Tissue and cytoplasm vitrification in cryopreservation monitored by low temperature scanning electron microscopy (cryo-SEM)

Abstract

Cryopreservation of cells and tissues frequently relies on inducing cytoplasm vitrification for storage with almost complete stoppage of both chemical reactions and physical processes and without ice crystal biological damage. The most frequently employed technique used for monitoring ice formation and vitrification in these systems is differential scanning calorimetry (DSC). This technique, which was used in this study to observe ice formation in cryoprotecting agent solutions and mint shoot tips, is fairly sensitive for detecting ice formation, but not so for directly observing glass transition. Besides, the possibility of coincident thermal phenomena obscuring the small glass transition signature in DSC and the lack of any spatial resolution, make valuable the information obtained from other sources. Low temperature scanning electron microscopy (cryo-SEM) is able to show ice crystals formed in the cooling process employed to introduce samples into the microscope, very similar to the cooling step of cryopreservation protocols. The images from samples that, after DSC evidence, have no ice crystals and are vitrified, appear completely unetched in cryo-SEM micrographs. Consequently, cryo-SEM is proposed as a suitable method to ascertain cells and tissues effective vitrification in cryopreservation.