Multi-lot analysis of custom collagenase enzyme blend in human islet isolations.

INTRODUCTION: Variability currently in Liberase HI from lot to lot limits the ability to effectively isolate islets with consistency. Roche Diagnostics Inc (Indianapolis, Ind, USA) has developed a Custom Collagenase enzyme blend in hopes that producing collagenase II and I and thermolysin separately will eliminate variability. In this study we examined the variability in Custom Collagenase lots in respect to isolation results and isolation success rates and compared those to Liberase HI. METHODS: We retrospectively analyzed records from 68 islet isolations where either Liberase HI (lot A: n = 23, Lot B: n = 20) or Custom Collagenase blend (Lot C: n = 10, Lot D: n = 15) was employed. Human islets were isolated from cadaveric pancreata using standardized methods performed in a controlled islet isolation facility. RESULTS: Analysis of Liberase HI and Custom Collagenase using Student t test showed no difference between the two groups. Comparison of the two Custom Collagenase lots using the t test showed a statistical difference between undigested pancreas weight and pancreas digestion times. Using chi-square test, no statistical significance was found in isolation success rates from lot to lot. CONCLUSION: Although the Custom Collagenase blend is comparable to Liberase HI in its ability to isolate human islets, variability still exists from lot to lot when used conventionally as Liberase HI is. The ability to predetermine doses is beneficial, and as techniques to manipulate the activity levels prior to isolations improve so to will the enzymes' ability to isolate islets on a consistent basis.

The standardization of pancreatic donors for islet isolation.

INTRODUCTION: Islet transplantation has proven to be a successful treatment for insulin-dependent diabetes mellitus (IDDM). The aim of this study was to establish an algorithm by which the combination of the donor quality and pancreas quality was given a numerical score from 0 to 100 for use in determining the quality of a pancreas for islet isolation. METHODS: In this study we retrospectively analyzed 326 pancreata and the outcomes of their respective isolations. Specific donor variables and physical characteristics were identified and weighted according to their influence on the success of the isolation. For each variable, ranges and point weightings were established based on our laboratory experience and literature review. RESULTS: Analysis of the data showed a strong association of the donor point with isolation outcome. Pancreata with lower donor point scores had lower transplant success rates, while higher donor point scores in turn produced higher transplant rates. CONCLUSION: This scoring system has proven to be effective in assessing the potential of pancreata for a favorable isolation outcome. By analyzing the final score of the pancreas, a standardized decision can be made on whether to accept or decline the pancreas. Another benefit of the scoring system is that it is a quick and efficient way to trend the quality of donor organs.

Variation in human islet viability based on different membrane integrity stains.

Membrane integrity fluorescent staining is used routinely to evaluate islet viability. Results are used as one of the determining factors in islet product release criteria, and are used to assess the efficacy of different culture conditions. Recently, it has been observed that there is variation in the viability staining of freshly isolated islets based on which viability assay is used. This investigation compares three membrane integrity stains for the viability assessment of isolated human islets. Fluorescein diacetate/propidium iodide (FDA/ PI), the current standard method for assessing islet viability, demonstrates intense extracellular fluorescence, reducing the differential staining of intact islets. We further evaluated SYTO-13/ethidium bromide (SYTO/ EB) and calcein AM/ethidium homodimer (C/EthD) as alternative viability assays, and found considerable variation between FDA/PI and either SYTO/EB or C/EthD staining. Preparations of human islets were obtained from cadaveric pancreata after collagenase digestion, mechanical separation, and purification by continuous Ficoll gradient centrifugation. For each preparation, two replicate samples of 50 islets were counted for each stain, and the percent viability calculated. The results for SYTO/EB and C/EthD were nearly identical [57.6 +/- 7.3% and 57.9 +/- 7.2%, respectively (mean +/- SEM), N = 11]. FDA/PI-stained islets, however, showed consistently elevated values when compared to SYTO/EB. Accurate assessment of islet viability remains a critical determinant of islet product release. The discrepancies found between FDA/PI scoring and visual quality, compared with alternative stains, suggests that the FDA/PI stain may not be the optimal approach to assess islet viability.

Methods of Human Islet Culture for Transplantation.

The ability to maintain isolated human islet preparations in tissue culture has recently been adopted by most islet transplant centers, and improves the safety as well as the practicality of islet transplantation. Maintaining islet viability and recovery, however, remains challenging in a clinical setting, due to stringent conditions required for culture. Islet culture is further complicated by the fact that islets do not form a monolayer. This review aims to clarify media, supplementation, and conditions that have been shown to be relevant to human islets, as well as to offer avenues of future research. Factors examined that may influence islet survival include base medium, glucose concentration, vitamin, inorganic ion, lipid, hormone, growth factor, amino acid, and binding protein composition and concentration, as well as culture temperature and seeding density. In addition, this article reviews novel techniques, such as coculture and matrices, that have been employed in an attempt to improve islet survival and functional viability.

Methods of human islet culture for transplantation.

The ability to maintain isolated human islet preparations in tissue culture has recently been adopted by most islet transplant centers, and improves the safety as well as the practicality of islet transplantation. Maintaining islet viability and recovery, however, remains challenging in a clinical setting, due to stringent conditions required for culture. Islet culture is further complicated by the fact that islets do not form a monolayer. This review aims to clarify media, supplementation, and conditions that have been shown to be relevant to human islets, as well as to offer avenues of future research. Factors examined that may influence islet survival include base medium, glucose concentration, vitamin, inorganic ion, lipid, hormone, growth factor, amino acid, and binding protein composition and concentration, as well as culture temperature and seeding density. In addition, this article reviews novel techniques, such as coculture and matrices, that have been employed in an attempt to improve islet survival and functional viability.