Desiccation of nucleated mammalian cells in nanoliter droplets

This paper reports on the development and testing of two techniques for nebulizing cell solutions into aerosols that contain living cells in nanoliter droplets: one pneumatic technique and one ultrasonic technique. NIH 3T3 fibroblasts were tested for viability and the ability to attach and grow post-spraying. Viabilities for the two techniques ranged from 50 to 70% and virtually all cells that maintained membrane integrity post-spraying were able to attach and divide. The cellular aerosols were used to study the effect of drying rates and drying temperature on the post-rehydration survival of fibroblasts that were loaded with trehalose. Nanoliter droplets were developed for this study so that non-uniformities in moisture content and temperature within the samples would be minimized, and thus the drying rates experienced by the cells could be inferred. The main findings of this study are that the relative humidity of the drying environment does have a strong effect on the post-rehydration viability of desiccated cells, but that effect is entirely explained by differences in the residual moisture content experienced by the cells dried at different humidities. The post-rehydration viability of the cells was found to be dependent only on the residual moisture content achieved during drying and on whether or not trehalose was loaded inside the cell, and was not dependent on the drying rate or on the temperature at which drying occurred. A comparison of viabilities for cells dried in nanoliter droplets and cells dried in more commonly used 10 μL drops suggested that cells in larger drops were not experiencing the average water content for the drop as a whole during critical phases of the drying process. The apparent non-uniformity of moisture inside larger drops may contribute to difficulties in understanding and establishing successful long-term storage techniques in a dried state.