Abstract
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.
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