Date Published: March 5, 2018
Publisher: Public Library of Science
Author(s): Jason Solocinski, Quinn A. Osgood, Eric Rosiek, Lukas Underwood, Oleg Zikanov, Nilay Chakraborty, Xiaoming He.
Dry state preservation at ambient temperatures (lyopreservation) is a biomimetic alternative to low temperature stabilization (cryopreservation) of biological materials. Lyopreservation is hypothesized to rely upon the creation of a glassy environment, which is commonly observed in desiccation-tolerant organisms. Non-uniformities in dried samples have been indicated as one of the reasons for instability in storage outcome. The current study presents a simple, fast, and uniform surface tension based technique that can be implemented for lyopreservation of mammalian cells. The technique involves withdrawing cells attached to rigid substrates to be submerged in a solution of lyoprotectant and then withdrawing the samples at a specific rate to an inert environment. This creates a uniform thin film of desiccated lyoprotectant due to sudden change of surface tension. The residual moisture contents at different locations in the desiccated film was quantified using a spatially resolved Raman microspectroscopy technique. Post-desiccation cellular viability and growth are quantified using fluorescent microscopy and dye exclusion assays. Cellular injury following desiccation is evaluated by bioenergetic quantification of metabolic functions using extracellular flux analysis and by a Raman microspectroscopic analysis of change in membrane structure. The technique developed here addresses an important bottleneck of lyoprocessing which requires the fast and uniform desiccation of cellular samples.
Storage of biologics and cellular material using lyopreservation has the potential to simplify logistics and transportation by reducing the need for cold-chain logistics. Development of such a technique for mammalian cells can have a significant impact in clinical application of advanced cell-based therapies, particularly in resource limited regions [1, 2]. The success of lyopreservation has been theorized to rely upon the creation of a high viscosity extra and intracellular environment at an advanced state of desiccation, where low molecular mobility prevents any degradative reactions [3, 4]. This mechanism of preservation is frequently observed in nature among a wide variety of bacteria , animals, and plants (anhydrobiotes) , suggesting that this ability, developed by ancient cell types, may have been a critical factor of successful colonization of terrestrial earth [7, 8].
Desiccated storage of biological samples is an alternative to low temperature preservation. Desiccated state storage is widely observed in nature among the cryptobiotic organisms and development of comparable biopreservation strategies can facilitate easy and economic transport of stored biologics including cells. In recent years, several lyoprocessing techniques have been developed for desiccated-state preservation of cells including freeze-drying , spray drying , and isothermal desiccation . Freeze drying has been successfully implemented to stabilize germ cells  and platelets . Isothermal drying techniques have been extensively studied due to their operational simplicity over techniques such as freeze drying and spray drying . Fast lyoprocessing techniques have the added advantage of limiting the exposure time to non-physiological states during processing [11, 13]. The primary goal of this study was to develop a viable lyoprocessing technique that can be used to desiccate cells and cellular materials rapidly with high uniformity.