| Autor: |
De Beuckeleer S; Laboratory of Cell Biology and Histology, Faculty of Biomedical, Pharmaceutical and Veterinary sciences, University of Antwerp, Universiteitsplein 1, Antwerp, Belgium. Winnok.DeVos@uantwerpen.be., Vanhooydonck A; Faculty of Design Sciences, Department of Product Development, University of Antwerp, Paardenmarkt 94, 2000 Antwerp, Belgium. Regan.Watts@uantwerpen.be., Van Den Daele J; Laboratory of Cell Biology and Histology, Faculty of Biomedical, Pharmaceutical and Veterinary sciences, University of Antwerp, Universiteitsplein 1, Antwerp, Belgium. Winnok.DeVos@uantwerpen.be., Van De Looverbosch T; Laboratory of Cell Biology and Histology, Faculty of Biomedical, Pharmaceutical and Veterinary sciences, University of Antwerp, Universiteitsplein 1, Antwerp, Belgium. Winnok.DeVos@uantwerpen.be., Asselbergh B; VIB-UAntwerp Center for Molecular Neurology, VIB, Universiteitsplein 1, Antwerp, Belgium., Kim H; Department of Molecular Medicine, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, 0372 Oslo, Norway., Campsteijn C; Department of Molecular Medicine, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, 0372 Oslo, Norway., Ponsaerts P; Laboratory of Experimental Hematology, Vaccine and Infectious Disease Institute (Vaxinfectio), University of Antwerp, Belgium., Watts R; Faculty of Design Sciences, Department of Product Development, University of Antwerp, Paardenmarkt 94, 2000 Antwerp, Belgium. Regan.Watts@uantwerpen.be., De Vos WH; Laboratory of Cell Biology and Histology, Faculty of Biomedical, Pharmaceutical and Veterinary sciences, University of Antwerp, Universiteitsplein 1, Antwerp, Belgium. Winnok.DeVos@uantwerpen.be.; Antwerp Centre for Advanced Microscopy, University of Antwerp, Belgium.; μNEURO Centre of Research Excellence, University of Antwerp, Belgium. |
| Abstrakt: |
Modern cell and developmental biology increasingly relies on 3D cell culture systems such as organoids. However, routine interrogation with microscopy is often hindered by tedious, non-standardized sample mounting, limiting throughput. To address these bottlenecks, we have developed a pipeline for imaging intact organoids in flow, utilizing a transparent agarose fluidic chip that enables efficient and consistent recordings with theoretically unlimited throughput. The chip, cast from a custom-designed 3D-printed mold, is coupled to a mechanically controlled syringe pump for fast and precise sample positioning. We benchmarked this setup on a commercial digitally scanned light sheet microscope with cleared glioblastoma spheroids. Spheroids of varying sizes were positioned in the field of view with micrometer-level stability, achieving a throughput of 40 one-minute recordings per hour. We further showed that sample positioning could be automated through online feedback microscopy. The optical quality of the agarose chip outperformed FEP tubing, glass channels and PDMS casts for the clearing agents used, as demonstrated by image contrast profiles of spheroids stained with a fluorescent nuclear counterstain and further emphasized by the resolution of fine microglial ramifications within cerebral organoids. The retention of image quality throughout 500 μm-sized spheroids enabled comprehensive spatial mapping of live and dead cells based on their nuclear morphology. Finally, imaging a batch of LMNA knockout vs. wildtype astrocytoma spheroids revealed significant differences in their DNA damage response, underscoring the system's sensitivity and throughput. Overall, the fluidic chip design provides a cost-effective, accessible, and efficient solution for high-throughput organoid imaging. |
