To understand the human brain and its neurodegenerative diseases, as well as to develop appropriate treatments, we need to investigate human tissue. Human organotypic culture provides an excellent opportunity to examine cellular and network-level activities, as well as molecular changes at long-term, as this technique enables the maintenance of the brain tissue for several weeks with a preserved tissue structure and cellular connections. However, due to the slice preparation and culture technique, considerable changes occur in the cultured tissue. In this study, we explored neuronal degeneration and loss, as well as the degree of gliosis along the culture period.We evaluated the preservation of the cultures in artificial culturing medium (AM) and human cerebrospinal fluid (hCSF) as well as the effect of distinct antibiotics and micotics (penicillin, streptomycin, amphotericin) in preventing infections. Neuronal densities were calculated based on the neuronal marker NeuN staining, Fluoro-Jade C (FJC) staining-based pixel intensity histograms showed the amounts of degenerating cells, while astrocytic involvement, including soma and cell process coverage of the samples were relieved by anti-GFAP staining.Our data shows that considerable changes occur during the culturing period of the organotypic tissue slices, highlighting the importance of anatomical examinations of the cultures to better understand these modifications and their relation with the physiological data from these slices.