Characterizing the Efficacy of Ice Recrystallization Inhibitors as a Novel Cryoprotectant for Lung Cryopreservation

  • Author / Creator
    Lautner, Larissa J.
  • Although lung transplant remains the only option for patients suffering from end-stage lung disease, donor lung supply is currently insufficient to meet demand. While many lungs become available for transplant, most are discarded due to failure to meet physiologic or compatibility criteria. Many lungs are currently unutilized due to short preservation times, with lungs surviving only 6-8 hours on ice. Successful cryopreservation of lungs would allow for extended storage to help ameliorate this problem; however, many challenges must be overcome before this can occur. The growth of existing ice at the expense of new ice nucleation is called ice recrystallization, and it is one of the major causes of freezing injury, as it results in osmotic stress and mechanical damage to cellular membranes, intercellular connections, and intracellular organelles. Previous research has demonstrated that ice recrystallization can be controlled through the use of small-molecule ice recrystallization inhibitors (IRIs). Therefore, the research performed for this thesis aimed to assess the utility of these IRIs as novel cryoprotectants for lung cryopreservation. It was hypothesized that IRIs would not be cytotoxic and would be capable of controlling intra- and extra-cellular ice growth resulting in improved cellular survival following cryopreservation of type II pneumocyte monolayers and rat lung tissue, when compared to DMSO-treated controls. The first objective of this work assessed the ability for IRIs to reduce intracellular ice grain size and improve post thaw survival, without cytotoxicity, in the immortalized type 2 pneumocyte cell line BEAS-2B. Short- and long-term cytotoxicity of two IRI compounds was compared to the commonly used cryoprotectant, DMSO, through the use of the metabolic assay reagent alamarBlue. Intracellular ice grain size was quantified after cells treated with IRIs, with or without the addition of DMSO, were cryofixed and stained with SYTO13 and observed under fluorescent microscopy. Post-thaw membrane integrity and metabolism was assessed following intracellular ice nucleation and recrystallization in samples treated with DMSO, with or without the addition of one IRI. These experiments indicated that one IRI, (2-fluorophenyl)-C6-azido-D-gluconamide (2FA), was non-toxic and reduced intracellular ice grain size when used alone. However, no reduction of intracellular ice grain size was observed when 2FA was added to DMSO, which corroborated with the finding that post-thaw survival was not improved by 2FA addition to DMSO. The second objective of this work set out to develop low-cost subnormothermic techniques for ex vivo rat lung perfusion to assess the cytotoxicity of 2FA and ability for 2FA to reduce extracellular ice growth resulting in improved post-thaw cell membrane integrity and tissue structural integrity, when compared to DMSO-treated controls. After rat lungs were perfused subnormothermically with 2FA in STEEN solution, toxicity was assessed through the perfusion of 0.4% (w/v) trypan blue prior to fixation, paraffin-embedding, sectioning, eosin staining, and visualization under light microscopy. The ability for 2FA to reduce extracellular ice grain size was assessed using tissue cryofixation after a 1 h hold at -20°C. Post-thaw cell membrane integrity and tissue morphology was assessed by perfusing rat lungs with 0.4% (w/v) trypan blue and 10% (v/v) DMSO, with or without the addition of 2FA, freezing to -20°C, and thawing in a 37 °C water bath. The addition of 2FA was found to improve post-thaw alveolar cell membrane integrity and tissue morphology when compared to DMSO treatment alone. This work successfully demonstrated that one IRI, 2FA, is not cytotoxic and reduces intra- and extra-cellular ice grain size in pneumocyte monolayers and rat lungs, respectively, when used alone. While previous work has demonstrated intracellular ice control by IRIs, this work expands on previous findings by demonstrating a reduction in extracellular ice grain size within lung tissue and revealing the limitations of intracellular ice control by IRIs when used with DMSO. In addition, this is the first work to assess ice-allowing cryopreservation of lungs. Given the underrepresentation of lungs in the whole organ cryopreservation literature, the techniques described here may be utilized by other researchers to assess cryoprotectant efficacy, in terms of cytotoxicity, ice control, and post-thaw cell membrane integrity and tissue morphology. Therefore, this work contributes to the fields of cryobiology and organ preservation, as it expanded upon the uses and limitations of IRIs and developed low-cost techniques for the analysis of whole rat lung cryopreservation.

  • Subjects / Keywords
  • Graduation date
    Fall 2020
  • Type of Item
  • Degree
    Master of Science
  • DOI
  • License
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