of cells, allowing direct cytosolic delivery of uncomplexed membrane-impermeable cargo. Using this platform, which is termed Hydrogel-enabled nanoPoration or HyPore, the delivery of fluorescein isothiocyanate (FITC)-dextran macromolecules in various cancer cell lines and primary bovine corneal epithelial cells is convincingly demonstrated. Of note, HyPore demonstrates efficient FITC-dextran delivery in primary human T cells, outperforming state-of-the-art electroporation-mediated delivery. Moreover, the HyPore platform enables cytosolic delivery of functional proteins, including a histone-binding nanobody as well as the enzymes granzyme A and Cre-recombinase. Finally, HyPore-mediated delivery of the MRI contrast agent gadobutrol in primary human T cells significantly improves their T1 -weighted MRI signal intensities compared to electroporation. Taken together, HyPore is proposed as a straightforward, highly versatile, and cost-effective technique for high-throughput, ex vivo manipulation of primary cells and cell lines.
- Published
- 2021
4. Technical implementations of light sheet microscopy
- Author
-
Stefaan C. De Smedt, Brans Toon, Braeckmans Kevin, Zagato Elisa, Neyts Kristiaan, and Remaut Katrien
- Subjects
0301 basic medicine ,SELF-RECONSTRUCTING BEAMS ,Histology ,Microscope ,Optical sectioning ,Computer science ,Nanotechnology ,law.invention ,03 medical and health sciences ,CELL 3D SUPERRESOLUTION ,law ,Microscopy ,Peak intensity ,Medicine and Health Sciences ,Fluorescence microscope ,HIGH-SPEED ,BESSEL BEAMS ,Instrumentation ,light sheet microscopy ,FLOW-CYTOMETRY ,FLUORESCENCE MICROSCOPY ,Biology and Life Sciences ,Photobleaching ,Visualization ,PLANE ILLUMINATION MICROSCOPY ,CROSS-CORRELATION SPECTROSCOPY ,Medical Laboratory Technology ,SPIM ,030104 developmental biology ,Light sheet fluorescence microscopy ,ZEBRAFISH DEVELOPMENT ,Anatomy ,THICK BIOLOGICAL SAMPLES - Abstract
Fluorescence-based microscopy is among the most successful methods in biological studies. It played a critical role in the visualization of subcellular structures and in the analysis of complex cellular processes, and it is nowadays commonly employed in genetic and drug screenings. Among the fluorescence-based microscopy techniques, light sheet fluorescence microscopy (LSFM) has shown a quite interesting set of benefits. The technique combines the speed of epi-fluorescence acquisition with the optical sectioning capability typical of confocal microscopes. Its unique configuration allows the excitation of only a thin plane of the sample, thus fast, high resolution imaging deep inside tissues is nowadays achievable. The low peak intensity with which the sample is illuminated diminishes phototoxic effects and decreases photobleaching of fluorophores, ensuring data collection for days with minimal adverse consequences on the sample. It is no surprise that LSFM applications have raised in just few years and the technique has been applied to study a wide variety of samples, from whole organism, to tissues, to cell clusters, and single cells. As a consequence, in recent years numerous set-ups have been developed, each one optimized for the type of sample in use and the requirements of the question at hand. Hereby, we aim to review the most advanced LSFM implementations to assist new LSFM users in the choice of the LSFM set-up that suits their needs best. We also focus on new commercial microscopes and "do-it-yourself" strategies; likewise we review recent designs that allow a swift integration of LSFM on existing microscopes.
- Published
- 2018
5. Water-soluble monofunctional perylene and terrylene dyes: powerful labels for single-enzyme tracking
- Author
-
Peneva, Kalina, Mihov, Gueorgui, Nolde, Fabian, Rocha, Susana, Hotta, Jun-ichi, Braeckmans, Kevin, Hofkens, Johan, Uji-i, Hiroshi, Herrmann, Andreas, Muellen, Klaus, Müllen, Klaus, Polymer Chemistry and Bioengineering, Zernike Institute for Advanced Materials, and Nanotechnology and Biophysics in Medicine (NANOBIOMED)
- Subjects
MOLECULE FLUORESCENCE SPECTROSCOPY ,Kinetics ,single-molecule studies ,Tracking (particle physics) ,DONOR ,Catalysis ,chemistry.chemical_compound ,ACCEPTOR ,fluorescent probes ,Organic chemistry ,INFECTION PATHWAY ,Animals ,Humans ,Perylene ,Fluorescent Dyes ,chemistry.chemical_classification ,Anthracenes ,Chemistry ,General Medicine ,General Chemistry ,Acceptor ,Enzymes ,Enzyme ,Water soluble ,Microscopy, Fluorescence ,kinetics ,dyes/pigments - Published
- 2008
6. Photobleaching with the confocal laser scanning microscope for mobility measurements and the encoding of microbeads
- Author
-
Braeckmans, Kevin, Demeester, J, and De Smedt, S
- Published
- 2004
7. Membrane vesicle secretion and prophage induction in multidrug-resistant Stenotrophomonas maltophilia in response to ciprofloxacin stress
- Author
-
Devos, Simon, Van Putte, Wouter, Vitse, Jolien, Van Driessche, Gonzalez, Stremersch, Stephan, Van Den Broek, Wim, Raemdonck, Koen, Braeckmans, Kevin, Stahlberg, Henning, Kudryashev, Misha, Savvides, Savvas N., and Devreese, Bart
- Subjects
biochemical phenomena, metabolism, and nutrition ,bacterial infections and mycoses - Abstract
Several bacterial species produce membrane vesicles (MVs) in response to antibiotic stress. However, the biogenesis and role of MVs in bacterial antibiotic resistance mechanisms have remained unclear. Here, we studied the effect of the fluoroquinolone ciprofloxacin on MV secretion by Stenotrophomonas maltophilia using a combination of electron microscopy and proteomic approaches. We found that in addition to the classical outer membrane vesicles (OMV), ciprofloxacin-stimulated cultures produced larger vesicles containing both outer and inner membranes termed outer-inner membrane vesicles (OIMV), and that such MVs are enriched with cytosolic proteins. Remarkably, OIMV were found to be decorated with filamentous structures identified as fimbriae. In addition, ciprofloxacin stress leads to the release of bacteriophages and phage tail-like particles. Prophage induction by ciprofloxacin has been linked to pathogenesis and horizontal gene transfer in several bacterial species. Together, our findings show that ciprofloxacin treatment of S. maltophilia leads to the secretion of a heterogeneous pool of MVs and the induction of prophages that are potentially involved in adverse side-effects during antibiotic treatment.
8. Fluorescence recovery after photobleaching in material and life sciences: putting theory into practice
- Author
-
Kevin Braeckmans, Marcel Ameloot, James G. McNally, Magnus Nydén, Francesca Cella-Zanacchi, Mats Rudemo, Anne-Marie Hermansson, Jenny Jonasson, Diana Bernin, Niklas Lorén, Hendrik Deschout, Joel H Hagman, Alberto Diaspro, Nick Smisdom, Lorén, Niklas, Hagman, Joel, Jonasson, Jenny K, Deschout, Hendrik, Bernin, Diana, Cella-Zanacchi, Francesca, Diaspro, Alberto, McNally, James G, Ameloot, Marcel, Smisdom, Nick, Nydén, Magnus, Hermansson, Anne-Marie, Rudemo, Mats, and Braeckmans, Kevin
- Subjects
LATERAL DIFFUSION ,Micrometer scale ,Anomalous diffusion ,3-DIMENSIONAL DIFFUSION MEASUREMENTS ,Diffusion ,Biophysics ,Nanotechnology ,photobleaching ,SINGLE-PARTICLE TRACKING ,Fluorescence ,Photobleaching ,Fluorescence microscope ,Medicine and Health Sciences ,FRAP ANALYSIS ,IMAGE CORRELATION SPECTROSCOPY ,ANOMALOUS DIFFUSION ,LASER-SCANNING MICROSCOPE ,Chemistry ,CELL-SURFACE RECEPTORS ,Fluorescence recovery after photobleaching ,NUCLEAR PROTEINS ,PLASMA-MEMBRANE ,fluorescence ,Concentration gradient - Abstract
Fluorescence recovery after photobleaching (FRAP) is a versatile tool for determining diffusion and interaction/binding properties in biological and material sciences. An understanding of the mechanisms controlling the diffusion requires a deep understanding of structure–interaction–diffusion relationships. In cell biology, for instance, this applies to the movement of proteins and lipids in the plasma membrane, cytoplasm and nucleus. In industrial applications related to pharmaceutics, foods, textiles, hygiene products and cosmetics, the diffusion of solutes and solvent molecules contributes strongly to the properties and functionality of the final product. All these systems are heterogeneous, and accurate quantification of the mass transport processes at the local level is therefore essential to the understanding of the properties of soft (bio)materials. FRAP is a commonly used fluorescence microscopy-based technique to determine local molecular transport at the micrometer scale. A brief high-intensity laser pulse is locally applied to the sample, causing substantial photobleaching of the fluorescent molecules within the illuminated area. This causes a local concentration gradient of fluorescent molecules, leading to diffusional influx of intact fluorophores from the local surroundings into the bleached area. Quantitative information on the molecular transport can be extracted from the time evolution of the fluorescence recovery in the bleached area using a suitable model. A multitude of FRAP models has been developed over the years, each based on specific assumptions. This makes it challenging for the non-specialist to decide which model is best suited for a particular application. Furthermore, there are many subtleties in performing accurate FRAP experiments. For these reasons, this review aims to provide an extensive tutorial covering the essential theoretical and practical aspects so as to enable accurate quantitative FRAP experiments for molecular transport measurements in soft (bio)materials.
- Published
- 2015
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.