Ma Ya H, Qin Guo F, Li Jing, Ding Gui R, Xu Sheng L, Zhou Yan, Guo Guo Z
Department of Radiation Medicine, Faculty of Preventive Medicine, The Fourth Military Medical University , Xi'an, People's Republic of China .
Biopreserv Biobank. 2016 Feb;14(1):23-8. doi: 10.1089/bio.2015.0042. Epub 2016 Feb 1.
Slow freezing coupled with an AC electric field (ACEF) has been demonstrated to miniaturize the ice crystals of a 0.9% (w/v) NaCl solution in a prior study. The aim of this study was to assess the effect of ACEF on Sprague-Dawley (SD) rat liver in vitro during the slow cooling procedure. SD rat liver exposed to an oscillating electric field was frozen in a programmed freezer initially down to -30°C at a cooling rate of -1°C/min and continuing down to -80°C at a cooling rate of -5°C/min. The cryovials were finally transferred into liquid nitrogen for 7 days. The frequency range was 0-20 MHz, and peak field strength was 1,000 V/m. For the sham and electric-exposed groups, the freezing solution consisted of 0%, 2.5%, 5.0%, 7.5%, or 10% (v/v) dimethyl sulfoxide (DMSO) Dulbecco's modified Eagles' medium culture solution, and fresh tissue was selected as the control group. The changes in cell survival rate, adenosine triphosphate (ATP) content, and morphology of fresh and frozen-thawed liver tissue were examined. Compared with the sham group with 5.0% DMSO, the result showed that slow freezing coupled with 2.45 MHz or 5 MHz ACEF significantly increased the relative survival rate by 43.27% and 26.31% (P < 0.001), respectively. However, ACEF exposure increased the ATP content compared with the sham group. Especially in 5% and 10% DMSO with 2.45 MHz ACEF exposure, the ATP content approximated the fresh group (7.3 ± 2.7 nmol/piece), corresponding to 94.52% and 80.82%. In addition, the cellular membrane and some organelles (e.g., mitochondria) in the electric-exposed group appeared to be more intact according to the transmission electron microscopy images. The underlying mechanism might be that the ACEF affects the formation and growth of the ice crystallization, and thus inhibits cryoinjury. These results show that ACEF would provide an efficient method for cryopreservation banking with a low concentration of CPA during the slow freezing process.
在先前的一项研究中，已证明慢速冷冻结合交流电场（ACEF）可使0.9%（w/v）氯化钠溶液中的冰晶细化。本研究的目的是评估在慢速冷却过程中ACEF对体外斯普拉格-道利（SD）大鼠肝脏的影响。将暴露于振荡电场的SD大鼠肝脏在程序降温冰箱中冷冻，最初以-1°C/分钟的冷却速率降至-30°C，然后以-5°C/分钟的冷却速率继续降至-80°C。最后将冻存管转移至液氮中保存7天。频率范围为0 - 20MHz，峰值场强为1000V/m。对于假手术组和电场暴露组，冷冻溶液由0%、2.5%、5.0%、7.5%或10%（v/v）二甲基亚砜（DMSO）的杜尔贝科改良伊格尔培养基培养液组成，选取新鲜组织作为对照组。检测新鲜及冻融肝脏组织的细胞存活率、三磷酸腺苷（ATP）含量及形态变化。与含5.0%DMSO的假手术组相比，结果显示，慢速冷冻结合2.45MHz或5MHz的ACEF分别使相对存活率显著提高43.27%和26.31%（P < 0.001）。然而，与假手术组相比，ACEF暴露使ATP含量增加。特别是在含2.45MHz ACEF暴露的5%和10%DMSO中，ATP含量接近新鲜组（7.3±2.7nmol/块），分别相当于94.52%和80.82%。此外，根据透射电子显微镜图像，电场暴露组的细胞膜和一些细胞器（如线粒体）似乎更完整。潜在机制可能是ACEF影响冰晶的形成和生长，从而抑制冷冻损伤。这些结果表明，在慢速冷冻过程中，ACEF可为低浓度冷冻保护剂的低温保存库提供一种有效的方法。
