A Raman microspectroscopy study of water and trehalose in spin-dried cells

Abstract

Long-term storage of desiccated nucleated mammalian cells at ambient temperature may be accomplished in a stable glassy state, which can be achieved by removal of water from the biological sample in the presence of glass-forming agents including trehalose. The stability of the glass may be compromised due to a nonuniform distribution of residual water and trehalose within and around the desiccated cells. Thus, quantification of water and trehalose contents at the single-cell level is critical for predicting the glass formation and stability for dry storage. Using Raman microspectroscopy, we estimated the trehalose and residual water contents in the microenvironment of spin-dried cells. Individual cells with or without intracellular trehalose were embedded in a solid thin layer of extracellular trehalose after spin-drying. We found strong evidence suggesting that the residual water was bound at a 2:1 water/trehalose molar ratio in both the extracellular and intracellular milieus. Other than the water associated with trehalose, we did not find any more residual water in the spin-dried sample, intra- or extracellularly. The extracellular trehalose film exhibited characteristics of an amorphous state with a glass transition temperature of ?22°C. The intracellular milieu also dried to levels suitable for glass formation at room temperature. These findings demonstrate a method for quantification of water and trehalose in desiccated specimens using confocal Raman microspectroscopy. This approach has broad use in desiccation studies to carefully investigate the relationship of water and trehalose content and distribution with the tolerance to drying in mammalian cells.

Figure 1

Raman scattering spectra collected from (a) dry cell lysate, (b–e) mixtures of cell lysate/trehalose at ratios of 1:1, 1:2, 1:4, and 1:10 g trehalose/g dry weight. Inset: blow-up of the region 720–1800 cm⁻¹. Annotations – 1), 851 cm⁻¹ (saccharides, trehalose), 2), 920 cm⁻¹ (C-C stretch of saccharides), 3), 1583 cm⁻¹ (attributed to C=C stretch in L-tryptophan, various nucleic acids, guanine, phenylalanine), 4), 1667 cm⁻¹ (protein band, Amide I), 5), ∼2940 cm⁻¹ (C-H stretch of organic matter), 6), 3250–3350 cm⁻¹ (O–H symmetric stretch low energy band), 7), 3350–3500 cm⁻¹ (O–H antisymmetric stretch high energy band).

Figure 2

(A) Ratio of the Raman intensity of the peak at 1667 cm⁻¹, representing proteins, to the C-H stretch peak intensity at 2940 cm⁻¹, representing all the organic matter, at various LYS/TRE ratios against water content. (B) Calibration curve for (trehalose/protein) ratio based on intensity ratio of 851 cm⁻¹ to 1667 cm⁻¹ in oven-dried (0 g water/g dry weight) samples. (C) Ratio of O-H stretch intensities at 3430 cm⁻¹ to 3300 cm⁻¹ at various LYS/TRE ratios against water content (Inset: The intensity ratio 3430 cm⁻¹ to 3300 cm⁻¹ for nitrogen-dried samples plotted against TRE/LYS ratios. Dashed line denotes the background intensity not from water). (D) Ratio of the O-H stretch intensity at 3430 cm⁻¹ to that of C-H stretch at 2940 cm⁻¹ at various LYS/TRE ratios against water content.

Figure 3

(A1 and A2) Brightfield images of C3A and TRET cells, embedded in a trehalose layer after spin-coating and desiccation under nitrogen flow. The white dotted line represents the XZ plane of scan. (B) Area-averaged intracellular spectrum collected from C3A and TRET cells. (C) Area-averaged spectra collected from the extracellular thin layer of trehalose.

Figure 4

Reconstructed confocal Raman images of C3A (A1–A3) and TRET (B1–B3) cells, based on protein (row 1), trehalose (row 2), and O-H stretch (row 3) contrasts. Images show cross sections of cells in the XZ plane, with air at the top and CaF₂ substrate at the bottom. The thin gray lines constitute the approximate boundaries of the cells (C1–C3) Respective distributions of proteins, trehalose, and O-H stretch across the cell width along the thick dashed white line. (D and E) Estimated amount of trehalose/protein and water/dry weight ratios from I₈₅₁/I₁₆₆₇ and I₃₃₀₀/I₂₉₄₀ ratios, respectively.