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

Abstract

BackgroundThe 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 methodHere, we present an optimized work-up to use resected tissue from brain surgeries for live cell experiments in vitro. Comparison with existing methodsWe provide a reworked, detailed protocol of the production, culturing and viral transduction of human organotypic brain slice cultures for research purposes. ResultsWe 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. ConclusionsOrganotypic brain slice cultures are of great value for basic neuroscience and disease modeling over a time course of three weeks. HighlightsO_LILong-term human organotypic brain slice cultures are viable for 2-3 weeks and provide a framework for basic neuroscience and disease modeling C_LIO_LIWe provide a reworked, detailed protocol for organotypic human brain slice culture production and maintenance C_LIO_LIWe show results of long-term culturing of human organotypic brain slice cultures in terms of viral transduction, whole-cell Patch-Clamp recordings and multi-electrode array recordings C_LIO_LIStatistics of 16 surgeries show the correlation between the overall success rates of efficient culturing and viral transduction with the patient age and morbidity C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/561508v1_fig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@17c985aorg.highwire.dtl.DTLVardef@f651fdorg.highwire.dtl.DTLVardef@180d32eorg.highwire.dtl.DTLVardef@a896b5_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1, Graphical abstract.C_FLOATNO 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. C_FIG