The Efficacy of Using Small Molecule Ice Recrystallization Inhibitors to Enable the Frozen Storage of Livers

• Author / Creator

  William, Nishaka

• The limited storage times afforded by hypothermic preservation at 0 °C to 4 °C imposes several logistic constraints on the organ transplant network. In the case of livers, two-thirds of wait listed patients do not receive transplants in Canada, which is partially attributed to the 12 h storage period under hypothermic conditions. Over the past seven decades, extensive research has gone into the development of methods to store organs at sub-zero temperatures in order to improving preservation standards. Emphasis is often placed on the avoidance of ice formation within tissues stored at sub-zero temperatures; however, these approaches come with their own respective challenges. Ice is not categorically damaging in tissues as evidenced by the survival strategies of several freeze tolerant organisms. Therefore, a preferable approach to sub-zero organ preservation may involve controlling the mechanisms through which ice damages tissues. Migratory recrystallization, the process through which large, more thermodynamically stable ice crystals grow at the expense of small ice crystals, is a major driving force for the cellular and structural damage that ice imparts. Thus, we sought to evaluate the efficacy using small molecule carbohydrate-derived ice recrystallization inhibitors (IRIs) our group has developed to minimize both intra- and extracellular ice recrystallization. Hepatocytes comprise approximately 80% of the cellular mass of the liver and are highly prone to the formation of intracellular ice due to their high activation energy for water transport. Therefore,
  we evaluated the toxicity of three different IRIs as well as their ability to permeate and control intracellular ice recrystallization in HepG2 cells, a commonly used human liver cancer cell line. IRI 2 and IRI 3 demonstrated a significant reduction in the rate of intracellular ice recrystallization
  under a treatment condition where they did not prove to be toxic. The intracellular activity of each compound was further evaluated in the presence of permeating and non-permeating cryoprotective agents (CPAs), which are vital to the recovery of cryopreserved cells and tissues. Generally, there was a loss of IRI activity in the presence of the four most commonly used permeating CPAs: ethylene glycol, glycerol, DMSO, and propylene glycol. IRI 2 demonstrated complete loss of intracellular activity in all cases, whereas IRI 3 retained activity in glycerol, DMSO, and propylene glycol. Interestingly, in the presence of trehalose, a commonly used non-permeating CPA, the activity of IRI 1 was improved while that of IRI 2 was diminished. IRI 3 activity, on the other hand, did not appeared to be altered in the presence of trehalose. The liver consists of five different cell types, potentially leading to different toxicity outcomes than those seen in a single cell system. IRI 2 did not elicit any toxic effects following 30 min of subnormothermic machine perfusion (SNMP) in whole rat livers. Furthermore, this compound proved to be capable of permeating liver tissues and minimizing recrystallization, thus setting the stage for the implementation of this compound that has recently been designed to store livers in a frozen state at high sub-zero temperatures. The compound did not, however, improve post-thaw functional outcomes following 1 day of storage at -10 °C or 5 days of storage at -15 °C. Whether this is the result of a lack of IRI activity in the CPA solutions used or the result of other forms of freezing injury taking precedence over recrystallization injury, requires further investigation. This work does not definitively establish the efficacy of small molecule IRIs in the frozen storage of livers; however, it highlights key considerations that need to be taken in future evaluations. Furthermore, it provides a better understanding of the ability of these compounds to control of intracellular ice recrystallization, which may prove valuable in the development of freezing protocols where intracellular ice formation (IIF) is probable.

• Subjects / Keywords

  • Ice recrystallization
  • Liver cryopreservation
  • Cryofixation
  • Cryomicroscopy
  • Hepatocyte cryopreservation

• Graduation date

  Fall 2020

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-q44k-5993

• License

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