Investigation on recrystallization of cryoprotectant solutions during warming under alternating electric fields

Abstract

In the field of cryopreservation, recrystallization during rewarming is a significant factor leading to cryo-injury, causing ice crystal-induced mechanical injury to biological samples. Currently, methods, such as adding ice recrystallization inhibiting agents and increasing warming rates, have been adopted to reduce recrystallization injury. These methods also greatly increase the potential toxicity risk and engineering difficulty of cryopreservation. Accumulating research has progressively demonstrated that application of an electromagnetic field during cryopreservation can influence the preservation efficacy. However, the mechanism of action of the electric field has remained a subject of ongoing debate. We observed the recrystallization phenomenon of cryoprotectant solutions under a polarized light microscope and discovered promising experimental results. The electric field strength did not exhibit a discernible impact on the ice crystal size, while electric field frequency exerted a more significant impact. We found that at specific frequencies, the ice crystal size and growing rate were significantly affected during annealing. Compared to no electric field, an alternating electric field with a voltage of 10 V and a frequency of 5 × 104 Hz increased the recrystallization areas of a 15% (w/v) propylene glycol solution by 57%. And an alternating electric field with a voltage of 3 V and a frequency of 5 × 108 Hz decreased the recrystallization area of a 0.9% (w/v) NaCl aqueous solution by 9.9%. Different types of cryoprotectants exhibit varying responses to alternating electric fields frequency. These findings prompt us to reevaluate the role and mechanism of alternating electric fields in the cryopreservation of biological materials.