A new cryomacroscope device (Type III) for visualization of physical events in cryopreservation with applications to vitrification and synthetic ice modulators

Abstract

The objective of the current study is to develop a new cryomacroscope prototype for the study of vitrification in large-size specimens. The unique contribution in the current study is in developing a cryomacroscope setup as an add-on device to a commercial controlled-rate cooler and in demonstration of physical events in cryoprotective cocktails containing synthetic ice modulators (SIM)—compounds which hinder ice crystal growth. Cryopreservation by vitrification is a highly complex application, where the likelihood of crystallization, fracture formation, degradation of the biomaterial quality, and other physical events are dependent not only upon the instantaneous cryogenic conditions, but more significantly upon the evolution of conditions along the cryogenic protocol. Nevertheless, cryopreservation success is most frequently assessed by evaluating the cryopreserved product at its end states—either at the cryogenic storage temperature or room temperature. The cryomacroscope is the only available device for visualization of large-size specimens along the thermal protocol, in an effort to correlate the quality of the cryopreserved product with physical events. Compared with earlier cryomacroscope prototypes, the new Cryomacroscope-III evaluated here benefits from a higher resolution color camera, improved illumination, digital recording capabilities, and high repeatability in tested thermal conditions via a commercial controlled-rate cooler. A specialized software package was developed in the current study, having two modes of operation: (a) experimentation mode to control the operation of the camera, record camera frames sequentially, log thermal data from sensors, and save case-specific information; and (b) post-processing mode to generate a compact file integrating images, elapsed time, and thermal data for each experiment. The benefits of the Cryomacroscope-III are demonstrated using various tested mixtures of SIMs with the cryoprotective cocktail DP6, which were found effective in preventing ice growth, even at significantly subcritical cooling rates with reference to the pure DP6.

Thermal conductivity of the cryoprotective cocktail DP6 in cryogenic temperatures, in the presence and absence of synthetic ice modulators

“…Note that solute precipitation from DP6+SIMs in EC has been reported recently [18], which affected opacity of the vitrified material, but did not affect the likelihood of crystallization or fracturing. The current study suggests that the thermal conductivity is not affected by solute precipitation at cryogenic temperatures as well.…”

The scanning cryomacroscope – A device prototype for the study of cryopreservation

“…Consistent with previous studies [20,22,34,37], the thermal protocol in the current study comprises of four phases with respect to the cooling chamber temperature as the controlled variable:…”

“…A third cryomacroscope prototype (Type III) has been currently presented to investigate physical events associated with vitrification in the presence of synthetic ice modulators (SIMs) [37]. The main improvement in Type III over Type I is the cooling mechanism: while Type I used a tailor-made passive cooling mechanism, Type III is designed to replace the lid of a commercially available top-loading controlled-rate cooler.…”

“…Type III further benefits from an improved high-speed camera and illumination via fiber optics. Developed results in [37] indicate improved suppression of crystallization with the application of SIMs and unexpected precipitation of solutes during rewarming. Both Type I and Type III are designed to visualize physical events with a stationary camera in a similar arrangement to the cryomicroscope setup, which represents a challenge for the study of large size samples—larger than the field of view of the optical system.…”

Polarized light scanning cryomacroscopy, part I: Experimental apparatus and observations of vitrification, crystallization, and photoelasticity effects

“…Cryomacroscope III has been designed to investigate physical events associated with vitrification in the presence of synthetic ice modulators (SIMs) [33]. The main improvement in Cryomacroscope III over Cryomacroscope I is that it has been redesigned as an add-on to a commercially available top-loading controlled-rate cooler, thereby improving its potential dissemination for the benefit of the cryobiology community.…”