According to current estimates from the American Diabetes Association, there are approximately 16 million diabetics in the U.S. in 2000, with 600,000 to 800,000 afflicted with Type I insulin-dependent diabetes (IDD). Tissue engineered pancreatic substitutes have the potential of providing a more physiologic and less invasive treatment of IDD relative to daily insulin injections. The US market for tissue-engineered bioartificial pancreas is currently estimated to be about $2.5 billion. The market for pancreas regeneration products is predicted to expand at a 6.8% compound rate for the next 10 years due to increases in the incidence of diabetes. A significant hurdle for deployment of such constructs is an effective means for long-term storage and transportation to permit worldwide product distribution. Ice formation within tissue matrices causes matrix damage in three-dimensional structures during "traditional" cryopreservation by freezing. The purpose of this proposal is to evaluate an ice-free cryopreservation method, vitrification, using mouse insulinomas encapsulated in calcium alginate/poly-L-lysine as a model system. Vitrification has previously proven to be effective for storage of complex tissues such as blood vessels and articular cartilage. In this proposal the effects of vitrification and a "traditional" freezing method, employing dimethylsulfoxide and control rate cooling, upon encapsulated cell viability and functions will be investigated. Test methods will include cell viability assays, insulin secretion in response to glucose, and morphological evaluation of capsule integrity. A significant part of these studies will consist of determining the penetration rates of cryoprotectants into encapsulated cell systems by nuclear magnetic resonance (NMR) and the effects of such exposure upon cell viability and functions. These observations will permit optimization of the existing procedure to be used for the vitrification of encapsulated cells, incorporating a novel synthetic ice blocking compound to promote vitrification at lower concentrations of cryoprotectants, thereby, reducing osmotic stresses and cytotoxicity. If vitrification is shown to be effective for storage of encapsulated cell constructs, these studies will be extended in a Phase II SBlR proposal to include in vivo testing of the mouse insulinoma cell model as well as alternative encapsulated cell systems.