Cryopreservation effects on the in vitro and in vivo function of a model pancreatic substitute

The use of encapsulated cell systems is widespread in the field of Tissue Engineering. They are primarily used to deliver cells that secrete therapeutic molecules. More specifically, encapsulated systems have shown promise in the delivery of insulin-secreting cells or cell clusters to revert diabetes. Although often overlooked, cryopreservation is critical in bringing these constructs from the laboratory to the clinic. Two main types of cryopreservation are currently being investigated for the preservation of tissue-engineered constructs: conventional freezing and vitrification. Conventional freezing is often used for preserving cell suspensions and uses low concentrations of cryoprotective agents (CPAs), slow cooling and rapid warming. Vitrification, or ice-free cryopreservation, utilizes high concentrations of CPAs paired with rapid cooling and warming to achieve a vitreous, or glassy, state. Our overall goal was to determine the effects of both types of cryopreservation on the in vitro and in vivo performance of an encapsulated cell system, a model tissue-engineered pancreatic substitute. The specific aims in this thesis were (1) to model CPA delivery and removal to encapsulated cells, and their effects on cell viability; (2) to characterize in vitro the effects of cryopreservation protocols on cell viability and the biomaterial structure and function of a pancreatic substitute; (3) to characterize in vivo the effects of cryopreservation on the biocompatibility and efficacy of a pancreatic substitute. This research addresses the systematic design of vitrification protocols and how these protocols and conventional freezing affect a tissue-engineered construct. Our results indicate that temperature of exposure is the most critical parameter for the proper design of vitrification protocols. Overexposure is another concern as it leads to a decrease in viable cell number. The use of a mathematical model is critical for the design of addition and removal protocols to ensure CPA equilibration and minimize CPA exposure. Results from in vitro studies indicate that both vitrification and conventionally frozen perform comparably to fresh. However, in vivo studies reveal that vitrification performs worse than both conventionally frozen and fresh beads. With adjustments, it may be possible to improve the performance of the vitrified beads. Nevertheless, for this encapsulated system, conventional freezing is the better method and allows successful cryopreservation.