Cell viability.

<p><b>A)</b> Optical images were acquired using Syto13/Ethidium Bromide fluorescent stains. 1–4 show in order; 50, 100, 150, 200 mm/min drawing rates, using a 1.2 M trehalose solution. The drawing rate dependence on cell survival is apparent and enumerated in <b>B</b>. <b>C)</b> Growth expressed as fold increase for 7 days after rehydration for the four processing conditions as well as a non-treated control group.</p

Survival of CHO-TRET1 cells spin-dried in solutions with or without trehalose, then stored in LN₂ for 1 h, and finally rehydrated.

<p>(A) Membrane integrity of spin-dried cells stored in LN₂ for 1 h and 45 min after thawing and rehydration (B) Micrograph of the spin-dried cells after thawing and rehydration. (C) Growth of spin-dried cells after thawing and rehydration. The values were normalized to the initial cell count (<i>n</i> = 10, ± SD).</p

Quantification of intracellular trehalose in wild-type CHO cells and CHO-TRET1 cells.

<p>Cells were incubated in fully complemented cell culture medium containing 400 mM trehalose for 4 hours (<i>n</i> = 3, ± SD).</p

Raman hyperspectral analysis of membrane functionality.

<p><b>A-C)</b> A mask (shown as green pixels) of cellular membrane spectral data was created to compare membrane functionality for dip coated cells. Scale bar = 5 μm. Cells are shown integrated on CH2 stretching band for spatial reference, with warmer colors indicating higher relative concentration. Cells were processed and then rehydrated immediately. Raman scanning was done on the third day after rehydration. The drawing rate of 100 mm/min <b>(C)</b> was compared to both non-treated cells <b>(A)</b> and cells processed at 50 mm/min <b>(B)</b>. Peak analysis consisted of deconvolution of CH2 stretching <b>(D-F)</b> [into Y: CH2sy and Z: CH2as peaks, shown with X: original smoothed spectra], and amide III <b>(G)</b> regions. <b>H)</b> The ratio of CH2 asymmetric (2940 rel. 1/cm) stretching peak intensity to amide III (1320 rel. 1/cm) peak intensity was calculated. Unprocessed cells and those at the optimal drawing rate had no statistical difference to each other <b>(*)</b>, but both were significantly different to the 50 mm/min condition <b>(**)</b>.</p

Metabolic characterization of dip coated cells.

<p>Bioenergetics were assessed with a Seahorse XFp for both untreated cells and cells which were processed via dip-coating technique (100 mm/min, 1.2 M trehalose solution), rehydrated immediately, and cultured for 14 days. <b>A)</b> Respiration (oxygen consumption rate) is shown, with legend titles referring to readings after specified reagent injections. <b>B)</b> Glycolysis (extracellular acidification rate) is shown similarly to A. <b>C)</b> Spare respiratory capacity is a (%) ratio between FCCP and Oligomycin groups in A. <b>D)</b> Glycolytic reserve is the (%) ratio between oligomycin and glucose groups in B. There was no statistical difference between treated and untreated groups. Error bars show +SD, n = 6.</p

Residual moisture analysis using Raman microspectroscopy.

<p><b>A)</b> Representative Raman spectrum of a 1.2 M trehalose-water binary solution. Significant peaks are detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193160#pone.0193160.t001" target="_blank">Table 1</a>. <b>B)</b> Correlation of intensity ratios and residual water content by weight of standardized trehalose solutions. <b>C)</b> Average water proportions for samples processed at 50–200 mm/min. <b>D)</b> Distribution of Raman spectral ratios and water content for water (3430 rel. 1/cm) and trehalose (923 rel. 1/cm) across a large area for an acellular sample processed at 100 mm/min with a 1.2 M trehalose solution.</p

Significant Raman bands for water-trehalose solution.

<p>Significant Raman bands for water-trehalose solution.</p

Glass transition temperature of the dried spinning solution (1.8 M trehalose, 10 mM KCl, 5 mM glucose, 20 mM HEPES, 120 mM choline chloride, pH 7.4) using FTIR technique.

<p>The figure indicates a characteristic peak-position of ν-OH stretch plotted against decreasing temperature.</p

Survival of CHO-TRET1 cells spin-dried in buffers with or without trehalose and rehydrated immediately following desiccation.

<p>(A) Membrane integrity of spin-dried cells 45 min after rehydration (B) Micrograph of the cell samples after spin drying and rehydration. (C) Growth of cells after spin-drying and rehydration. The values were normalized to the initial cell count (<i>n</i> = 10, ± SD).</p

FTIR analysis of the dried film obtained after spin drying of sample buffer (1.8 M trehalose, 10 mM KCl, 5 mM glucose, 20 mM HEPES, 120 mM choline chloride, pH 7.4).

<p>The ratio of I₁₆₅₀/I₁₁₅₀ was used to quantify local water contents.</p