Confocal live cell images of microglia in 3D cell culture from the dissertation ' 3D culture conditions instruct an in vivo-like phenotype in primary microgia' (doi:10.60507/FK2/FFPGJA)

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Confocal live cell images of microglia in 3D cell culture from the dissertation ' 3D culture conditions instruct an in vivo-like phenotype in primary microgia'

doi:10.60507/FK2/FFPGJA

bonndata

2025-03-25

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Tambe, Bertrand Agbor, 2025, "Confocal live cell images of microglia in 3D cell culture from the dissertation ' 3D culture conditions instruct an in vivo-like phenotype in primary microgia'", https://doi.org/10.60507/FK2/FFPGJA, bonndata, V1

Confocal live cell images of microglia in 3D cell culture from the dissertation ' 3D culture conditions instruct an in vivo-like phenotype in primary microgia'

doi:10.60507/FK2/FFPGJA

Tambe, Bertrand Agbor (University of Bonn)

bonndata

Tambe, Bertrand Agbor

Tambe, Bertrand

2025-03-24

https://doi.org/10.60507/FK2/FFPGJA

Medicine, Health and Life Sciences

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.

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Bertrand_Tambe_3D_microglia_readme.txt

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Figure 17A. Microglial cell-matrix interaction in 3D culture.

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Representative confocal time-lapse images of 3D matrix fibers (silver) and Cx3cr1-GFP microglia (green). 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.

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Figure 18A. Analysis of time-lapse confocal images of microglia in 2D and 3D culture

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Representative confocal time-lapse images of microglia culture in 2D culture over 10 minutes. 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 ).

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Figure 18B. Analysis of time-lapse confocal images of microglia in 2D and 3D culture

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Representative confocal time-lapse images of microglia culture in 3D over 10 minutes. 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 18B ).

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Figure 21A. Microglial phagocytic assay in 3D culture

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Representative confocal time-lapse images of microglia phagocytosis in 2D culture. Green = Cx3cr1-GFP microglia, blue = 2 um beads. 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). In contrast, 2D microglia phagocytosed beads mainly with their amoeboid cell bodies (Figure21 A).

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Figure 21B. Microglial phagocytic assay in 3D culture

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Representative confocal time-lapse images of microglia phagocytosis in 3D culture. Green = Cx3cr1-GFP microglia, blue = 2 um beads. 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 21B). In contrast, 2D microglia phagocytosed beads mainly with their amoeboid cell bodies (Figure21B).

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Figure 22A. Microglia motility during phagocytosis.

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Representative confocal time-lapse images of migrating microglia during phagocytosis for 24 hours in 2D culture. 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).

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Figure 22B. Microglia motility during phagocytosis.

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Representative confocal time-lapse images of migrating microglia during phagocytosis for 24 hours in 3D culture. 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 22B).

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