Analysis of calcium signaling in live human tongue cell 3D-cultures upon tastant perfusion
Bridging the gap between two-dimensional cell cultures and complex in vivo tissues, three-dimensional cell culture models are of increasing interest in the fields of cell biology and pharmacology. However, present challenges hamper live cell imaging of three-dimensional cell cultures. These include...
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| Main Authors: | , , , , , , , |
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| Format: | Article (Journal) |
| Language: | English |
| Published: |
23 January 2020
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| In: |
Cell calcium
Year: 2020, Volume: 87 |
| ISSN: | 1532-1991 |
| DOI: | 10.1016/j.ceca.2020.102164 |
| Online Access: | Verlag, lizenzpflichtig, Volltext: https://dx.doi.org/10.1016/j.ceca.2020.102164 Verlag, lizenzpflichtig, Volltext: http://www.sciencedirect.com/science/article/pii/S0143416020300063 
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| Author Notes: | Elena von Molitor, Elina Nürnberg, Torsten Ertongur-Fauth, Paul Scholz, Katja Riedel, Mathias Hafner, Rüdiger Rudolf, Tiziana Cesetti |
| Summary: | Bridging the gap between two-dimensional cell cultures and complex in vivo tissues, three-dimensional cell culture models are of increasing interest in the fields of cell biology and pharmacology. However, present challenges hamper live cell imaging of three-dimensional cell cultures. These include (i) the stabilization of these structures under perfusion conditions, (ii) the recording of many z-planes at high spatio-temporal resolution, (iii) and the data analysis that ranges in complexity from whole specimens to single cells. Here, we addressed these issues for the time-lapse analysis of Ca2+ signaling in spheroids composed of human tongue-derived HTC-8 cells upon perfusion of gustatory substances. Live cell imaging setups for confocal and light sheet microscopy were developed that allow simple and robust spheroid stabilization and high-resolution microscopy with perfusion. Visualization of spheroids made of HTC-8 cells expressing the G-GECO fluorescent Ca2+ sensor revealed Ca2+ transients that showed similar kinetics but different amplitudes upon perfusion of bitter compounds Salicine and Saccharin. Dose-dependent responses to Saccharin required extracellular Ca2+. From the border towards the center of spheroids, compound-induced Ca2+ signals were progressively delayed and decreased in amplitude. Stimulation with ATP led to strong Ca2+ transients that were faster than those evoked by the bitter compounds and blockade of purinergic receptors with Suramin abutted the response to Saccharin, suggesting that ATP mediates a positive autocrine and paracrine feedback. Imaging of ATP-induced Ca2+ transients with light sheet microscopy allowed acquisition over a z-depth of 100 μm without losing spatial and temporal resolution. In summary, the presented approaches permit the study of fast cellular signaling in three-dimensional cultures upon compound perfusion. |
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| Item Description: | Gesehen am 16.06.2020 |
| Physical Description: | Online Resource |
| ISSN: | 1532-1991 |
| DOI: | 10.1016/j.ceca.2020.102164 |