Establishment of a Simple and Versatile Evaporation Compensation Model for

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. In vitro and 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 at the start of the treatment. 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 of the treatment, ethanol concentration 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 hours in a cell-free system. Greater effects in ethanol-induced decrease in neuronal cell viability were observed with than without compensation. Our method effectively compensates for the evaporative loss of ethanol typically observed in the traditional method. This method provides an economic, simple and effective in vitro model system for ethanol treatment over an extended timeframe where maintenance of a relatively constant concentration of ethanol is desired.