Evaluation of collagenase gold plus BP protease in isolating islets from human pancreata.

Selection of enzymes for optimal pancreas digestion is essential for successful human islet isolations. The aim of this study was to evaluate the efficacy and outcome of using Collagenase Gold plus BP protease (VitaCyte) (n = 8) by comparing it to two commercially available enzymes, Liberase MTF C/T (Roche) (n = 48) and Collagenase NB1/NP (Serva) (n = 15). The isolation outcomes were assessed by islet counting, viability, glucose-stimulated oxygen consumption rate (OCR), and successful graft-rate following transplantation in diabetic NOD scid mice. The pancreas donor characteristics were not significantly different between the tested enzyme groups regarding their BMI, pancreas weight, cold ischemia time (CIT) and HbA1c. The results show that digested tissue volume was not statistically significant between the VitaCyte enzyme (34.25 ± 5.4 mL) and the Roche enzyme (55.25 ± 3.42 mL, p = 0.073), however, this was significant with Serva enzyme (64.07 ± 7.95 mL, p = 0.020). Interestingly, the islet yields were not statistically different between all enzyme groups. Moreover, when islets were transplanted into NOD scid mice, the reversal rate of diabetes for the VitaCyte enzyme group was similar to all enzyme groups. In conclusion, the effectiveness of Collagenase Gold plus BP protease is comparable to the MTF C/T and the Collagenase NB1/NP enzymes; the low cost could facilitate the use of more pancreata for islet isolations.

Encompassing ATP, DNA, insulin, and protein content for quantification and assessment of human pancreatic islets.

Islet transplantation has made major progress to treat patients with type 1 diabetes. Islet mass and quality are critically important to ensure successful transplantation. Currently, islet status is evaluated using insulin secretion, oxygen consumption rate, or adenosine triphosphate (ATP) measurement. These parameters are evaluated independently and do not effectively predict islet status post-transplant. Therefore, assessing human pancreatic islets by encompassing ATP, DNA, insulin, and protein content from a single tissue sample would serve as a better predictor for islet status. In this study, a single step procedure for extracting ATP, DNA, insulin, and protein content from human pancreatic islets was described and the biomolecule contents were quantified. Additionally, different mathematical calculations integrating total ATP, DNA, insulin, and protein content were randomly tested under various conditions to predict islet status. The results demonstrated that the ATP assay was efficient and the biomolecules were effectively quantified. Furthermore, the mathematical formula we developed could be optimized to predict islet status. In conclusion, our results indicate a proof-of-concept that a simple logarithmic formula can predict overall islet status for various conditions when total islet ATP, DNA, insulin, and protein content are simultaneously assessed from a single tissue sample.

Fluorogenic Peptide Substrate for Quantification of Bacterial Enzyme Activities.

A novel peptide substrate (A G G P L G P P G P G G) was developed for quantifying the activities of bacterial enzymes using a highly sensitive Fluorescence Resonance Energy Transfer (FRET) based assay. The peptide substrate was cleaved by collagenase class I, II, Liberase MTF C/T, collagenase NB1, and thermolysin/neutral protease, which was significantly enhanced in the presence of CaCl2. However, the activities of these enzymes were significantly decreased in the presence of ZnSO4 or ZnCl2. Collagenase I, II, Liberase MTF C/T, thermolysin/neutral protease share similar cleavage sites, L↓G and P↓G. However, collagenase NB1 cleaves the peptide substrate at G↓P and P↓L, in addition to P↓G. The enzyme activity is pH dependent, within a range of 6.8 to 7.5, but was significantly diminished at pH 8.0. Interestingly, the peptide substrate was not cleaved by endogenous pancreatic protease such as trypsin, chymotrypsin, and elastase. In conclusion, the novel peptide substrate is collagenase, thermolysin/neutral protease specific and can be applied to quantify enzyme activities from different microbes. Furthermore, the assay can be used for fine-tuning reaction mixtures of various agents to enhance the overall activity of a cocktail of multiple enzymes and achieve optimal organ/tissue digestion, while protecting the integrity of the target cells.

Prophylactically Decontaminating Human Islet Product for Safe Clinical Application: Effective and Potent Method.

BACKGROUND: Transplanting pancreatic islets into recipients must be safe and effective to treat Type 1 diabetes. Islet quality and quantity are important, however, the final product must also be free from microbial contamination and low endotoxin levels. METHODS: This study explored a method to eliminate contamination in manufacturing islets for transplantation. A simple (single antibiotic, n=164) and refined (triple antimicrobial agents, n=279) pancreas decontaminating methods were used to test their effects on reducing the contamination rates in the islet final product. A total of 443 pancreata were processed for islet isolations. Three samples for microbial tests (Gram stain, aerobic, and anaerobic culture) were taken at pre-process (pancreas preservation), post-isolation, and post-culture. Endotoxin levels were measured only for islets considered for transplantation. RESULTS: Out of 443 pancreata used for islet isolation, 79 (17.8%) showed signs of contamination in pre-process samples; 10 (2.3%) were contaminated in both pre-process and in the final product (post-isolation and post-culture) samples. Contamination rates in which pre-process and final product samples were positive for contamination was significantly lower using the refined method (refined vs. simple method: 5% vs. 20.5%, p=0.045). Identical microbial species were present in both pre-process and in the final product. CONCLUSIONS: This study demonstrated that the refined method reduces the rate of contamination of the islet final product and is safe for clinical application. Moreover, it may be used as a standard method during human islet manufacturing facilitating the application of a biological license agreement from United States Food and Drug Administration.

The Choice of Enzyme for Human Pancreas Digestion is a Critical Factor for Increasing the Success of Islet Isolation.

BACKGROUND: We evaluated three commercially available enzymes for pancreatic digestion by comparing key parameters during the islet isolation process, as well as islet quality post-isolation. METHODS: Retrospectively compared and analyzed islet isolations from pancreata using three different enzyme groups: Liberase HI (n=63), Collagenase NB1/Neutral Protease (NP) (n=43), and Liberase Mammalian Tissue Free Collagenase/Thermolysin (MTF C/T) (n=115). A standardized islet isolation and purification method was used. Islet quality assessment was carried out using islet count, viability, in vitro glucose-stimulated insulin secretion (GSIS), glucose-stimulated oxygen consumption rate (ΔOCR), and in vivo transplantation model in mice. RESULTS: Donor characteristics were not significantly different among the three enzyme groups used in terms of age, sex, hospital stay duration, cause of death, body mass index (BMI), hemoglobin A1c (HbA1c), cold ischemia time (CIT), and pancreas weight. Digestion efficacy (percentage of digested tissue by weight) was significantly higher in the Liberase MTF C/T group (73.5 ± 1.5 %) when compared to the Liberase HI group (63.6 ± 2.3 %) (p<0.001) and the Collagenase NB1/NP group (61.7 ± 2.9%) (p<0.001). The stimulation index for GSIS was significantly higher in the Liberase MTF C/T group (5.3 ± 0.5) as compared to the Liberase HI (2.9 ± 0.2) (p<0.0001) and the Collagenase NB1/NP (3.6 ± 2.9) (p=0.012) groups. Furthermore, the Liberase MTF C/T enzymes showed the highest success rate of transplantation in diabetic NOD Scid mice (65%), which was significantly higher than the Liberase HI (42%, p=0.001) and the Collagenase NB1/NP enzymes (41%, p<0.001). CONCLUSIONS: Liberase MTF C/T is superior to Liberase HI and Collagenase NB1/NP in terms of digestion efficacy and glucose-stimulated insulin secretion in vitro. Moreover, Liberase MTF C/T had a significantly higher success rate of transplantation in diabetic NOD Scid mice compared to Liberase HI and Collagenase NB1/NP enzymes.

Human Pancreatic Islets Isolated From Donors With Elevated HbA1c Levels: Islet Yield and Graft Efficacy.

UNLABELLED: The aim of this study was to investigate the effects of elevated donor HbA1c levels (type 2 diabetes, T2D) on the islet yield and functionality postisolation. In this retrospective analysis, donors for islet isolations were classified into two groups: T2D group (HbA1c ≥ 6.5%, n = 18) and normal group (HbA1c < 6.5%, n = 308). Optimum pancreas digestion time (switch time) was significantly higher in the T2D group compared to the normal group (13.7 ± 1.2 vs. 11.7 ± 0.1 min, respectively, p = 0.005). Islet yields were significantly lower in the T2D group compared to the control (T2D vs. control): islet equivalent (IEQ)/g (prepurification 2,318 ± 195 vs. 3,713 ± 114, p = 0.003; postpurification 1,735 ± 175 vs. 2,663 ± 89, p = 0.013) and islet particle number (IPN)/g (prepurification, 2,519 ± 336 vs. 4,433 ± 143, p = 0.001; postpurification, 1,760 ± 229 vs. 2,715 ± 85, p = 0.007). Islets from T2D pancreata had significantly lower viability (T2D vs. CONTROL: 91.9 ± 1.6 vs. 94.4 ± 0.3%, p = 0.004) and decreased oxygen consumption rate (ΔOCR) (T2D vs. CONTROL: 0.09 ± 0.01 and 0.21 ± 0.03 nmol O2 100 islets(-1) min(-1), p = 0.049). The islets isolated from T2D donor pancreata reversed diabetes in NOD-SCID mice in 9% (2/22) compared to islets from control donor pancreata, which reversed diabetes in 67% (175/260, p < 0.001). In conclusion, this study demonstrates that elevated HbA1c (≥ 6.5%) is associated with impairment of islet function and lower islet yield; however, these islets could not be suitable for clinical applications.

Sodium levels of human pancreatic donors are a critical factor for determination of islet efficacy and survival.

Organs from hypernatremia (elevated Na+) donors when used for transplantation have had dismal outcomes. However, islet isolation from hypernatremic donors for both transplantation and research applications has not yet been investigated. A retrospective analysis of in vivo and in vitro islet function studies was performed on islets isolated from hypernatremic (serum sodium levels≥160 meq/l) and normal control (serum sodium levels≤155 meq/l) donors. Twelve isolations from 32 hypernatremic and 53 isolations from 222 normal donors were randomly transplanted into diabetic NOD Scid mice. Sodium levels upon pancreas procurement were significantly elevated in the hypernatremia group (163.5±0.6 meq/l) compared with the normal control group (145.9±0.4 meq/l) (P<0.001). The postculture islet recovery rate was significantly lower in the hypernatremia (59.1±3.8%) group compared with the normal (73.6±1.8%) group (P=0.005). The duration of hypernatremia was inversely correlated with the recovery rate (r2=0.370, P<0.001). Furthermore, the percentage of successful graft function when transplanted into diabetic NOD Scid mice was significantly lower in the hypernatremia (42%) group compared with the normal control (85%) group (P<0.001). The ability to predict islet graft function posttransplantation using donor sodium levels and duration of hypernatremia was significant (ROC analysis, P=0.022 and 0.042, respectively). In conclusion, duration of donor hypernatremia is associated with reduced islet recovery postculture. The efficacy of islets from hypernatremia donors diminished when transplanted into diabetic recipients.