(485bi) The Effects of Cryopreservation On a Tissue Engineered Pancreatic Substitute

Within the field of Tissue Engineering, long-term preservation is a critical step in bringing constructs from the benchtop to the clinic. Cryopreservation is the most often used method of preservation and can be divided into two main types: conventional freezing and vitrification. As its name suggests, conventional freezing allows ice formation by using low concentrations of cryoprotectants (CPAs) during the preservation process. Vitrification utilizes high concentrations of CPAs and rapid cooling and warming profiles to achieve a vitreous, or glassy, state. Optimal cryopreservation would maintain all components of the construct, including the cells and the biomaterial. The ice that is formed during conventional freezing occurs preferentially in the extracellular matrix, or biomaterial, and may be damaging to the integrity of the biomaterial. Vitrification eliminates ice but introduces high levels of CPAs that may be cytotoxic to the cells. In this study, the effects of the different types of cryopreservation are investigated using a pancreatic substitute consisting of murine insulinomas encapsulated in calcium alginate/poly-L-lysine/alginate (APA) beads. The hypothesis is that optimized vitrification protocols maintain sufficient post-thaw viability and function while preserving the biomaterial integrity better than conventional freezing.

In vitrification, the high concentrations of CPAs can be detrimental to the cells due to excessive osmotic excursions or cytotoxicity. In order to minimize osmotic excursions, a previously developed mathematical model was used to determine the addition and removal protocols for vitrification (Mukherjee 2008). CPA addition and removal studies were conducted to find the most promising vitrification solutions. These solutions were then used to vitrify APA beads. Post-thaw, the viability and function of the vitrified encapsulated cells were compared to those in fresh and conventionally frozen capsules. Viable cell number was determined using alamarBlue® and insulin secretory function by subjecting the capsules to a step-change in glucose and measuring by ELISA the amount of insulin released in response to the change. Biomaterial integrity was assessed via histology and mechanical testing. Maintaining the biomaterial integrity is critical as the murine insulinomas within the capsule are proliferating cells and can be considered a regenerating portion of the substitute, whereas the biomaterial portion of the construct cannot repair or regenerate. Studies are currently under way to determine the in vivo immune acceptance and efficacy of cryopreserved capsules implanted in a small animal model. This study investigates the effects of cryopreservation, specifically vitrification and conventional freezing, on the biomaterial, cells within the biomaterial and the performance of the tissue substitute in an animal model.