Wang Shangping, Lee Pei-Chih, Elsayed Amanda, Zhang Fan, Zhang Yong, Comizzoli Pierre, Elliott Gloria D
Department of Mechanical Engineering and Engineering Sciences, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC 28223 USA.
Smithsonian Conservation Biology Institute, National Zoological Park, Washington, DC USA.
Cell Mol Bioeng. 2020 Jul 14;14(1):101-112. doi: 10.1007/s12195-020-00635-y. eCollection 2021 Feb.
Maintaining a stable dry state is critical for long-term preservation of live biomaterials at suprazero temperatures. The objective of the study was to characterize the effect of moisture content on DNA integrity within the germinal vesicle (GV) of feline oocytes following dehydration and storage at 22-24 °C.
Using microwave-assisted drying, conditions that led to a predictable and stable moisture content in trehalose solutions were determined. To explore moisture content stability during storage, trehalose samples were dried for 15 min and stored in glass vials at 11 or 43% RH for 8 weeks. To examine whether this condition allowed proper storage of GVs, permeabilized cat oocytes were incubated in trehalose for 10 min and dried for 15 or 30 min. Oocytes then were rehydrated to assess DNA integrity either directly after drying or after 8 weeks of storage in an 11% RH environment. Raman spectroscopy was used to identify the states of dried samples during storage.
Moisture content was stable during the storage period. There was no significant difference in DNA integrity between fresh and dried samples without storage. After 8 weeks of storage, DNA integrity was maintained in GVs dried for 30 min. Samples dried for 15 min and stored were compromised, suggesting crystallization of the preservation matrix during storage. Biostabilization was optimal when samples were directly processed to moisture contents consistent with storage in the glassy state.
Microwave-assisted drying processing and storage conditions were optimized to ensure stable long-term storage of structural and functional properties of genetic resources.
保持稳定的干燥状态对于在零上温度下长期保存活的生物材料至关重要。本研究的目的是表征脱水并在22 - 24°C储存后，水分含量对猫卵母细胞生发泡（GV）内DNA完整性的影响。
使用微波辅助干燥，确定了能使海藻糖溶液中水分含量可预测且稳定的条件。为探究储存期间水分含量的稳定性，将海藻糖样品干燥15分钟，并在相对湿度11%或43%的条件下于玻璃瓶中储存8周。为检验这种条件是否允许对GV进行适当储存，将通透处理的猫卵母细胞在海藻糖中孵育10分钟，然后干燥15或30分钟。卵母细胞随后复水，以评估干燥后或在11%相对湿度环境中储存8周后的DNA完整性。使用拉曼光谱法识别储存期间干燥样品的状态。
储存期间水分含量保持稳定。未储存的新鲜样品和干燥样品在DNA完整性方面无显著差异。储存8周后，干燥30分钟的GV中DNA完整性得以维持。干燥15分钟后储存的样品受到损害，表明储存期间保存基质发生了结晶。当样品直接处理至与玻璃态储存一致的水分含量时，生物稳定性最佳。
对微波辅助干燥处理和储存条件进行了优化，以确保遗传资源的结构和功能特性能够长期稳定储存。
