A role for microwave processing in the dry preservation of mammalian cells.

Dry preservation involves removing water from samples so that degradative biochemical processes are slowed and extended storage is possible. Recently this approach has been explored as a method for preserving living mammalian cells. The current work explores the use of microwave processing to enhance evaporation rates and to improve drying uniformity, thereby overcoming some of the challenges in this field. Mouse macrophage cells (J774) were pre-incubated in full complement media containing 50 mM trehalose, for 18-h, to allow for endocytosis of trehalose. Droplets of experimental and control (no intracellular trehalose) cell suspensions were placed on coverslips in a microwave cavity. Water was evaporated using intermittent microwave heating (600 W, 30 s intervals). Samples were dried to various moisture levels, rehydrated, and then survival was assessed after a 45-min recovery period using Calcein-AM/PI fluorescence and Trypan Blue exclusion assays. The metabolic activity of dried cells (4.3 gH(2)O/gdw) was assessed after rehydration using a resazurin reduction assay. Apoptosis levels were also measured. Post- rehydration survival correlated with the final moisture content achieved, consistent with other drying methods. Intracellular trehalose provided protection against injury associated with moisture loss. Metabolic assays revealed normal growth in surviving cells, and these survival levels were consistent with results from apoptosis assays (P > 0.05). Brightfield and fluorescence images of microwave-dried samples revealed a uniform distribution of cells within the dried matrix and profilometry analysis demonstrated that solids were uniformly distributed throughout the sample. Microwave-processing successfully facilitated rapid and uniform dehydration of cell-based samples.

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.

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.