Using the specified culture methods, non-islet pancreas cells can generate cell clusters resembling islets. These ICCs, obtained from fractions of the pancreas that are otherwise discarded, continue to differentiate after transplantation to become mature islets.

Background Assays for assessing human islet cell quality which provide results prior to transplantation would be very beneficial to improving outcomes for islet transplantation therapy. Parameters such as percent beta cell apoptosis and cell composition are found to vary markedly between different islet preparations, and may serve as markers of islet quality. We have developed fluorescence-based assays using laser scanning cytometry (LSC) for assessing beta cell apoptosis and islet cell composition on serial sections of intact isolated islets. Methods Isolated human islets were fixed in formalin and embedded in paraffin. Serial sections were immunostained for the pancreatic hormones, acinar and ductal cell markers. DNA fragmentation was used to label apoptotic cells. Stained cells were quantified using an iCys laser scanning cytometer. Results Islet preparations from 102 human pancreatic islet isolations were analyzed. For the whole set of islet preparations we found a mean islet cell composition of 54.5±1.2% insulin positive; 33.9±1.2% glucagon; 12.1±0.7% somatostatin and 1.5±0.2% pancreatic polypeptide positive cells. The apoptotic beta cells were 2.85±0.4% with a range of 0.27% to 18.3%. The percentage of apoptotic beta cells correlated well (p<0.0001, n=59) with results obtained in vivo by transplantation of the corresponding islets in diabetic NODscid mice. Conclusions The analysis of whole, non-dissociated islets for cell composition and beta cell apoptosis using LSC is giving reliable and reproducible results and could be done both before islet transplantation, as well as on preserved cell blocks at any future time. Thus, they can be a powerful tool for islet quality assessment.

The activation of p38 mitogen-activated protein kinase (MAPK) has been shown to cause ischemia/reperfusion injury of several organs used for transplantation and also to play a significant role in primary islet graft nonfunction. Activation of p38 MAPK may also occur during islet cryopreservation and thawing. In this study, a p38 MAPK inhibitor (p38IH) was applied to human islet cryopreservation to improve islet yield and quality after thawing. Under serum-free conditions, human islets were cryopreserved, thawed and cultured using our standard procedures. Three types of solutions were tested: conventional RPMI1640 medium (RPMI), a newly developed islet cryopreservation solution (ICS), and ICS supplemented with a p38IH, SD-282 (ICS-p38IH). Activation or inhibition of p38 MAPK was demonstrated by the diminished phosphorylation of HSP27 substrate. Islet recovery on day 2 after thawing was highest with ICS-p38IH and islet viability was not significantly different in the three groups. b Cell numbers and function were the highest in islets cryopreserved with ICS-p38IH. Glucosestimulated human C-peptide levels were 86% of that of the nonfrozen islets when measured 4 weeks after transplantation into NODscid mice. This improvement may provide an opportunity to establish islet banks and allow the use of cryopreserved islets for clinical transplantation.

Prior to transplantation into individuals with type 1 diabetes, in vitro assays are used to evaluate the quality, function and survival of isolated human islets. In addition to the assessments of these parameters in islet, they can be evaluated by multiparametric morphological scoring (0–10 points) and grading (A, B, C, D, and F) based on islet characteristics (shape, border, integrity, single cells, and diameter). However, correlation between the multiparametric assessment and transplantation outcome has not been fully elucidated. In this study, 55 human islet isolations were scored using this multiparametric assessment. The results were correlated with outcomes after transplantation into immunodeficient diabetic mice. In addition, the multiparametric assessment was compared with oxygen consumption rate of isolated islets as a potential prediction factor for successful transplantations. All islet batches were assessed and found to score: 9 points ( n = 18, Grade A), 8 points ( n = 19, Grade B), and 7 points ( n = 18, Grade B). Islets that scored 9 (Grade A), scored 8 (Grade B) and scored 7 (Grade B) were transplanted into NOD/SCID mice and reversed diabetes in 81.2%, 59.4%, and 33.3% of animals, respectively ( P < 0.0001). Islet scoring and grading correlated well with glycemic control post-transplantation ( P < 0.0001) and reversal rate of diabetes ( P < 0.05). Notably, islet scoring and grading showed stronger correlation with transplantation outcome compared to oxygen consumption rate. Taken together, a multiparametric assessment of isolated human islets was highly predictive of transplantation outcome in diabetic mice.

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