On the Effects of Thermal History on the Development and Relaxation of Thermo-Mechanical Stress in Cryopreservation.

This study investigates the effects of the thermal protocol on the development and relaxation of thermo-mechanical stress in cryopreservation by means of glass formation, also known as vitrification. The cryopreserved medium is modeled as a homogeneous viscoelastic domain, constrained within either a stiff cylindrical container or a highly compliant bag. Annealing effects during the cooling phase of the cryopreservation protocol are analyzed.

Can thermal expansion differences between cryopreserved tissue and cryoprotective agents alone cause cracking?

One of the limiting factors in large-scale cryopreservation is the formation of fractures. The prevalence of cracking in cryopreserved bulky tissues is frequently associated with temperature gradients, which lead to non-uniform thermal contraction of the tissue. With new cryoprotectants available, it may be possible to reduce temperature gradients to much lower levels, in which case other contributions to mechanical stress development and cracking will become more significant.

Viscosity of cryoprotective agents near glass transition: a new device, technique, and data on DMSO, DP6, and VS55.

The low strain-rate viscosity of glass-forming cryoprotective agents (CPAs) in the vicinity of the glass transition is studied experimentally. Data on the mechanical behavior in this regime is necessary to the long-term goal of developing planning tools for cryopreservation via vitrification (vitreous means glassy in Latin); such tools will provide guidelines for reducing thermal stress with its devastating effects. While the flow behavior of some glass-forming CPAs is well documented in the literature for the upper part of the cryogenic temperature range (where the CPA has a comparatively low viscosity), it is the flow behavior near the glass transition temperature (where the CPA behaves as nearly a solid with an extremely high viscosity) which is critical to the analysis of stress that develops in the cryopreserved material.

Continuum mechanics analysis of fracture progression in the vitrified cryoprotective agent DP6.

As part of an ongoing effort to study the continuum mechanics effects associated with cryopreservation, the current report focuses on the prediction of fracture formation in cryoprotective agents. Fractures had been previously observed in 1 ml samples of the cryoprotective agent cocktail DP6, contained in a standard 15 ml glass vial, and subjected to various cooling rates. These experimental observations were obtained by means of a cryomacroscope, which has been recently presented by the current research team.

The Effect of Temperature Gradients on Stress Development During Cryopreservation via Vitrification.

This study addresses the problem of thermal stress development in bulky specimens during cryopreservation via vitrification (vitreous means glassy in Latin). While this study is a part of an ongoing effort to associate the developing mechanical stress with the relevant physical properties of the cryopreserved media and to its the thermal history, the current paper focuses exclusively on the role of temperature gradients. Temperature gradients arise due to the high cooling rates necessary to facilitate vitrification; the resulting non-uniform temperature distribution leads to differential thermal strain, possibly resulting in cracking.

Stress-strain measurements and viscoelastic response of blood vessels cryopreserved by vitrification.

To gain increased insight into thermo-mechanical phenomena during cryopreservation, tensile stress relaxation experiments were conducted on vitrified blood vessels (vitreous in Latin means Glassy), and the results compared with various viscoelastic models. Using a recently presented device, isothermal stress-relaxation results were obtained for a bovine carotid artery model, permeated with the cryoprotectant cocktail VS55 and a reference solution of 7.05 M DMSO.

Analysis of the effect of partial vitrification on stress development in cryopreserved blood vessels.

Thermal stress development in blood vessels, during processes associated with vitrification (vitreous means glassy in Latin), is studied. This paper addresses the limiting case where the specimen completely crystallizes, while the cryoprotectant medium (CPA) completely vitrifies. This case is expected to provide upper boundary estimates for stresses for the more common problem of a partially vitrified sample.

Cryomacroscopy of vitrification, Part II: Experimental observations and analysis of fracture formation in vitrified VS55 and DP6.

A new imaging device, termed a "cryomacroscope", was used to observe macrofractures in the cryoprotectant cocktails DP6 and VS55. Details of the design and construction of the cryomacroscope were presented in Part I of this report, which focused on describing the apparatus and observations of crystallization. Part I and the current paper (Part II) describe events that occur as 1 mℓ of cryoprotectant contained in a glass vial is cooled from room temperature down to cryogenic temperatures (∼ -135°C).

Fracture formation in vitrified thin films of cryoprotectants.

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 cryoprotective 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.

Cryomacroscopy of vitrification, Part I: A prototype and experimental observations on the cocktails VS55 and DP6.

A new imaging device, termed a "cryomacroscope", is presented in this report. This device is designed to assist in exploring thermal and mechanical effects associated with large-scale vitrification and crystallization, with the current setup aimed at the range of 50 μm to 2 cm. The cryomacroscope is not intended as a substitute for the cryomicroscope, but as a complementary tool for the cryobiologist.