In comparison with UW organ preservation, exposure of pancreata to the TLM result in greater islet yields and extended preservation times.

Previous reports indicate that the two-layer method (TLM) of human pancreas preservation is superior to University of Wisconsin solution (UW) when pancreata are preserved for extended periods (i.e., >24 h) prior to islet isolation. In this study, the efficacy of using the TLM for preserving pancreata for short periods (i.e., <13 h) was evaluated using both nonhuman primate and human pancreata preserved with a TLM kit precharged with oxygen. An oxygen precharged TLM (static TLM) was established and compared with the original TLM with continuous oxygen supply. For the static TLM, the perfluorochemical was fully oxygenated and the oxygen supply removed prior to pancreas preservation. In the primate model, pancreata were preserved by the static TLM, the original TLM, and UW for 5 h prior to islet isolation. In the human model, pancreata were preserved with the static TLM or the original TLM or UW for 4–13 h. Both primate and human pancreata were processed by intraductal collagenase injection and digestion followed by continuous density gradient purification to isolate islets. Islets were assessed for islet yield, purity, viability, and in vitro functionality. In the primate model, islet yield, viability, and in vitro functionality were significantly improved by both the static TLM and the original TLM with similar results. Postculture islet yields were 23,877 ± 3619 IE/g in the static TLM, 21,895 ± 3742 IE/g in the original TLM, and 6773 ± 735 IE/g in UW. In the human model, both the static TLM and the original TLM significantly increased islet yield compared with UW with postculture islet yields of 2659 ± 549 IE/g in the static TLM, 2244 ± 557 IE/g in the original TLM, and 1293 ± 451 IE/g in UW. Nonhuman primate and human pancreata stored in the static TLM, immediately upon procurement, yield isolated islets of a substantially higher quantity than when pancreata are stored in UW. Thus, the use of the static TLM should replace the use of UW for storage of pancreata during transport prior to islet isolation.

Investigations indicate that an extract of green tea, polyphenol, can significantly increase the culture survival rate of rat islets without deteriorating their functionality. In this study, we examined the effect of adding polyphenol to islets isolated from human pancreata and nonhuman primate pancreata. Islets were isolated from human pancreata that did not meet criteria for clinical transplantation (n = 6) and from nonhuman primate pancreata (n = 5). The islets were cultured in CMRL-1066 + 10% FCS with the addition of 0, 30, 60, 125, 250, or 500 µg/ml of polyphenol. After 24 or 48 h of culture, islet yield, viability, purity, morphology, and stimulation index was assessed. RT-PCR and Western blot analysis were also performed to assess the expression levels of the apoptotic related genes, Bcl-2 and BAX. After 24 h of culture, islet yields were significantly higher in cultures supplemented with 30-250 µg/ml of polyphenol than in cultures without polyphenol. After 48 h of culture, significant differences in islet numbers were observed with polyphenol concentrations of 125 µg/ml ( p < 0.01) and 250 µg/ml ( p < 0.01). However, no significant differences were noted in islet viability, purity, morphology, and stimulation index at each time point with or without polyphenol. RT-PCR and Western blot analysis of the islets indicated that Bcl-2 levels increased by 2.5-fold and BAX levels decreased by twofold in cultures supplemented with polyphenol. This resulted in BAX/Bcl-2 ratios that were lower in polyphenol-supplemented cultures than with control cultures. Polyphenol increases culture recovery rates by precluding islet apoptosis.

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