Connexins form gap junction channels and hemichannels that participate in the formation of functional physiological units from individual cells and therefore are essential for many tissue functions. Tissue-engineered models can be used to analyse the integration of connexin channels in general signalling mechanisms in a tissue and thereby to understand the role of connexin channels in the physiology of a tissue and their involvement in pathological conditions. However, analysing connexin channels in tissue-like 3D cell culture is challenging. Simple and convenient methods to analyse gap junction coupling usually rely on dye transfer, e. g. after microinjection or scrape loading. These techniques are invasive and almost impossible to apply to complex cell culture systems. We used the GNOME LP technique (Heinemann et al. 2013) to establish an improved method for gap junction coupling analysis via dye transfer (Begandt et al. 2015). The method is less invasive, allows a semi-automatic analysis of gap junction coupling and is applicable to complex cell culture models as shown for 3D cell culture of vascular endothelial cells in vessel-like structures (Begandt et al. 2015). Furthermore, a blood-brain barrier model with co-culture of cerebral microvascular endothelial cells, pericytes and astrocytes can be analysed. Adding a medium flow to mimic the circulating blood will allow to analyse the role of connexin channels in the blood-brain barrier function and their participation in pathological events such as ischemia or stroke. The connexin channels in each cell type involved in the formation of the blood-brain barrier can be analysed, while at the same time following intercellular cross-signalling based on other mediators such as purinergic signalling or inflammatory-related responses. The GNOME LP/dye transfer technique was also applied to lung epithelial cells cultured in transwell inserts, allowing a simultaneous analysis of connexin channels and transepithelial electrical resistance or permeability. Furthermore, implementation of air-liquid interface culture in this model allows to polarise the cells with separated apical and basolateral sides. Within this model, a crosstalk between connexin channels and signalling pathways activated from either side of the cells can be analysed. Connexin channels were shown to be involved in pathological events in tissue. It was proposed to target these channels for treatment of various pathologies such as inflammation or hypoxia-related impairments. Application of the GNOME LP/dye transfer technique to analyse connexin channels in tissue-engineered models will improve our understanding of the pivotal role of connexin channels in tissue physiology. It can further create a testing platform for pharmacological intervention of various pathologies.