Development of a surface tension mediated technique for dry stabilization of mammalian cells.

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.

A technique for lyopreservation of Clostridium ljungdahlii in a biocomposite matrix for CO absorption.

A system capable of biocatalytic conversion of distributed sources of single carbon gases such as carbon monoxide into hydrocarbons can be highly beneficial for developing commercially viable biotechnology applications in alternative energy. Several anaerobic bacterial strains can be used for such conversion. The anaerobic carbon monoxide-fixing bacteria Clostridium ljungdahlii OTA1 is a model CO assimilating microorganism that currently requires cryogenic temperature for storage of the viable strains. If these organisms can be stabilized and concentrated in thin films in advanced porous materials, it will enable development of high gas fraction, biocomposite absorbers with elevated carbon monoxide (CO) mass transfer rate, that require minimal power input and liquid, and demonstrate elevated substrate consumption rate compared to conventional suspended cell bioreactors. We report development of a technique for dry-stabilization of C. ljungdahlii OTA1 on a paper biocomposite. Bacterial samples coated onto paper were desiccated in the presence of trehalose using convective drying and stored at 4°C. Optimal dryness was ~1g H2O per gram of dry weight (gDW). CO uptake directly following biocomposite rehydration steadily increases over time indicating immediate cellular metabolic recovery. A high-resolution Raman microspectroscopic hyperspectral imaging technique was employed to spatially quantify the residual moisture content. We have demonstrated for the first time that convectively dried and stored C. ljungdahlii strains were stabilized in a desiccated state for over 38 days without a loss in CO absorbing reactivity. The Raman hyperspectral imaging technique described here is a non-invasive characterization tool to support development of dry-stabilization techniques for microorganisms on inexpensive porous support materials. The present study successfully extends and implements the principles of dry-stabilization for preservation of strictly anaerobic bacteria as an alternative to lyophilization or spray drying that could enable centralized biocomposite biocatalyst fabrication and decentralized bioprocessing of CO to liquid fuels or chemicals.