A simple mechanistic way to increase the survival of Mammalian cells during processing for dry storage

Recently, there has been considerable interest in developing processing methods that enable storage of cells in a dry state. Most of these studies describe cell viability following processing as a function of the final moisture content reached or the duration of drying. Recently, a cumulative osmotic stress model has been proposed, which takes both final moisture content and duration of drying into consideration in an effort to account for the effects of cumulative processing stresses. The present study demonstrates the applicability of this approach and elucidates a simple mechanistic technique to reduce cumulative osmotic stress during processing. Mouse macrophage cells (J774) were exposed to increasing concentrations of trehalose-containing 0.33× phosphate-buffered saline (PBS) by step-changing the extracellular solution in 2 increments of 0.7 Osm, using only trehalose as the additive solute. Three minutes was provided for equilibration prior to drying in a traditional low-humidity chamber. The data were compared with that of cells dried directly in isotonic 0.2 M trehalose in 0.33× PBS. Following dehydration, cells were rehydrated and viability was assessed 45 min postrehydration using a combination of trypan blue staining for membrane integrity of detached cells and calcein AM-propidium iodide fluorescence assay for live-dead staining of the attached cells. Cells that were preprocessed to higher trehalose concentrations in step changes prior to drying had higher viability scores at comparable final moisture levels when compared with cells dried in iso-osmotic solution, up to a limit of ∼8 Osm, at which point cells processed by both methods approached zero viability.