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

Abstract

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 Spin-Drying Technique for Lyopreservation of Mammalian Cells

“…2 Recent studies indicated that the slow rate of drying in conventional techniques may be a reason for the failure, 5,41,48 and to develop a successful processing technique for lyopreservation, it is important to minimize the exposure time of the cells to the deleterious effects of supersaturated solutions. 5,28 For this reason, researchers have looked at developing several fast desiccation techniques for dry processing of cells including vacuum drying, 9 microwave drying, 16 and nebulization. 48 However, these fast drying techniques, depending on their drying mechanism, frequently expose cells to additional injury during processing.…”

Advancing Microwave Technology for Dehydration Processing of Biologics

“…13 This approach yielded a more uniform dried product, and the drying rates achieved were very reproducible. However, processing was limited to a single sample at a time and the drying approach required manual stop and start operation to control temperature.…”

“…While this thermal titration effect could theoretically also be achieved by contact heating of the sample, microwave energy input has been shown to effectively interrupt gradient-driven mass transfer processes that lead to sample inhomogeneity, likely due to the volumetric nature of this energy delivery. 13 It is not clear whether or not contact heating would yield this same beneficial overall effect.…”

“…Achieving a homogeneous sample with a uniform moisture content can also be a challenge. 13 Numerous drying approaches have been investigated to achieve a dried specimen suitable for storage in a dry state, including lyophilization, 5,7 drying in controlled humidity desiccators, 14 or the use of high flow nitrogen gas for convective drying.…”

“…13 The current work expands upon the original proof-of-concept, and details advances in instrumentation and process methodology to enable higher throughput drying with more process control.…”