Fracture formation in vitrified thin films of cryoprotectants

Abstract

As a part of an ongoing effort to study the continuum mechanics effects associated with cryopreservation, the current report focuses on fracture formation in vitrified thin films of cryoprotectant agents. The current study combines experimental observations with continuum mechanics analysis. Experimental results have been developed using a new imaging device, termed a "cryomacroscope", which has been recently presented by the current research team. A newly developed liquid nitrogen-based cooling stage is presented in this paper. The samples under investigation are 0.5 ml droplets of cryoprotective agents, having a characteristic diameter of 20 mm and a characteristic thickness of 1.5 mm. Tested samples included dimethyl sulfoxide (DMSO) in a concentration range from 6M to 8.4M, and the cryoprotectant cocktails VS55 and DP6. Some samples contained small bovine muscle segments, having a characteristic dimension of 1 mm, in order to study stress concentration effects. Experimental results show that the onset of fracturing in vitrified films of cryoprotectants is very consistent, occurring over a small temperature range. Fracture pattern, however, was affected by the cooling rate. The presence of tissue segments did not affect the onset temperature of fracture, but affected the fracture pattern. The continuum mechanics analysis solidified the hypothesis that fracture is driven by thermal stress, not by temperature per se, and allowed fracture strain to be inferred from observed fracture temperature. In conjunction with the current report, additional photos of fracture formation in thin films are available at

The Effect of Temperature Gradients on Stress Development during Cryopreservation via Vitrification

“…While the physical properties of DMSO are concentration dependent, a reference concentration of 7.05M DMSO has been selected in the current study. This concentration has been demonstrated to have similar physical properties to two CPA cocktails currently under investigation in parallel studies: DP6 (3M DMSO, 3M propylene glycol, and 2.4 g/ℓ HEPES in EuroCollins solution) and VS55 (3.1M DMSO, 2.2M propylene glycol, 3.1M formamide, and 2.4 g/ℓ HEPES in EuroCollins solution) [12,16,17]. Nevertheless, results presented in the current study are rather general, and the conclusions presented in the current study are not limited to a specific CPA and concentration.…”

“…In a previous experimental study [12], droplets of CPA were cooled on a very stiff substrate to low temperatures at which fractures occurred. Due to the higher thermal expansion of the CPA compared to the substrate, the CPA droplet develops tensile stress.…”

“…While T g is commonly measured with differential calorimetry techniques (DSC), an alternative definition of glass transition is the temperature at which the viscosity reaches a value of 10 12 Pa-s (10 13 Poise). In the absence of viscosity data on cryoprotectants at low temperatures, the following functional behavior is assumed in the current study [15]: ) (1) where η is the shear viscosity, and the parameters b = 8.607 K and T 0 = 111.1 K are selected to best-fit the available experimental data on 7.05M DMSO at higher temperatures [12], and the glass transition temperature was taken to be −132°C, measured with DSC for 7.05M DMSO [16].…”

“…Previous work focused on the consequences of constraining the thermal contraction of the vitrifying specimen; theoretical and experimental studies of a CPA droplet vitrifying on a substrate gave insight into the role of viscosity and the level of strain at which vitrified CPA may crack [12]. The constraining effect of vial walls on the thermal contraction of the contained CPA medium was later studied and used to rationalize crack propagation patterns in the CPA [10].…”

“…However, pure vitrification can only be associated with pure CPAs, while in the tissue-CPA system partial vitrification is expected, and the quality of vitrification is the subject matter of many cryobiology studies [6][7][8]. Analysis of mechanical stress during vitrification has been paid very little attention over the years, and only recent studies have begun to address modeling and worst case scenarios of mechanical stress during cryopreservation via vitrification [9][10][11][12]. The resulting mechanical stresses during vitrification can potentially exceed the strength of the material, leading to its fracturing, which is a significant barrier to the cryopreservation of three-dimensional multi-cellular tissues.…”