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 (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 Ca 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 Ca sensor revealed Ca transients that showed similar kinetics but different amplitudes upon perfusion of bitter compounds Salicine and Saccharin. Dose-dependent responses to Saccharin required extracellular Ca. From the border towards the center of spheroids, compound-induced Ca signals were progressively delayed and decreased in amplitude. Stimulation with ATP led to strong Ca 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 Ca 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.