Organ donation procurement, and preservation

Functional integrity of the rat liver after subzero preservation under high pressure☆

This study investigates the feasibility of ice-free isochoric vitrification for cryopreservation applications using mathematical modeling, computation tools, and the underlying principles of thermo-mechanics. This study is triggered by an increasing interest in the possibility of isochoric vitrification, following promising experimental results of isochoric cryopreservation. In general, isochoric cryopreservation is the preservation of biological materials in subzero temperatures in a rigid-sealed container, where some ice crystallization creates favorable pressure elevation due to the anomaly of water expansion upon ice Ih formation. Vitrification on the other hand is the transformation of liquid into an amorphous solid in the absence of any crystals, which is typically achieved by rapid cooling of a highly viscous solution. The current study presents a mathematical model for vitrification under variable pressure conditions, building upon a recently published thermo-mechanics modeling approach for isochoric cryopreservation. Using the physical properties of dimethyl sulfoxide (DMSO) as a representative cryoprotective agent (CPA), this study suggests that vitrification under isochoric conditions is not feasible, essentially since the CPA solution contracts more than the isochoric chamber by an order of magnitude. This differential contraction can lead to absolute zero pressure in the isochoric chamber, counteracting the premise of the isochoric cryopreservation process. It is concluded that the only alternative to prevent ice formation while benefiting from the potential advantages of higher pressures is to create the required pressures by external means, and not merely by passively enclosing the specimen in an isochoric chamber.

We introduce an isochoric (constant-volume) supercooling cryomicroscope (ISCM), enabling the ice-free study of biological systems and biochemical reactions at subzero temperatures at atmospheric pressure absent ice. This technology draws from thermodynamic findings on the behavior of water in isochoric systems at subfreezing temperatures. A description of the design of the ISCM and a demonstration of the stability of the supercooled solution in the ISCM is followed by an illustration of the possible use of the ISCM in the preservation of biological matter research. A comparison was made between the survival of HeLa cells in the University of Wisconsin (UW) solution in the ISCM at +4 °C under conventional atmospheric conditions and at −5 °C under isochoric supercooled conditions. Continuous real-time monitoring at cryopreservation temperature via fluorescence microscopy showed that after three days of isochoric supercooling storage, the percentage of compromised cells remained similar to fresh controls, while storage at +4 °C yielded approximately three times the mortality rate of cells preserved at −5 °C.

Isochoric (constant volume) preservation at subfreezing temperatures is being investigated as a novel method for preserving cells and organs. This study is a first initial effort to evaluate the efficacy of this method for heart preservation, and to provide a preliminary outline of appropriate preservation parameters. To establish a baseline for further studies, rat hearts were preserved in a University of Wisconsin (UW) intracellular solution for one hour under isochoric conditions at: 0 °C (atmospheric pressure - 0.1 MPa), - 4 °C (41 MPa), - 6 °C (60 MPa) and – 8 °C (78 MPa). The viability of the heart was evaluated using Langendorff perfusion and histological examination. The physiological performance of hearts preserved at – 4 °C (41 MPa) was comparable to that of a heart preserved on ice at atmospheric pressure, with no statistically significant difference in histological injury score. However, hearts preserved at −4 °C displayed substantially reduced interstitial edema compared to hearts preserved by conventional hypothermic preservation in UW on ice at atmospheric pressure, suggesting significant protection from increased vascular permeability following preservation. Hearts preserved at – 6 °C (60 MPa) suffered injury from cellular swelling and extensive edema, and at – 8 °C (78 MPa) hearts experienced significant morphological disruption. To the best of our knowledge, this is the first publication showing that a mammalian organ can survive low subfreezing temperatures without the use of a cryoprotective additive. Lowering the preservation temperature reduces metabolism and improves preservation quality, and these results suggest that improvements in preservation are possible at subzero temperatures with low to moderate pressures observed at −4 °C. Notably, tissue damage was observed at lower temperatures (−6 °C or below) accompanying further elevation of pressure associated with isochoric preservation that may prove detrimental. Therefore, subfreezing temperature isochoric preservation protocols should optimize, a combination of temperature and pressure that will minimize the negative effects of elevated pressure while retaining the beneficial effect of lower temperatures and reduced metabolism.

Isochoric (constant volume) preservation is an alternative to traditional cryopreservation methods because it requires less cryoprotectant and is simple to operate. In order to validate that this method automatically minimizes the pressure for a given temperature, pressure and temperature data were collected from a specially designed pressure vessel. This vessel was then used to examine the effect of an isochoric environment on freezing point nucleation in an aqueous antifreeze protein solution, and to generate pressure–temperature phase diagrams for various cryoprotectant solutions. Our results show that the isochoric pressure vessel follows the pressure–temperature phase diagram of water, thereby minimizing the pressure for the given temperature. We also show that the nucleation temperature of the antifreeze protein in an isochoric vessel is lower than that of the isobaric method. Furthermore, the nucleation temperature decreased with increasing concentration in the isochoric vessel while the isobaric nucleation temperature showed no change. These results indicate that the isochoric environment imposes additional constraints on ice formation and warrants further study as these results may change when a different type of cryoprotectant is used. Finally, all of the cryoprotectant phase diagrams exhibited a similar pressure–temperature slope indicating that, regardless of the cryoprotectant used or the mechanism by which it suppresses freezing, isochoric freezing affects the molecules in the same manner. Together, all of these results indicate that the isochoric method of preservation is a valuable tool for characterizing the thermodynamic properties of cryoprotectants and has great potential as a cryopreservation method in the field of cryobiology.

The goal of this study is to introduce the fundamental thermodynamic principles of isochoric (constant volume) cryopreservation for low temperature preservation of biological materials. Traditionally, cryopreservation is performed in an isobaric process (constant pressure) at 1 atm, because this is our natural environment and it is most convenient experimentally. More than half a century of studies on cryopreservation shows that the major mechanism of damage during isobaric cryopreservation is the increase in intracellular ionic concentration during freezing, which presumably causes chemical damage to the components of cells. Cryoprotectants as well as hyperbaric pressures have been developed as methods to reduce the extent of chemical damage during freezing. The theoretical studies in this paper show that in isochoric cryopreservation, the increase in solution concentration during freezing is lower at each temperature by almost an order of magnitude from that in isobaric cryopreservation. This suggests that isochoric cryopreservation could be a preferential alternative to isobaric cryopreservation. The technology for isochoric cryopreservation is very simple; freezing in a constant volume chamber. Using a simple isochoric cryopreservation device, we confirm the theoretical thermodynamic predictions.