------------------------------------------------------------------------------------------------------------- This file was generated on 2025-03-24 by Bertrand Agbor Tambe A GENERAL INFORMATION 1. Title of the dataset: Confocal live cell images of microglia in 3D cell culture from the dissertation '3D culture conditions instruct an in vivo-like phenotype in primary microglia' 2. Brief description of the research project and its aims: Microglia are innate immune cells of the central nervous system. They possess a diverse range of morphological features that enable them to perform different functions, such as surveillance, phagocytosis and immune response. In response to changes in physiological conditions within the CNS, microglia actively alter their shape in order to maintain brain homeostasis. Previous studies on the morphology and function of microglia have been conducted in 2D cell culture systems, which have been associated with an artificial morphology in microglia and altered microglial functions. Consequently, there is an increasing imperative to develop 3D cell culture systems that can more accurately replicate the complex microenvironment of the brain. The present study aims to investigate the impact of defined 3D cell culture conditions on gene expression, morphology, cell motility, innate immune response, and electrophysiological properties of cultured primary murine microglia. To this end, live cell confocal microscopy imaging was employed to visualise microglia within a 3D cell culture environment, thereby enabling the study of their motility, morphology and phagocytosis.The findings of this study demonstrate that microglia develop a more in vivo-like morphology and surveillance motility when cultured under 3D compared to 2D conditions. 3. Author Information A. Investigator Contact Information Name:Bertrand Agbor Tambe Institution: Deutsches Zentrum für Neurodegenerative Erkrankungen e. V. (DZNE) Address: Venusberg-Campus 1, Gebäude 99, Venusberg Campus 1/Gebäude 99, 53127 Bonn Email: drbertrands@yahoo.com B. Project Supervisor (Principal Investigator) Contact Information Name: Annett Halle Institution: Deutsches Zentrum für Neurodegenerative Erkrankungen e. V. (DZNE) Address: Venusberg-Campus 1, Gebäude 99, Venusberg Campus 1/Gebäude 99, 53127 Bonn Email: annett.halle@dzne.de C. In case of questions related to this dataset, please contact: Name: Bertrand Agbor Tambe Institution: Deutsches Zentrum für Neurodegenerative Erkrankungen e. V. (DZNE) Address: Venusberg-Campus 1, Gebäude 99, Venusberg Campus 1/Gebäude 99, 53127 Bonn Email: drbertrands@yahoo.com 6. Language of the dataset:English 7. Geographic location of data collection : Bonn, NRW, Germany B DATA & FILE OVERVIEW 1. File List: Figure 17. Microglial cell-matrix interaction in 3D culture. To explore possible microglial cell-matrix interactions in our 3D culture, we utilized a combination of fluorescent and confocal reflection microscopy techniques to visualize the interaction between microglia and the ECM. Time-lapse confocal z-stack images of eGFP microglia with z-slice of 0.5 um over 10 minutes with a time frame of 30 s were obtained. Confocal reflection microscopic grayscale images of the 3D gel matrix structure were simultaneously imaged using a 633 nm laser. We observed interactions between microglial cell branching processes and 3D matrix fibers (Figure 17A). Microglia constantly extended their branching processes to make several contacts with matrix fibers in 3D culture (Figure 17A). This illustrates a bidirectional cell-matrix fiber interaction; potentially promoting the more branched and ramified morphology of microglia seen in our 3D cell culture system. Figure 18A and B. Analysis of time-lapse confocal images of microglia in 2D and 3D culture We investigated microglial motility in 3D culture. To achieve this, we obtained time-lapse images of microglia using a high-resolution spinning disk confocal microscope equipped with a 60x water immersion objective. A time frame of 30s per image over 10 min with a z-slice interval of 0.5 µm was used to record the motility of microglia in 2D and 3D cultures. We observed that microglia in 3D culture showed a ramified morphology with constant extensions and retractions of processes reminiscent of in vivo-like microglial surveillance (Figure 18A and B). In contrast, microglia in 2D culture showed an amoeboid morphology with membrane ruffling (Figure 18A and B). Figure 21A and B. Microglia phagocytic assay in 3D culture To establish a microglial phagocytic assay in 3D culture, microglial cells were cultured with 2 µm polystyrene beads in Matrigel-collagen gel. The cell-matrix mixture was allowed to solidify at room temperature for 15 min and then at 37°C for 15 min. Confocal z-stack time-lapse images were obtained for a period of 24 h in 15 min intervals. In 3D culture, microglia phagocytosed beads through their ramified main branching processes and cell bodies (Figure 21A and B). In contrast, 2D microglia phagocytosed beads mainly with their amoeboid cell bodies (Figure 21 A and B). Figure 22A and B. Microglia motility during phagocytosis To investigate whether microglial cells migrate during phagocytosis in 3D culture, confocal z-stack time-lapse images were obtained for a period of 24 h at 15 min intervals. We observed that microglial cells migrated less during phagocytosis in 3D cultures compared to 2D cultures (Figure 22A and B). C SHARING/ACCESS INFORMATION 1. Was data derived from another source? yes/no : no 2. Licenses/restrictions placed on the data: CC BY 4.0 3. Links to publications that cite or use the data : https://nbn-resolving.org/urn:nbn:de:hbz:5-81722 4. Links to other publicly accessible locations of the data : https://doi.org/10.60507/FK2/FFPGJA 5. Links/relationships to ancillary datasets :