In this study, we investigated the use of a novel oxygen biosensor system to detect changes in oxygen consumption rates (OCRs) by islets in response to glucose. Islets from non-human primate and human pancreata were seeded into an oxygen biosensor system microplate and exposed to basal (2·8 or 5·6 mM) or high (16·7 or 33·3 mM) glucose over either a long-term or a short-term culture. Our data clearly demonstrated that non-human primate islets cultured in high glucose conditions exhibited significant increases in OCRs over a 168 h extended culture period (P<0·05), which indicates an accelerated rate of -cell metabolism triggered by glucose over time. Significant increases in OCRs (P<0·01) were also attained in both non-human primate and human islets exposed to high glucose conditions in a 120 min short-term incubation period. OCRs exhibited by human islets exposed to different glucose concentrations correlated with insulin secretion (r 2 =0·7681, P<0·01). Moreover, the OCR stimulation index (i.e. OCR at high glucose/OCR at basal glucose) was significantly greater in human islets displaying high viabilities as opposed to islets exhibiting low viabilities (P<0·05). Together these data demonstrate that this novel oxygen biosensor system documents significant increases in islet oxygen consumption upon acute and chronic exposure to high glucose concentrations. Importantly, this methodology rapidly and robustly detects changes in OCRs by islets in response to high glucose stimulation that correlate well with the metabolic activities and functional viability of islets and clearly delineates significant differences in OCR stimulation index between high and low viability human islets, and therefore may prove to be an effective approach for quickly assessing the functional viability of islets prior to transplantation.
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.
Despite improvements and recent attempts to standardize techniques to isolate islets from human donor pancreata, there still exists the problem of consistently recovering sufficient quantities of high quality islets. Moreover, achieving consistent recoveries of high numbers of good quality islets becomes even more challenging from marginal grade human donor pancreata with prolonged cold ischemic times. In this study, we investigate whether addition of Pefabloc SC, a serine protease inhibitor, in combination with Pulmozyme, a recombinant human DNase I, to Liberase HI improves islet isolation outcome from marginal grade human donor pancreata (cold ischemic time > 12 h). Twenty-three marginal grade human donor pancreata were randomly digested using four different enzyme preparations: (1) Liberase alone (n = 6), (2) +Pefabloc (n = 7), (3) +Pefabloc/Pulmozyme (n = 5), and (4) +Pulmozyme (n = 5). Overall, there were no significant differences in donor age, body mass index (BMI), pancreas weight, and cold ischemic time. After purification, significantly higher islet yields (3,281 +/- 590 IE/g) were obtained with the Pefabloc/Pulmozyme group as compared to the Liberase alone (1,615 +/- 305 IE/g) or the Pefabloc group (1,255 +/- 261 IE/g) (P < 0.05). Significant improvements in islet viability were also noted from the Pefabloc/Pulmozyme group (87.3 +/- 4.4%) as opposed to islets isolated from the Pefabloc group (75.2 +/- 3.9%) (P < 0.05). No significant differences in insulin secretory response to glucose stimulation among the four groups were observed, which indicates that the addition of Pefabloc and/or Pulmozyme does not have a detrimental effect on the functionality of islets. It is concluded that the addition of Pefabloc in combination with Pulmozyme to the Liberse HI significantly improves islet isolation outcome and potentially impacts the viability and morphology of the islets obtained from marginal grade human donor pancreata with prolonged cold ischemic times.
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