Abstract
This study explores physical effects associated with the application of cryopreservation via vitrification using a class of compounds which are defined here as synthetic ice modulators (SIMs). The general classification of SIMs includes molecules that modulate ice nucleation and growth, or possess properties of stabilizing the amorphous state, by virtue of their chemical structure and at concentrations that are not explained on a purely colligative basis. A sub-category of SIMs, referred to in the literature as synthetic ice blockers (SIBs), are compounds that interact directly with ice nuclei or crystals to modify their structure and/or rate of growth. The current study is part of an ongoing effort to characterize thermo-mechanical effects during vitrification, with emphasis on measuring the physical property of thermal expansion—the driving mechanism to thermo-mechanical stress. Materials under investigation are the cryoprotective agent (CPA) cocktail DP6 in combination with one of the following SIMs: 12% polyethylene glycol 400, 6% 1,3 cyclohexanediol, and 6% 2,3 butanediol. Results are presented for the CPA-SIM cocktail in the absence and presence of bovine muscle and goat artery specimens. This study focuses on the upper part of the cryogenic temperature range, where the CPA behaves as a fluid for all practical applications. Results of this study indicate that the addition of SIMs to DP6 allows lower cooling rates to ensure vitrification and extends the range of measurements. It is demonstrated that the combination of SIM with DP6 increases the thermal expansion of the cocktail, with implications for the likelihood of fracture formation—the most dramatic outcome of thermo-mechanical stress.
Highlights
The role of cryopreservation for tissue banking is undisputable, being the only practical alternative for long-term storage of high quality biomaterial
The two components are connected by means of a flexible tube, which is filled with the same cryoprotective agent (CPA) solution under investigation, but has a negligible volume compared with the volume of the cooling chamber
It is established that both the concentration needed to vitrify and the toxicity can be reduced by incorporating a wide variety of non-permeating disaccharides or polymeric compounds [2,36]; the selection of polyethylene glycol 400 (PEG400), which we have previously reported to elevate the glass transition temperature, reduce the energy associated with the transition, and reduce the risk of devitrification in a concentration dependent manner [4]. 1,3-CHD falls within the sub-classification of synthetic ice blockers (SIBs) that are designed to lattice-match with the basal plane of an ice crystal by hydrogen bonding and thereby inhibit ice crystal growth [10,40]
Summary
The role of cryopreservation for tissue banking is undisputable, being the only practical alternative for long-term storage of high quality biomaterial. Techniques for successful cryopreservation have been developed over the years for several tissue types. Successful cryopreservation techniques are generally related to small specimens in the scalerange of cells to small-organized tissues, with the stem cells [12,29], corneas [11,40], and pancreatic islets [41] as examples. Classical cryopreservation, which makes use of low concentrations of cryoprotective agents (CPAs), such as dimethyl sulfoxide (DMSO), does a reasonable job of cell preservation by prevention of intracellular ice formation, but a poor job of tissue preservation. There have been several hypotheses on the mechanisms of freezing-induced injury based upon a variety of factors [19,21], but experience with mammalian tissues shows that the disadvantages of classical cryopreservation revolve primarily around ice formation [22,23,37]. The formation of extracellular ice in particular (generally regarded as innocuous for cells in suspension) is known to be hazardous to structured tissues and organs [13,14,22,23,37]
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