Multi-view cryomacroscopy: A new device for study of cryopreservation by vitrification

This study presents an innovative multi-view cryomacroscope (MVC) designed to advance the study of cryopreservation by vitrification in large samples. Over the past two decades, cryomacroscopy has evolved through five different devices to observe key physical phenomena during cryopreservation, including vitrification, crystallization, contamination, material flow, deformation, mechanical strain, and fracturing. Accompanying modeling was used to infer material properties based on those observations, such as the strength of the material, thermal conductivity, buoyancy, and critical cooling rates to facilitate vitrification in large samples. The MVC addresses key limitations inherent to previous cryomacroscopes, which led to the development of a standalone thermo-optical device. It accommodates an array of up to 12 cameras in various vantage positions, and its operation is demonstrated on vitrification in a 60 ml container. Observed physical effects include crystallization at the cryoprotective agent (CPA)-air interface during cooling, fracturing in the vitrified CPA, and rewarming phase crystallization (RPC). An artifact of air suction into the center of the CPA through the thermocouple wires is also demonstrated, driven by nonuniform contraction of the CPA. The resulting air bubbles could adversely affect the ability to cool the container, may promote crystallization on the CPA-air interfaces, and may create areas with high stress concentration, making the vitrified CPA more prone to fracturing. While air suction can be easily prevented, observing this effect and thereby identifying the need to prevent it could not have been done without the MVC, which further underscores the unmet needs for cryomacroscopy in the study of vitrification. • Cryomacroscopy captures key physical events along the thermal protocol. • Fracturing, crystallization, air bubble formation visualization in real time. • Air bubbles are formed due to thermal contraction, which may trigger ice nucleation. • Multi-angle cryomacroscopy views reveal effects otherwise overlooked with earlier, single view devices. • New insights may improve cryopreservation protocols for large samples.