Cryopreservation of biological materials has many useful applications and is currently the most effective long term storage method used across a variety of fields. The success of freezing products or biological materials, however, varies because of the process' complexity and related cryo-injuries. One of the primary issues is the ice recrystallization induced development of extracellular and intracellular ice throughout the freezing and thawing process. Ice recrystallization is a significant contributor to freezing damage, ultimately reducing post-thaw viability and function. To address this issue, the Ben laboratory has developed and synthesized a variety of classes of small molecule carbohydrate-based ice recrystallization inhibitors (IRIs). These compounds act as supplements or alternatives to current cryoprotectants, such as trehalose, DMSO, or glycerol, which do not address ice recrystallization and can be cytotoxic. This thesis focuses on the comprehensive chemical property assessment of N-Aryl-β-D-aldonamides and N-Benzyl-β-D-gluconamides, as well as optimization of biopreservation protocols for tissue products and freeze-dried proteins.
Utilizing a 5-minute modified splat cooling assay, dose-response curves of five N-Benzyl-β-D-gluconamides were generated. All compounds produced ice recrystallization inhibition active IC50 values comparable to previously investigated active compounds such as, N-Octyl-β-D-gluconamide and N-4-Bromophenyl-β-D-glucopyranoside. Furthermore, validation that the dose-response curves follow a 4-parameter logistic (4PL) or 5PL sigmodal trend depending on symmetry was obtained. In addition, all tested compounds had lower cytotoxicity than N-4-Bromophenyl-β-D-glucopyranoside and higher solubility than N-Octyl-β-D-gluconamide. Overall, N-Benzyl-β-D-gluconamides proved to be a promising class of compounds with the para derivatives being the most IRI active.
The second part of this work involved the examination of IRIs' ability to cryopreserve two different biological materials using different biopreservation protocols. The first being proteins and master mix (enzymes and oligonucleotides) during RT-qPCR after the freeze-drying process. The data showed that the IRIs did not interfere and were effective during both the lyophilization and qPCR processes. When compared to most effective concentration of the current industry standard, N-4-Bromophenyl-β-D-glucopyranoside increased the protein activity by ~30%, reducing the number of cycles to reach threshold value. The most significant contribution of this work was the discovery that carbohydrate-based small molecules may be working in more than one mechanism, as both cryoprotectants and lyoprotectants. In addition to proteins, the ability of IRIs to cryopreserve tissue products was investigated. Cell media supplemented with IRIs indicated that they can increase viability and reduce mortality in both cell suspension and single dermal sheets. With N-4-Methylbenzyl-β-D-gluconamide and N-Octyl-β-D-gluconamide being the most effective at reducing the damage associated with freezing and increasing recovery of the cells within the system of a simple one cell type thin tissue matrix.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/44963 |
| Date | 17 May 2023 |
| Creators | Mangan, Sophia |
| Contributors | Ben, Robert |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Attribution 4.0 International, http://creativecommons.org/licenses/by/4.0/ |
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