Background. Current techniques for isolating islets require that pancreata stored with University of Wisconsin solution (UW) are processed within 12 hours of cold storage. In this study, we hypothesized that the two-layer method (TLM) could extend the acceptable preservation period of pancreata before islet isolation and increase islet yields. Methods. In the first experimental set, eight pancreata were maintained for an average of 8.3±1.2 hours in UW and transferred into the TLM for an additional 14.3±1.1 hours for a total cold ischemic period of 22.6±1.6 hours (prolonged TLM). Four pancreata were maintained as a control group in UW alone for a total of 21.3±2.0 hours. In the second experimental set, six pancreata were maintained for an average of 6.4±1.8 hours in UW followed by 4.8±0.8 hours with the TLM for a total preservation time of 11.3±2.5 hours (short TLM). The control organs for the short TLM group were stored for an average of 9.5±1.3 hours in UW alone. Islets were isolated and evaluated according to the Edmonton protocol. Results. Between each group of the two experimental sets, there was no significant difference in donor-related factors (i.e. gender, age, body mass index [BMI], etc.). The TLM as compared with UW preservation resulted in a significant increase in islet yields postpurification for both short (3,353±394 islet equivalents [IE] vs. 2,027±415 IE; mean±SEM) and prolonged (2,404±503 IE vs. 514±180 IE) periods of storage. Furthermore, islet yields after prolonged storage with the TLM were not significantly different from organs maintained for only a short period with UW (P=0.17). The quality of islets as assessed by size, postculture viability, survival rates, insulin content, and insulin secretion were similar for each of the four groups. Conclusion. In comparison with UW organ preservation, exposure of pancreata to the TLM result in greater islet yields and extended preservation times.