Isochoric Cryopreservation as a Possible Path to Reversible Organ Preservation

An important goal in cryopreservation research is to find an efficient way to reversibly cryopreserve whole organs and then larger masses without ice crystal formation and other structural damage, leading up to whole body preservation. Being able to take donated organs and provide them with an indefinite shelf life would revolutionize the logistics of the transplant industry. Later, it would enable an efficient industry for manufacture of new organs, as tissue engineering capabilities increase. The eventual goal for this technological capability is to greatly improve the ability to preserve individuals at clinical death, maintaining them in cold storage indefinitely, with minimal additional damage, until technological progress allows for repair and revival.

The start of the modern field of cryobiology is thought to have happened in 1948, with the discovery of the cryoprotective effects of glycerol, a cryoprotective agent (CPA) that prevents ice crystal formation through the creation of bonds with free water molecules. Since then, a huge aspect of cryobiology and cryopreservation technologies was that we can modulate a given system's chemistry by involving CPAs, which could, in theory, allow us to preserve a live biologic sample for a long time. Many more CPAs, like dimethyl sulfoxide (DMSO), appeared on the scene afterwards, revolutionizing the subfield of human sperm cryopreservation. In 1972, scientists published evidence of the first-ever successful cryopreservation of mammalian embryos using slow-freezing. Eleven years later, the first-ever human embryo was cryopreserved.

A turning point in cryobiology happened in the 1980s, the so-called golden era of cryopreservation. Researchers introduced the process of vitrification to medical cryopreservation. Vitrification is a process of rapid cooling of liquid medium until it becomes a glass-like non-crystalline amorphous solid. It requires the protective effect of CPAs, which lower the freezing point of water, as a major part of biological systems. In its vitrified state, water is locked in place, preventing the formation of ice crystals, and the entire sample becomes a glass-like solid. Vitrification is used widely today in the cryopreservation of very small biological samples (specifically in in vitro fertilization and other reproductive applications), and many cryobiologists believe it could eventually be applied to freeze any biological materials, even organs and whole organisms.

One of the major focus in cryobiology research is, in fact, centered around the process of vitrification and how much and which CPAs to add during this stage, or how to remove these often toxic compounds in the rewarming stages. But, so far, CPA-aided vitrification only enabled the routine preservation of cells and cell suspensions and failed to produce any clinically translatable technique on how to reversibly preserve any complex biological systems like organs outside of the human body.

Methods in cryopreservation haven't changed much in the last few years but there is a different approach currently available called isochoric cryopreservation. The term stands for cryopreservation of biological tissues at a constant volume, versus the more "traditional" way of cryopreservation that's done at constant pressure, called isobaric cryopreservation. During isochoric preservation, the cooling process happens in a confined, constant-volume chamber, representing one of the biggest differences between isochoric and isobaric conditions. Another difference is minimized role of CPAs, which are very much needed in the classical isobaric cryopreservation, but not in several modes of isochoric cryopreservation.

The advantage of isochoric freezing is that it completely avoids the question of the toxicity associated with CPA usage as well as the amount of CPAs needed to be present in the biological sample you might want to freeze. Even if there is a need to use CPAs, their concentrations would be dramatically decreased. Under isochoric conditions, a biological sample is confined within a container with high rigidity and strength, usually made out of titanium. The container is completely absent of the bulk gas phase, and is denied any access to the atmosphere, which changes both the thermodynamic equilibrium and the ice nucleation kinetics within the system inside.


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