Under-oil neuronal cell culture: Enhanced system stability, yield, and modulated oxygen environment.

BACKGROUND: Culturing neuronal cells in vitro, especially at smaller scales with reduced media volumes, has been challenging due to the limited proliferation of mature neurons and the inherent high sensitivity of neuronal cells to environmental fluctuations. NEW METHOD: In this study, we report a neuronal cell culture method that leverages oil overlay and an autonomously regulated oxygen microenvironment (AROM), in which primary rat cortical cells and human neural progenitor cells (NPCs) were cultured in standard well plates with an oil overlay on top of the media layer. The oil overlay prevents evaporation and achieves in vivo-like oxygen concentrations without the use of glove boxes or hypoxic chambers. RESULTS: This oil overlay method achieved > 95 % yield of viable replicates after up to 30 days. Human NPCs cultured under the oil overlay for 15 days exhibited sustained viability without requiring media change. Additionally, oil overlays create a modulated oxygen microenvironment (i.e., AROM) that mimics in vivo conditions, capable of maintaining and restoring optimal oxygen concentrations after disturbances. COMPARISON WITH EXISTING METHOD: In contrast, existing method (no-oil controls) resulted in < 20 % yield, low viability for human NPCs (11 % versus 89 % with oil overlay), and oxygen concentrations that returns to ambient levels (21 % oxygen). CONCLUSION: Overall, these results support the oil overlay method as a robust small-scale neuronal cell culture system, offering improved stability and higher yield. The results also underscore the critical role of the oxygen microenvironment in supporting neuronal cell viability, maintenance, and growth.