Anhydrous preservation of Mammalian cells: cumulative osmotic stress analysis

Cumulative osmotic stress models have been previously used to successfully describe the dehydration kinetics of recalcitrant seeds. For example, Liang and Sun demonstrated that a cumulative stress model effectively described the dehydration rate-dependence of recalcitrant seed viability under various drying conditions. In contrast, most studies describing the functionality of mammalian cells following drying conditions have been end-point oriented, describing cell viability as a function of the final moisture content reached or the duration of drying. This study applies a thermodynamics-based water metric similar to the Liang and Sun model to describe the viability of J774 mouse macrophage cells as a function of both moisture content and time of drying. Cells were incubated in full-complement DMEM media containing 50 mM trehalose, to enable trehalose loading by endocytosis. Treated cells and untreated controls that were not previously incubated in trehalose were dried in hypertonic (508 mOsm) and isotonic (308 mOsm) solutions of trehalose (200 mM) in phosphate-buffered saline (PBS). Various levels of dehydration were achieved by placing droplets of cell suspension in a desiccator for time periods up to 2 h. Cells were then immediately rehydrated and viability was assessed 45-min after rehydration. Cell viability was evaluated using a combination of Trypan Blue staining for membrane integrity of detached cells and Calcein AM - ethidium bromide fluorescence as a live-dead assay for attached cells. The cellular response was then evaluated as a function of cumulative osmotic stress, defined as the integral of the deviation in osmolality from isotonic conditions as a function of time. The results of this modeling suggested that significant cell injury was occurring in a moderate osmolality range, and that modulation of osmotic stresses in this range could lead to improved processing outcomes.