Date of Award

Spring 2022

Project Type

Dissertation

Program or Major

Microbiology

Degree Name

Doctor of Philosophy

First Advisor

Krisztina Varga

Second Advisor

Feixia Chu

Third Advisor

Sherine Elsawa

Abstract

Antifreeze proteins (AFPs) are specialized evolutionary adaptations by certain cold climate organisms, including fish, plants, fungi, bacteria, and insects, to prevent cryo-injury. AFPs contribute to freeze resistance or tolerance by binding to the surface of ice crystals and preventing their growth, as well as inhibiting ice recrystallization (i.e., small crystals restructuring into large crystals) during large temperature fluctuations and constant sub-zero temperatures. AFPs have potential applications in cryopreservation and in the development of cryo-functional materials for medicine, the food industry, and agriculture. We investigated the ability of an antifreeze protein from the desert beetle Anatolica polita, ApAFP752, to confer cryoprotection in human embryonic kidney (HEK) 293T cells. HEK 293T cells were transfected with ApAFP752, and/or ApAFP752 was added to extracellular media prior to freezing at cryogenic temperature. AFP was assessed for its cryoprotective effects both intra- and extracellulary and both simultaneously at different concentrations with and without DMSO at different concentrations. After freezing and thawing, comparisons were made to DMSO or medium alone. ApAFP752 significantly improved cryopreserved cell survival when used with DMSO intracellularly. Extracellular ApAFP752 also significantly improved cryopreserved cell survival when included in the DMSO freezing medium. Intra- and extracellular ApAFP752 used together demonstrated the most significantly increased cryoprotection compared to DMSO alone at all DMSO concentrations. Together, these findings present potential methods for using an insect antifreeze protein to confer cryoprotection and improve the viability of cryopreserved mammalian cells.

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