Publication date: 25th July 2016
Stem cells are broadly studied and envisioned for use as model systems in regenerative medicine, developmental and disease modelling, as well as for evaluating the effectiveness and safety of therapeutic drugs. However, conventionally used planar cell cultivation (monolayer) lacks spatial organization of cells, which leads to aberrant behaviors: flattened shape, abnormal polarization, altered response to pharmaceutical reagents and loss of differentiated phenotype. Therefore 3D cell culture methods have been developed in efforts to produce the most possible biologically relevant 3D models in vitro.
The complex three-dimensional environment provides the cell-cell and cell-extracellular matrix (ECM) signaling, and also establishes structural features, such as morphology and spatial organization of cells and subcellular compartments. Still, 3D cell culture is not yet optimized due to lack of standardized analytical tools for quantifying biological response. Utilization of microscopy and imaging for morphological characterization of aggregates, individual cells and ultrastructural compartments with subsequent 3D visualization could overcome some of these limits.
High resolution 3D imaging utilizing electron microscopy of cellular ultrastructure represents excellent method to reveal in situ morphological characteristics, cell-cell interactions, organelle distribution, niches in which cells reside, and many more. Here, we present serial block-face scanning electron microscopy of stem cell spheroids, which provides scanning of relatively large overviews (typically 500 to 5000 μm2) and volumes. Staining procedures and further image processing were suited to electron dense contrasting of DNA and membranes for 3D modeling and visualization of nuclei, membranes, and organelles (e.g. Golgi apparatus, mitochondria). This method is effective for detailed morphological analysis of stem cell spheroids, organoids as well as organotypic cell cultures ex vivo.
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