Chakraborty Nilay, Biswas Debasree, Parker Wesley, Moyer Pat, Elliott Gloria D
Department of Mechanical Engineering and Engineering Sciences, University of North Carolina, Charlotte, 9201 University City Blvd., Charlotte, North Carolina 28223-0001, USA.
Biotechnol Bioeng. 2008 Jul 1;100(4):782-96. doi: 10.1002/bit.21801.
Dry preservation involves removing water from samples so that degradative biochemical processes are slowed and extended storage is possible. Recently this approach has been explored as a method for preserving living mammalian cells. The current work explores the use of microwave processing to enhance evaporation rates and to improve drying uniformity, thereby overcoming some of the challenges in this field. Mouse macrophage cells (J774) were pre-incubated in full complement media containing 50 mM trehalose, for 18-h, to allow for endocytosis of trehalose. Droplets of experimental and control (no intracellular trehalose) cell suspensions were placed on coverslips in a microwave cavity. Water was evaporated using intermittent microwave heating (600 W, 30 s intervals). Samples were dried to various moisture levels, rehydrated, and then survival was assessed after a 45-min recovery period using Calcein-AM/PI fluorescence and Trypan Blue exclusion assays. The metabolic activity of dried cells (4.3 gH(2)O/gdw) was assessed after rehydration using a resazurin reduction assay. Apoptosis levels were also measured. Post- rehydration survival correlated with the final moisture content achieved, consistent with other drying methods. Intracellular trehalose provided protection against injury associated with moisture loss. Metabolic assays revealed normal growth in surviving cells, and these survival levels were consistent with results from apoptosis assays (P > 0.05). Brightfield and fluorescence images of microwave-dried samples revealed a uniform distribution of cells within the dried matrix and profilometry analysis demonstrated that solids were uniformly distributed throughout the sample. Microwave-processing successfully facilitated rapid and uniform dehydration of cell-based samples.
干燥保存是指从样本中去除水分，从而减缓降解性生化过程，并实现长期保存。最近，这种方法已被探索作为一种保存活的哺乳动物细胞的方法。当前的工作探索了利用微波处理来提高蒸发速率并改善干燥均匀性，从而克服该领域的一些挑战。将小鼠巨噬细胞（J774）在含有50 mM海藻糖的完全培养基中预孵育18小时，以使海藻糖内吞。将实验性和对照性（无细胞内海藻糖）细胞悬液的液滴置于微波腔内的盖玻片上。使用间歇微波加热（600 W，间隔30 s）蒸发水分。将样品干燥至不同的水分含量，再水化，然后在45分钟恢复期后使用钙黄绿素-AM/PI荧光和台盼蓝排斥试验评估存活率。使用刃天青还原试验在再水化后评估干燥细胞（4.3 gH₂O/gdw）的代谢活性。还测量了凋亡水平。再水化后的存活率与最终达到的水分含量相关，这与其他干燥方法一致。细胞内海藻糖提供了针对与水分流失相关损伤的保护。代谢试验显示存活细胞生长正常，并且这些存活水平与凋亡试验结果一致（P>0.05）。微波干燥样品的明场和荧光图像显示细胞在干燥基质中分布均匀，轮廓测定分析表明固体在整个样品中均匀分布。微波处理成功地促进了基于细胞的样品的快速和均匀脱水。
