Background: Pancreatic islets are subjected to stresses throughout isolation procedures that account for considerable variation in quality and yield. Multiparameter methods are needed to monitor islet quality in terms of damage, viability, and potency. We have demonstrated that miR-375 is a highly sensitive and reliable marker for acute islet cell damage in islet transplant patient plasma samples. Here, we report a method to monitor islet damage by analyzing miRNA-375 content from islet-free supernatant samples collected at multiple stages throughout the islet isolation process up to the final product.

Methods: Human pancreata were obtained from cadaveric donors and chronic pancreatitis patients. Islet isolation procedures were performed using a modified Ricordi method. C-peptide and miR-375 and miR-200c concentrations were measured by ELISA and QPCR respectively, in supernatant samples collected at each stage of islet isolation procedures, including enzyme perfusion, pancreas digestion, tissue recombination, and post purification.

Results: MiRNA-375 and miR-200c were detected with high resolution within 10-minute intervals during pancreatic digestion compared to C-peptide. In contrast, increasing levels of C-peptide were detected in later stages of isolation procedures, due in part by secretion of C-peptide from functional islets rather than islet damage. Most miRNA-375 detection profiles among islet isolations were bimodal with higher peak damage measured during pancreatic digestion stages and post-purification than during islet recombination procedures (139.6 ± 33.2 vs 42.4 ± 51.4 vs 146.3 ± 5.5 fmol). Maximal damage was consistently observed during 10-20 minof enzymatic digestion. The level of miRNA-375 from supernatants provided higher (10⁶-10⁷ fold) resolution detection than miR-200c.

Conclusion: MicroRNA-375 can be used to detect beta-cell damage during islet isolation procedures with high selectivity and sensitivity compared to miRNA-200c and C-peptide profiling which may be difficult to distinguish between islet function versus cell damage. , This method can potentially be used to monitor clinical isolation procedures to identify areas for improving islet quality and quantity during processing.

CITATION INFORMATION: Prathab Balaji S, Kanak M, Yoshimatsu G, Chang C, Levy M, Lawrence M, Kim P, Onaca N, Naziruddin B. High Resolution Detection of Human Pancreatic Islet Damage During and After Isolation Procedure Using MicroRNAs. Am J Transplant. 2016;16 (suppl 3).

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