Implication of Mitochondrial Cytoprotection in Human Islet Isolation and Transplantation

Islet transplantation is a promising therapy for type 1 diabetes mellitus; however, success rates in achieving both short- and long-term insulin independence are not consistent, due in part to inconsistent islet quality and quantity caused by the complex nature and multistep process of islet isolation and transplantation. Since the introduction of the Edmonton Protocol in 2000, more attention has been placed on preserving mitochondrial function as increasing evidences suggest that impaired mitochondrial integrity can adversely affect clinical outcomes. Some recent studies have demonstrated that it is possible to achieve islet cytoprotection by maintaining mitochondrial function and subsequently to improve islet transplantation outcomes. However, the benefits of mitoprotection in many cases are controversial and the underlying mechanisms are unclear. This article summarizes the recent progress associated with mitochondrial cytoprotection in each step of the islet isolation and transplantation process, as well as islet potency and viability assays based on the measurement of mitochondrial integrity. In addition, we briefly discuss immunosuppression side effects on islet graft function and how transplant site selection affects islet engraftment and clinical outcomes

Implication of Mitochondrial Cytoprotection in Human Islet Isolation and Transplantation

Islet transplantation is a promising therapy for type 1 diabetes mellitus; however, success rates in achieving both short-and longterm insulin independence are not consistent, due in part to inconsistent islet quality and quantity caused by the complex nature and multistep process of islet isolation and transplantation. Since the introduction of the Edmonton Protocol in 2000, more attention has been placed on preserving mitochondrial function as increasing evidences suggest that impaired mitochondrial integrity can adversely affect clinical outcomes. Some recent studies have demonstrated that it is possible to achieve islet cytoprotection by maintaining mitochondrial function and subsequently to improve islet transplantation outcomes. However, the benefits of mitoprotection in many cases are controversial and the underlying mechanisms are unclear. This article summarizes the recent progress associated with mitochondrial cytoprotection in each step of the islet isolation and transplantation process, as well as islet potency and viability assays based on the measurement of mitochondrial integrity. In addition, we briefly discuss immunosuppression side effects on islet graft function and how transplant site selection affects islet engraftment and clinical outcomes

Islet Preconditioning via Multimodal Microfluidic Modulation of Intermittent Hypoxia

Simultaneous stimulation of ex vivo pancreatic islets with dynamic oxygen and glucose is a critical technique for studying how hypoxia alters glucose-stimulated response, especially in transplant environments. Standard techniques using a hypoxic chamber cannot provide both oxygen and glucose modulations, while monitoring stimulus-secretion coupling factors in real-time. Using novel microfluidic device with integrated glucose and oxygen modulations, we quantified hypoxic impairment of islet response by calcium influx, mitochondrial potentials, and insulin secretion. Glucose-induced calcium response magnitude and phase were suppressed by hypoxia, while mitochondrial hyperpolarization and insulin secretion decreased in coordination. More importantly, hypoxic response was improved by preconditioning islets to intermittent hypoxia (IH, 1 min/1 min 5–21% cycling for 1 h), translating to improved insulin secretion. Moreover, blocking mitochondrial K_ATP channels removed preconditioning benefits of IH, similar to mechanisms in preconditioned cardiomyocytes. Additionally, the multimodal device can be applied to a variety of dynamic oxygen-metabolic studies in other ex vivo tissues

Histidine-Tryptophan-Ketoglutarate and University of Wisconsin Solution Demonstrate Equal Effectiveness in the Preservation of Human Pancreata Intended for Islet Isolation: A Large-Scale, Single-Center Experience

We previously reported a small-scale study on the efficacy of histidine-tryptophan-ketoglutarate (HTK) solution versus University of Wisconsin (UW) solution on pancreas preservation for islet isolation. In this large-scale, retrospective analysis (n=252), we extend our initial description of the impact of HTK on islet isolation outcomes and include pancreatic digestion efficacy, purification outcomes, and islet size distribution. Multivariable linear regression analysis, adjusted for donor age, sex, BMI, cold ischemia time, and enzyme, demonstrated similar results for the HTK group (n=95) and the UW group (n=157), including postpurification islet yields (HTK: 289,702 IEQ vs. UW: 283,036 IEQ; p=0.76), percentage of digested pancreatic tissue (HTK: 66.9% vs. UW: 64.1%; p=0.18), and islet loss from postdigestion to postpurification (HTK: 24,972 IEQ vs. MAT: 39,551 IEQ; p=0.38). Changes in islet size between the postdigestion and postpurification stages were comparable within each islet size category for HTK and UW (p=0.14-0.99). Tissue volume distribution across purification fractions and islet purity in the top fractions were similar between the groups; however, the HTK group had significantly higher islet purity in the middle fractions (p=0.003-0.008). Islet viability and stimulation indices were also similar between the HTK and the UW groups. In addition, we analyzed a small sample of patients transplanted either with HTK (n=7) or UW (n=8) preserved islets and found no significant differences in posttransplant HbA(1e), beta-score, and frequency of insulin independence. This study demonstrates that HTK and UW solutions offer comparable pancreas preservation for islet transplantation. More in vivo islet outcome data are needed for a complete analysis of the effects of HTK on islet transplantation

Implication of Mitochondrial Cytoprotection in Human Islet Isolation and Transplantation

Islet transplantation is a promising therapy for type 1 diabetes mellitus; however, success rates in achieving both short- and longterm insulin independence are not consistent, due in part to inconsistent islet quality and quantity caused by the complex nature and multistep process of islet isolation and transplantation. Since the introduction of the Edmonton Protocol in 2000, more attention has been placed on preserving mitochondrial function as increasing evidences suggest that impaired mitochondrial integrity can adversely affect clinical outcomes. Some recent studies have demonstrated that it is possible to achieve islet cytoprotection by maintaining mitochondrial function and subsequently to improve islet transplantation outcomes. However, the benefits of mitoprotection in many cases are controversial and the underlying mechanisms are unclear. This article summarizes the recent progress associated with mitochondrial cytoprotection in each step of the islet isolation and transplantation process, as well as islet potency and viability assays based on the measurement of mitochondrial integrity. In addition, we briefly discuss immunosuppression side effects on islet graft function and how transplant site selection affects islet engraftment and clinical outcomes

Highly Purified versus Filtered Crude Collagenase: Comparable Human Islet Isolation Outcomes

This study was designed to retrospectively compare the impact of crude Sigma V collagenase (Sigma V, n=52) with high-purified Serva NB1 collagenase (Serva NB1, n=42) on human islet isolation outcomes. A three-step filtration was applied to the crude Sigma V to eliminate endotoxin contamination and impurities; in addition this process was used as a lot prescreening tool. Isolation outcomes were determined by digestion efficacy, islet yields, purity, viability, glucose-stimulated insulin release, and endotoxin content. The difference of the digestion efficacy between Sigma V and Serva NB1 was very small, however, statistically significant (Sigma V: 64.71% vs. Serva NB1: 69.71%, p <0.05). Islet yields were similar (Sigma V: 23422.58 vs. Serva NB1: 271097 IEq, p>0.05) between groups. No significant purity differences were observed for fractions with purities greater than 75%. Viability (Sigma V: 93.3% vs. Serva NB1: 94.8%, p>0.05), and stimulation indexes (Sigma V: 3.41 vs. Serva NB1: 2.74, p>0.05) were similar between the two groups. The impact of cold ischemia and age on the isolation outcome in the Sigma V group was comparable to the Serva NB1 group. However, we were intrigued to find that the endotoxin content of the final products in the filtered Sigma V group was significantly less than that in the high-purified Serva NB1 group (0.022 EU/ml vs. 0.052 EU/ml, p<0.05). In addition, we found that there was minimal lot to lot variation after filtration and no significant loss of enzymatic activity. These finding indicate that using this or other crude enzyme blends for research pancreata is warranted to reduce isolation costs and increase the amount of islets available for critical islet research. These findings also validate the need for a systematic enzyme analysis to resolve these inconsistencies in overall enzyme quality once and for all