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

Abstract

The effects of two types of cryopreservation, conventional freezing and vitrification, on the in vitro and in vivo function of a pancreatic substitute were investigated. Conventional freezing uses low concentrations of cryoprotective agents (CPAs), slow cooling and rapid warming and allows ice formation. Vitrification requires high concentrations of CPAs coupled with rapid cooling and warming to achieve a vitreous, or ice-free, state. A previously published mathematical model describing the mass transfer of CPAs through the alginate matrix of the substitute and the cell membrane was expanded to incorporate heat transfer as well as CPA cytotoxicity. Our results indicate that temperature of exposure is the most critical parameter for the proper design of CPA addition and removal protocols. The use of a mathematical model is critical to ensure CPA equilibration and minimize CPA exposure. Properly designed CPA addition and removal protocols were used for vitrification. The effects of cryopreservation on the biomaterial and the cellular function of a pancreatic substitute consisting of murine insulinomas encapsulated in calcium alginate/poly-L-lysine/alginate beads were assessed. In vitro results indicate that both vitrification and conventionally frozen perform comparably to fresh. However, in vivo studies reveal that vitrified beads perform 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 pancreatic substitute, conventional freezing is the better method and allows successful cryopreservation.