Conference paper Open Access

3D live cell imaging: study of inter- and intracellular signalling induced by bitter molecules in tongue cell spheroids

von Molitor, Elena; Nürnberg, Elina; Fauth, Torsten; Scholz, Paul; Riedel, Katja; Krohn, Michael; Hafner, Mathias; Rudolf, Rüdiger; Cesetti, Tiziana

Tissue complexity is not well represented in a flat 2D environment. In particular, proliferation rates and cellular phenotypes are altered, intercellular communication is lost or severely affected and signalling molecules do not have to diffuse through several cell layers to reach their target. Spheroids allow to investigate cell signalling in a 3D structure with intact cell-to-cell communication. However, there are challenges hampering 3D live-cell imaging including: (i) the stabilization of spheroids under perfusion conditions, (ii) the recording of many z-planes at high spatio-temporal resolution, and (iii) the complexity of data analysis. Here, we addressed these issues using spheroids made with human-papillae derived tongue cells (HTC-8), immortalized and engineered to express a green-fluorescent genetically-encoded Ca2+ (G-GECO) sensors. By confocal and Light Sheet Fluorescence Microscopy (LSFM) we measured quantitatively intracellular Ca2+ changes upon acute application of gustatory bitter substances. We developed a simple and robust live-cell imaging perfusion setup for confocal as well as LSFM that granted spheroid stabilization and high-resolution microscopy in many z-planes at high spatio-temporal resolution.  With this approach we could show that Ca2+ transients induced by saccharin appeared in the whole spheroid and required extracellular Ca2+. However, the responses differed between the border and the centre of the spheroids, with larger and faster response in the outer region. Furthermore, ATP-mediated intercellular communication was found to play a major role in the generation of compound-evoked Ca2+ transients, suggesting the presence of an ATP-mediated positive autocrine/paracrine communication in HTC-8 spheroids. In summary, the presented approach permits the study of fast inter- and intracellular signalling in 3D spheroid cultures upon compounds perfusion.

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