C-37: Measurement of intracellular water and trehalose in desiccated cells

Abstract

Stability of intracellular glass, a requirement for long-term storage in the dry state, largely depends on the residual water content and the heterogeneity in trehalose and water distributions. In light of studies on the slowed rate of desiccation in sessile droplets due to glassy skin formation at the air-liquid interface, we investigated the potential heterogeneity in water and trehalose distributions at the single-cell level in spin-dried samples. We utilized label-free detection capabilities of Raman microspectroscopy for quantitative analysis and measurement of water and trehalose concentrations in the intra- and extracellular milieu. The results confirmed the previous measurements of water content in trehalose thin film (∼0.1 g water/g trehalose). Intracellular trehalose resulted in a marginally higher water content compared to non-loaded cells. Nonetheless, we were able to show that the retained water was bound, and we observed no free intracellular water in the absence or presence of intracellular trehalose. Also, cross-sectional analysis of the desiccated cells showed that the concentrations of intracellular trehalose and water were the highest at the cell membrane adjacent to the extracellular milieu, and both followed a decreasing trend toward the middle of the cell. This was the first study to probe into desiccated cells at the single-cell level and to quantify intracellular water and trehalose contents and distributions, which are essential information for studies on storage stability of desiccated cells. The results suggest that, for optimum stabilization of trehalose-loaded cells, further desiccation for increasing the Tg of the desiccated cells and the trehalose layer is required, or otherwise, storage at below-ambient temperature is recommended. Also, our results warrant further studies on the observed uneven intracellular distribution of trehalose and investigation of its intracellular glassy/crystalline state, which may adversely impact the biostabilization properties of trehalose.