Long-term human organotypic brain slice cultures: a detailed protocol to provide a comprehensive framework for single-neuron and neuronal network investigations

Background The investigation of the human brain at cellular and microcircuit level remains challenging due to the fragile viability of neuronal tissue, inter- and intra-variability of the samples and limited availability of human brain material. New method Here, we present an optimized work-up to use resected tissue from brain surgeries for live cell experiments in vitro. Comparison with existing methods We provide a reworked, detailed protocol of the production, culturing and viral transduction of human organotypic brain slice cultures for research purposes. Results We highlight the critical pitfalls of the culturing process of the human brain tissue and present results on viral expression, single-cell Patch-Clamp recordings, as well as multi-electrode array recordings over a prolonged period of time. Additionally, our statistics show that brain tissue from patients of any age and morbidity can be used for organotypic brain slice cultures if carefully selected. Conclusions Organotypic brain slice cultures are of great value for basic neuroscience and disease modeling over a time course of three weeks. Highlights ![Figure 1, Graphical abstract.][1] Figure 1, Graphical abstract. Schematic overview of preparation, maintenance and experimental workup of human organotypic brain slice cultures. After surgical resection, human brain tissue is prepared, sliced and cultured at air-liquid-interface between human cerebrospinal fluid and defined incubator atmosphere, allowing for week-long viability. This enables extensive experimental workup such as viral transduction, single cell and multi-electrode array electrophysiological recordings. ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes