Concentration of glycerol in aqueous microdroplets by selective removal of water

A major roadblock to the vitrification of cells is the requirement of high concentrations of cryoprotectant (CPA) chemicals and the damage caused by prolonged exposure of cells to these high concentrations above the glass transition temperature. These effects are minimized with controlled CPA loading. Certain organic oils, such as soybean oil, are made of triacylglycerols and are capable of dissolving small amounts of water, a property which is enhanced significantly as temperature is increased. This phenomenon was exploited here to accomplish temperature-controlled concentration of glycerol in single water droplets dispersed in the organic phase. Emulsions of aqueous solutions of glycerol in soybean oil were made and subjected to a temperature increase of 10 degrees C from room temperature. Upon increasing temperature, water dissolved into the oil, rendering the 15-20 microm droplets concentrated an average of 3.6 times and 2.6 times for 1 and 2 M starting concentrations, respectively, with the oil-insoluble glycerol in 90-110 s. This phenomenon could be used to dynamically concentrate CPAs within cell-containing droplets which may then be vitrified before being exposed to high temperatures for fatally long times.