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Alcohol overconsumption is a major cause of preventable mental disorders and death in the United States and around the world. The pathogenesis of alcohol dependence, abuse, and toxicity to the central nervous system remains incompletely understood. Cell culture-based models have been highly valuable in studying the molecular and cellular mechanisms underlying the contribution of individual CNS cell types to ethanol’s effects on the brain. However, conventional cell culture model systems carry the inherent disadvantage of rapid loss of ethanol due to evaporation following a bolus addition of ethanol at the start of the treatment. In this study, we have established a multi-well cell culture plate-based ethanol evaporation compensation model that utilizes the inter-well space as a reservoir to compensate for the evaporative loss of ethanol in the cell treatment wells. Following a single bolus addition at the start, ethanol concentration in the treatment wells rapidly decreased over time. Through compensation using the multi-well plate platform, maintenance of ethanol concentrations ranging from 10–100 mM was achieved for up to 72 h in a cell-free system. Furthermore, greater effects on ethanol-induced decrease in the viability of human dopaminergic neuronal cells were observed with than without evaporation compensation. Our method effectively compensates for the evaporative loss of ethanol typically observed in the traditional treatment method and provides a simple and economic in vitro model system for ethanol treatment over an extended timeframe where maintenance of a relatively constant concentration of ethanol is desired.

Establishment of a Simple and Versatile Evaporation Compensation Model for In Vitro Chronic Ethanol Treatment: Impact on Neuronal Viability