Enhancing neuronal viability: The protective role of 10% human cerebrospinal fluid in primary neuronal cultures

Human cerebrospinal fluid (hCSF) is a physiologically rich medium containing neurotrophic factors, signaling molecules, and essential metabolites that support neuronal development, survival, and function. While its neuroprotective properties have been demonstrated in organotypic brain slices and human iPSC-derived models, its application in primary rodent cortical neuron cultures-a foundational system for studying synaptic development and neurodegeneration-remains underexplored. In this study, we systematically evaluated the effects of hCSF supplementation on neuronal viability in primary cortical cultures derived from embryonic day 18 (E18) rat embryos. To determine the optimal concentration, we tested a range of media:hCSF ratios and identified 90:10 (i.e., 10% hCSF) as the most effective for enhancing neuronal survival. Cell viability was assessed using two complementary assays: SYTOX Green for detecting dead cells and Calcein AM/Ethidium Homodimer-2 (EthD2) dual-staining for quantifying live/dead cell populations. Our results show that 10% hCSF supplementation significantly reduces cell death and improves overall neuronal health under standard in vitro conditions. This optimized approach offers a reproducible and physiologically relevant strategy for improving dissociated cortical neuron cultures and has important implications for in vitro modeling of neurodegenerative diseases, neurotoxicity screening, and regenerative neuroscience research.