Honors College Thesis
 

Enhanced Delivery of Cryoprotectant Chemicals to Cultured Neurons

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  • Due to the many advances in the areas of biotechnology and medicine, the need for long- term storage and stabilization of biological materials is rapidly increasing. The field of biopreservation is attempting to address these issues by finding ways to maintain the integrity and functionality of proteins, cells, and organs while storing them outside of their native environment. Neurons have been chosen for investigation based on their potential as electrically active sensors and promise for use in cell-based devices. This thesis will focus on the delivery of cryoprotectant chemicals (CPAs), used to prevent damage to the cells during freezing, to cultured neurons prior to cryopreservation. The addition and removal of CPAs can cause serious cell damage due to their creation of an anisotonic environment for cells. Therefore, the development of successful cryopreservation procedures is critically dependent on the method used for addition and removal of CPAs. In order to determine the optimal way to deliver CPAs to neurons, the membrane permeability parameters and osmotic tolerance limits of the cells were determined. The permeability coefficients for water and the cryoprotectants DMSO, propylene glycol, and ethylene glycol were determined using a fluorescence quenching method. Results of the osmotic tolerance experiments showed that at least 50% cell viability was maintained between 25 and 3000 mOsm. Initial feasibility studies of a method of adding CPAs to cells by gradually increasing the concentration (rather than a typical stepwise procedure) were also performed.
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