Stress-Strain Measurements in Vitrified Arteries Permeated With Synthetic Ice Modulators.

There is an unmet need for high-quality tissues and organs for transplantation medicine. The typically short time window between organ donation and transplantation creates a severe supply chain management problem, where the duration of this window is dramatically affected by the available preservation methods and technology. Cryopreservation—the preservation of biological material at very low temperatures—is generally considered the only plausible method for preserving tissues and organs for long periods of time. Cryopreservation by vitrification (vitreous means glassy in Latin) involves using high concentrations of CPAs and high cooling rates to suppress crystallization and promote the transformation into an amorphous (arrested fluid) state. Vitrification can potentially prevent the devastating effects of cryoinjury [1] associated with ice crystallization and can potentially be applied to a wide spectrum of tissues. Vitrification appears to present the only alternative for cryopreservation of large-size specimens. However, the development of cryopreservation via vitrification for implantable tissues of large size needs to overcome critical obstacles, before it can become a wide-spread and accessible application. First and foremost, CPAs tend to be toxic at the high concentrations necessary for vitrification [2–4]. Lowering the CPA concentration requires higher cooling rates in order to suppress crystal formation, leading to large temperature gradients which can give rise to a host of devastating effects. One potentially harmful effect is associated with the tendency of the material to contract as it cools, where thermomechanical stress may develop if different areas in the material contract differently as a result of the temperature gradients. When the resultant mechanical stress exceeds the strength of the material, permanent damage will follow, with fracture formation as its most dramatic outcome [5–9]. Based on the above, there is an obvious desire to reduce the CPA concentration, and hence its toxicity. Additionally, there is a need to increase the CPA's effectiveness, thus reducing the needed cooling rate to prevent crystallization and reducing mechanical stress. 1,3-cyclohexanediol (1,3-CHD) was designed to bind to ice crystal nuclei and prevent their growth and has been designated as a synthetic ice blocker (SIB) [10,11]. It was shown that a lower concentration CPA with the addition of 1,3-CHD could be as effective as a higher concentration (and hence more toxic) CPA [12]. More recently, other compounds have been discovered that also seem to increase CPA effectiveness in very small doses. The mechanisms by which these compounds achieve this are mostly unknown, and so a broader term than SIB has been proposed to describe them, SIM [13–17]. Clearly the SIB, 1,3-CHD, would fall under this general SIM designation as well. The present study includes three contrasting SIMs: 1,3-CHD represents the best published example of an SIB [10,11], and polyethylene glycol (PEG400), and 2,3-butanediol (2,3-BD) which are included as compounds known to facilitate the stability of the amorphous state by virtue of their interactions with water (reviewed in Ref. [12]). A defining feature of ice modulators is that they have a greater effect on influencing ice nucleation and growth and stabilizing the amorphous state than accounted for on a purely colligative basis [11]. Success in cryopreservation requires a balance between thermal, mechanical, and chemical properties, as they pertain to the material interaction with living cells. It is generally accepted that toxicity is dependent upon concentration and temperature. SIMs allow for the reduction of the toxicity of the CPA cocktail by reducing the total concentration of solutes while retaining the ability to promote vitrification [10]. The application of cryopreservation with the aid of SIMs has been investigated [12,17,18], but engineering concepts related to these processes are still in their early stages. The combination of ordinary CPA cocktails with SIMs has been shown to reduce the high cooling rate necessary for vitrification [17], which is critical to scale-up cryopreservation for two primary reasons: (i) regardless of the cooling mechanism at the outer surface of a bulky specimen, the maximum achievable cooling rate at its center is always lower—limited by principles of heat conduction through the tissue, a problem which becomes more pronounced as the specimen size increases and (ii) lower cooling rates often mean lower thermomechanical stress and diminishing risk to structural damage. The first patent for an SIM to be used for cryopreservation was awarded in 2004. However, very few reports in this context are available in the literature of cryobiology. One encouraging study demonstrated significantly improved crystallization suppression of the CPA cocktail, DP6, with the addition of SIMs [17]. Another study found that the addition of 1,3-CHD to DP6 significantly improved its tendency to vitrify in the preservation of pancreatic islets [12]. That study showed comparable islet viability for islets preserved in DP6 + 1,3-CHD and VS55, where the latter is a reference solution with a significantly higher concentration of permeating CPAs. This study is the continuation of an ongoing effort to characterize thermal and mechanical properties of CPAs and tissues in cryogenic temperatures. Earlier studies have measured the thermal expansion property of CPAs and SIMs in the presence and absence of biological tissues [17], thermal conductivity of CPAs [21], and viscosity of CPAs [22]. Additionally, experiments have investigated Young's modulus of vitrified tissues permeated with 7.05 M DMSO and VS55 as well as relaxation properties of tissues permeated with those CPAs near glass transition [23,24]. The current study represents the first attempt to measure Young's modulus of tissues permeated with CPA-SIM cocktails. In general, the Young's modulus is a measure of the stiffness of an elastic material and can be directly used to predict the onset of structural damage in brittle materials.