Honors College Thesis


The Effects of Toxicity Inhibitors on the Cytotoxicity of Cryoprotective Agents using a High-Throughput Experimental Set-up Público Deposited

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  • Even with advanced medical technology, there is still an unmet need for organs to reduce the transplant waiting list and to advance organ-related research. Cryopreservation through vitrification is one possible solution to increase organ availability. A major challenge with cryopreservation of organs is the toxicity of the cryoprotectants (CPAs) that are added to prevent freezing. We hypothesized that additives that mitigate different mechanisms of toxicity will reduce the cytotoxicity of the five tested CPAs and that there will be increased cell viability as a result. Five commonly used CPAs including, glycerol (Gly), dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), and formamide (FA) were tested. The 14 treatments include antioxidants, a calcium chelator, a calcium channel blocker, an iron chelator, an apoptosis inhibitor, sodium-free buffers, a metabolic inhibitor, a heat shock protein, altering the buffering capacity, a combination of calcium chelator and channel blocker, and the inclusion of polyethylene glycol (PEG). We used an automated liquid handler to test all 14 of the combinations of CPA treatments. The cell viability after treatment was determined using a Presto Blue assay and plate reader. We found that none of the inhibitors had a significant effect on cell viability when cells were exposed to CPA solutions. Since the mechanisms of cell death caused by each CPA is still relatively unknown, our data could provide some insight as to which mechanism is causing cytotoxic damage to the cells and a method to reduce the damage.
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