Live-cell physiology in human brain tissue culture—the potential, the challenges, and the lessons learned

A growing number of studies are now utilizing human organotypic brain slice cultures (OBSC), enabled by improved long-term culture protocols that support the investigation of human-specific cellular physiology. Human OBSC offer the unique advantage, that they preserve the connectivity and microenvironment of human brain tissue while remaining experimentally tractable for live functional assays. However, methods established in rodent brain slice cultures, such as viral labeling, genetic manipulation, and long-term transduction strategies, do not readily translate to human tissue due to differences in viral tropism, tissue heterogeneity, and limited survival in culture. In this review, we highlight key historical developments and provide an overview of current best practices of human brain tissue culture. We provide an overview of live cell-specific labeling strategies, including viral approaches, as well as current and emerging technologies for functional manipulation and for investigating disease-related pathologies. Throughout, we integrate insights gained from mouse models and discuss how these technologies could potentially be adapted and leveraged for human OBSCs. Beyond methodological approaches, we also address the challenges and limitations inherent to working with human brain tissue. Together, these advances position human OBSCs as a powerful platform to bridge the gap between reductionist in vitro systems and the in vivo living human brain.