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Visualizing Cell-Laden Fibrin-Based Hydrogels using Cryogenic Scanning Electron Microscopy and Confocal Microscopy.
Journal of Tissue Engineering and Regenerative Medicine 2019 January 19
The present investigation explores the microscopic aspects of cell-laden hydrogels at high resolutions, using three-dimensional cell cultures in semi-synthetic constructs that are of very-high water content (>98% water). The study aims to provide an imaging strategy for these constructs, while minimizing artifacts. Constructs of PEG-fibrinogen (PEG-Fb) and fibrin hydrogels containing embedded mesenchymal cells (human dermal fibroblasts) were first imaged by confocal microscopy. Next, high resolution scanning electron microscopy (HR-SEM) was used to provide images of the cells within the hydrogels, at submicron resolutions. Because it was not possible to obtain artifact-free images of the hydrogels using room-temperature HR-SEM, a cryogenic HR-SEM (cryo-HR-SEM) imaging methodology was employed to visualize the sample while preserving the natural hydrated state of the hydrogel. The ultrastructural details of the constructs were observed at subcellular resolutions, revealing numerous cellular components, the biomaterial in its native configuration, and the uninterrupted cell membrane as it relates with the biomaterial in the hydrated state of the construct. Constructs containing microscopic albumin microbubbles were also imaged using these methodologies to reveal fine details of the interaction between the cells, the microbubbles and the hydrogel. Taken together with the confocal microscopy, this imaging strategy provides a more complete picture of the hydrated state of the hydrogel network with cells inside. As such, this methodology addresses some of the challenges of obtaining this information in amorphous hydrogel systems containing a very-high water content (>98%) with embedded cells. Such insight may lead to better hydrogel-based strategies for tissue engineering and regeneration.
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