Center for Engineering in Medicine, BioMEMS Resource Center, Massachusetts General Hospital, Harvard Medical School, Shriners Burns Hospital for Children, 51 Blossom Street, Boston, MA 02114, USA.
Ann Biomed Eng. 2011 May;39(5):1582-91. doi: 10.1007/s10439-011-0253-1. Epub 2011 Feb 4.
Stabilization of cellular material in the presence of glass-forming sugars at ambient temperatures is a viable approach that has many potential advantages over current cryogenic strategies. Experimental evidence indicates the possibility to preserve biomolecules in glassy matrices of low-molecular mobility using "glass-forming" sugars like trehalose at ambient temperatures. However, when cells are desiccated in trehalose solution using passive drying techniques, a glassy skin is formed at the liquid/vapor interface of the sample. This glassy skin prevents desiccation of the sample beyond a certain level of dryness and induces non-uniformities in the final water content. Cells trapped underneath this glassy skin may degrade due to a relatively high molecular mobility in the sample. This undesirable result underscores the need for development of a uniform, fast drying technique. In the present study, we report a new technique based on the principles of "spin drying" that can effectively address these problems. Forced convective evaporation of water along with the loss of solution due to centrifugal force leads to rapid vitrification of a thin layer of trehalose containing medium that remains on top of cells attached to the spinning glass substrate. The glassy layer produced has a consistent thickness and a small "surface-area-to-volume" ratio that minimizes any non-homogeneity. Thus, the chance of entrapping cells in a high-mobility environment decreases substantially. We compared numerical predictions to experimental observations of the drying time of 0.2-0.6 M trehalose solutions at a variety of spinning speeds ranging from 1000 to 4000 rpm. The model developed here predicts the formation of sugar films with thicknesses of 200-1000 nm, which was in good agreement with experimental results. Preliminary data suggest that after spin drying cells to about 0.159 ± 0.09 gH₂O/gdw (n = 11, ±SE), more than 95% of cells were able to preserve their membrane integrity. Membrane integrity after spin drying is therefore considerably higher than what is achieved by conventional drying methods; where about 90% of cells lose membrane integrity at 0.4 gH₂O/gdw (Acker et al. Cell Preserv. Technol. 1(2):129-140, 2002; Elliott et al. Biopreserv. Biobank. 6(4):253-260, 2009).
在环境温度下，用玻璃形成糖稳定细胞物质是一种可行的方法，与当前的低温策略相比，它具有许多潜在的优势。实验证据表明，使用“玻璃形成”糖（如海藻糖）可以将生物分子保存在低分子流动性的玻璃基质中。然而，当细胞在海藻糖溶液中通过被动干燥技术干燥时，在样品的液体/蒸汽界面会形成一层玻璃状外皮。这种玻璃状外皮阻止样品在一定干燥程度以下进一步干燥，并导致最终含水量不均匀。被困在这种玻璃状外皮下面的细胞可能会由于样品中相对较高的分子流动性而降解。这种不理想的结果强调了开发均匀、快速干燥技术的必要性。在本研究中，我们报告了一种基于“旋转干燥”原理的新技术，该技术可以有效地解决这些问题。水的强制对流蒸发以及离心力导致的溶液损失导致附着在旋转玻璃基底上的细胞顶部的含有海藻糖的薄层迅速玻璃化。产生的玻璃层具有一致的厚度和小的“表面积-体积”比，最大限度地减少任何非均质性。因此，将细胞困在高流动性环境中的机会大大降低。我们将数值预测与在 1000 至 4000 rpm 多种旋转速度下 0.2-0.6 M 海藻糖溶液干燥时间的实验观察结果进行了比较。这里开发的模型预测了厚度为 200-1000nm 的糖膜的形成，这与实验结果非常吻合。初步数据表明，在旋转干燥后，细胞的水含量约为 0.159 ± 0.09 gH₂O/gdw（n = 11，±SE），超过 95%的细胞能够保持其膜的完整性。因此，旋转干燥后的膜完整性明显高于传统干燥方法；在 0.4 gH₂O/gdw 时，约 90%的细胞失去膜完整性（Acker 等人，细胞保存技术 1(2):129-140，2002；Elliott 等人，生物保存和生物库 6(4):253-260，2009）。
