Previously we have introduced a single cell system that allows the culturing of adult rat cardiac myocytes for more than 6 days with minimized cellular dedifferentiation. The aim of the present study was to investigate possible morphological changes of cell structures within the culture period. We constructed adenoviral vectors encoding for fluorescent proteins fused to the subunit VIII of human cytochrome C oxidase, Calreticulin, GPI and ts045G in order to visualize mitochondria, ER, membrane and Golgi, respectively. In parallel, organelles of naïve cells were labelled using inorganic dyes. The mitochondrial membrane potential sensitive dye JC-1 was used to monitor the state of mitochondrial activity. High resolution confocal z-stacks were recorded and 3D- rendered at DIV0, 3 and 6. Expression of plasma membrane targeted GPI-YFP indicated the prolonged presence of T-tubular structures. Plasma membrane probing with Di-8-Anepps displayed a lack of T-tubules in cells beyond DIV 3. This indicates the intracellular prolonged presence of T-tubular structures that progressively detach from the outer membrane. These findings strongly support earlier findings in that our culture conditions largely prevented myocyte dedifferentiation. In conclusion, long term studies of the physiology of cardiac myocytes can be performed without major alteration of subcellular structures.