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Balamurugan A.N.,University of Minnesota | Breite A.G.,Vitacyte, Llc | Anazawa T.,University of Minnesota | Loganathan G.,University of Minnesota | And 5 more authors.
Transplantation | Year: 2010

Background. Purified tissue dissociation enzymes (TDEs) are critical to successful human islet isolation required for clinical transplantation, but little is known about the characteristics of the key enzymes-class I (C1) and class II (C2) collagenase from Clostridium histolyticum-used in these procedures. Here, we show the differences between the C1 collagenase found in purified collagenase products manufactured by three suppliers and the impact of differences in C1 between two suppliers on human islet yield. Methods. Collagenase from Roche, Serva/Nordmark (Uetersen, Germany), and VitaCyte (Indianapolis, IN) were analyzed by analytical high-performance liquid chromatography and collagen degradation activity (CDA), an assay that preferentially detects intact C1 collagenase. Human islet isolations were performed using current standard practices. Results. These studies showed that the highest amount of intact C1 that correlated with a high specific CDA (CDA unit per milligram of protein). The highest specific CDA was found in VitaCyte product followed by the Roche and Serva/Nordmark products. The products of VitaCyte were used successfully for human islet isolation (n=14) with an average final islet yield obtained was 419,100±150,900 islet equivalent number (IEQ) (4147±1759 IEQ/g pancreas). Four of these preparations were used successfully in clinical transplantation procedures. These TDEs gave significantly better results when compared with earlier data where 27 isolations were performed using Serva NB1 collagenase and NB neutral protease where the final islet yield was 217,500±152,400 IEQ (2134±1524 IEQ/g pancreas). Conclusions. These data indicate the importance of intact C1 and the use of the appropriate analytical assays to correlate biochemical characteristics of TDEs to islet quality and yield. © 2010 by Lippincott Williams & Wilkins.

Balamurugan A.N.,University of Minnesota | Loganathan G.,University of Minnesota | Bellin M.D.,University of Minnesota | Wilhelm J.J.,University of Minnesota | And 10 more authors.
Transplantation | Year: 2012

Background. The optimal enzyme blend that maximizes human islet yield for transplantation remains to be determined. In this study, we evaluated eight different enzyme combinations (ECs) in an attempt to improve islet yield. The ECs consisted of purified, intact or truncated class 1 (C1) and class 2 (C2) collagenases from Clostridium histolyticum (Ch), and neutral protease (NP) from Bacillus thermoproteolyticus rokko (thermolysin) or Ch (ChNP). Methods. We report the results of 249 human islet isolations, including 99 deceased donors (research n=57, clinical n=42) and 150 chronic pancreatitis pancreases. We prepared a new enzyme mixture (NEM) composed of intact C1 and C2 collagenases and ChNP in place of thermolysin. The NEM was first tested in split pancreas (n=5) experiments and then used for islet autologous (n=21) and allogeneic transplantation (n=10). Islet isolation outcomes from eight different ECs were statistically compared using multivariate analysis. Results. The NEM consistently achieved higher islet yields from pancreatitis (P<0.003) and deceased donor pancreases (P<0.001) than other standard ECs. Using the NEM, islet products met release criteria for transplantation from 8 of 10 consecutive pancreases, averaging 6510±2150 islet equivalent number/gram (IEQ/g) pancreas and 694,681±147,356 total IEQ/transplantation. In autologous isolation, the NEM yielded more than 200,000 IEQ from 19 of 21 pancreases (averaging 422,893±181,329 total IEQ and 5979±1469 IEQ/kg recipient body weight) regardless of the severity of fibrosis. Conclusions. A NEM composed of ChNP with CIzyme high intact C1 collagenase recovers higher islet yield from deceased and pancreatitis pancreases while retaining islet quality and function. © 2012 by Lippincott Williams & Wilkins.

PubMed | University of Minnesota, University of Louisville and Vitacyte, Llc
Type: Journal Article | Journal: Transplantation direct | Year: 2016

Isolation following a good manufacturing practice-compliant, human islet product requires development of a robust islet isolation procedure where effective limits of key reagents are known. The enzymes used for islet isolation are critical but little is known about the doses of class I and class II collagenase required for successful islet isolation.We used a factorial approach to evaluate the effect of high and low target activities of recombinant class I (rC1) and class II (rC2) collagenase on human islet yield. Consequently, 4 different enzyme formulations with divergent C1:C2 collagenase mass ratios were assessed, each supplemented with the same dose of neutral protease. Both split pancreas and whole pancreas models were used to test enzyme targets (n = 20). Islet yield/g pancreas was compared with historical enzymes (n = 42).Varying the Wunsch (rC2) and collagen degradation activity (CDA, rC1) target dose, and consequently the C1:C2 mass ratio, had no significant effect on tissue digestion. Digestions using higher doses of Wunsch and CDA resulted in comparable islet yields to those obtained with 60% and 50% of those activities, respectively. Factorial analysis revealed no significant main effect of Wunsch activity or CDA for any parameter measured. Aggregate results from 4 different collagenase formulations gave 44% higher islet yield (>5000 islet equivalents/g) in the body/tail of the pancreas (n = 12) when compared with those from the same segment using a standard natural collagenase/protease mixture (n = 6). Additionally, islet yields greater than 5000 islet equivalents/g pancreas were also obtained in whole human pancreas.A broader C1:C2 ratio can be used for human islet isolation than has been used in the past. Recombinant collagenase is an effective replacement for the natural enzyme and we have determined that high islet yield can be obtained even with low doses of rC1:rC2, which is beneficial for the survival of islets.

Breite A.G.,Vitacyte, Llc | Dwulet F.E.,Vitacyte, Llc | McCarthy R.C.,Vitacyte, Llc
Transplantation Proceedings | Year: 2010

Neutral proteases, essential components of purified tissue dissociation enzymes required for successful human islet isolation, show variable activities and effects of substrate on their activities. Initially we used a spectrophotometric endpoint assay with azocasein substrate to measure neutral protease activity. After critical review of the results, we observed these data to be inconsistent and not correlating expected differences in specific activities between thermolysin and Bacillus polymyxa proteases. This observation led to the development of a fluorescent microplate assay using fluorescein isothyocyanateconjugated bovine serum albumin (FITC-BSA) as the substrate. This simpler, more flexible method offered a homogeneous, kinetic enzyme assay allowing determination of steady state reaction rates of sample replicates at various dilutions. The assay had a linear range of 4- to 8-fold and interassay coefficients of variation for B polymyxa protease and thermolysin of <9% and <15%, respectively, which were lower than those using the spectrophotometric endpoint assay, namely, 54% and 36%, respectively. This format allowed for incorporation of enzyme inhibitors, as illustrated by addition of sulfhydryl protease inhibitors, which, consistent with earlier reports, strongly indicated that the main contaminant in purified collagenase preparations was clostripain. Determination of the specific activities for several purified neutral proteases showed that the B polymyxa and Clostridium histolyticum proteases had approximately 40% and 15% specific activities, respectively, of those obtained with purified thermolysin, indicating the different characteristics of neutral protease enzymes for cell isolation procedures. © 2010 Elsevier Inc.

Stull N.D.,Indiana University | Breite A.,Vitacyte, Llc | McCarthy R.,Vitacyte, Llc | Tersey S.A.,Indiana University | Mirmira R.G.,Indiana University
Journal of Visualized Experiments | Year: 2012

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

McCarthy R.C.,Vitacyte, Llc | Breite A.G.,Vitacyte, Llc | Green M.L.,Vitacyte, Llc | Dwulet F.E.,Vitacyte, Llc
Transplantation | Year: 2011

Tissue dissociation enzymes are critical reagents that affect the yield and quality of human pancreatic islets required for islet transplantation. The United States Food and Drug Administration's oversight of this procedure recommends laboratories to set acceptance criteria for enzymes used in the manufacture of islet products for transplantation. Currently, many laboratories base this selection on personal experience because biochemical analysis is not predictive of success of the islet isolation procedure. This review identifies the challenges of correlating results from enzyme biochemical analysis to their effectiveness in human islet isolation and suggests a path forward to address these challenges to improve control of the islet manufacturing process. Copyright © 2011 by Lippincott Williams & Wilkins.

Objectives: Clostridium histolyticum expresses two classes of collagenases, C1 and C2. However, degradation of these enzymes by proteases during the fermentation or purification process may lead to numerous molecular forms that lead to inconsistent release of islets from human pancreata. This report defines the amino acid sequence of the truncated forms of C1 (C1b or C1c) that contain a single collagen-binding domain (CBD) and investigates the synergy between the different forms of C1 collagenase and C2 to degrade native collagen. Methods: Highly purified collagenase isoforms were purified from C. histolyticum culture supernatants using established column chromatography techniques and analyzed using high-pressure liquid chromatograph (HPLC), sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS). The collagen-degrading activity (CDA) assay was used to investigate the synergy between different collagenase molecular forms. Results: MS was used to confirm the sequence of full-length C2 and C1 from the reported gene sequence. These results were correlated with the molecular weights observed on the SDS- PAGE and elution after analytical anion-exchange HPLC. HPLC peaks designated as C1b and C1c were both confirmed to be C1 lacking the terminal CBD. The only difference being the cleavage site leading to a 12 amino acid difference between the two forms. A non-additive synergy in CDA relative to activity of individual collagenases was observed for C2 with each of the three C1 molecular forms. The C1 molecular forms did not display this synergy in the absence of C2. Conclusions: These observations support earlier reports that suggest the two collagenases bind to different portions of the collagen and have different specificities to cut native collagen. Although the implications of this are not yet understood, they are fundamental in advancing the understanding of how collagenases work together along with the neutral protease to breakdown the extracellular matrix for islet isolation. © 2011 Published by Elsevier Inc.

PubMed | The Rogosin Institute, Vitacyte, Llc and The Rogosin Institute Xenia Division
Type: Journal Article | Journal: Transplantation direct | Year: 2016

Human allogeneic islet transplantation for treatment of type 1 diabetes provides numerous clinical benefits, such as fewer episodes of hypoglycemic unawareness and tighter control of blood glucose levels. Availability of human pancreas for clinical and research use, however, is severely limited. Porcine pancreas offers an abundant source of tissue for optimization of islet isolation methodology and future clinical transplantation, thereby increasing patient access to this potentially lifesaving procedure.Porcine islet isolations were performed using varying amounts of collagenase (7.5, 3.75, or 2.5 Wunsch units per gram tissue) and neutral protease activity (12 000, 6000, or 4000 neutral protease units per gram tissue) and perfusion volumes (1.7 or 0.85 mL/g tissue) to assess their effects on isolation outcomes. Retention of dissociative enzymes within the pancreas during perfusion and digestion was evaluated, along with distribution of the perfusion solution within the tissue.Reducing enzyme usage by as much as 67% and perfusion volume by 50% led to equally successful islet isolation outcomes when compared with the control group (48 7% of tissue digested and 1088 299 islet equivalents per gram of pancreas vs 47 11% and 1080 512, respectively). Using margin-marking dye in the perfusion solution to visualize enzyme distribution demonstrated that increasing perfusion volume did not improve tissue infiltration.Current protocols for porcine islet isolation consume excessive amounts of dissociative enzymes, elevating cost and limiting research and development. These data demonstrate that islet isolation protocols can be optimized to significantly reduce enzyme usage while maintaining yield and function and thus accelerating progress toward clinical application.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 232.33K | Year: 2011

DESCRIPTION (provided by applicant): The demonstration by the Edmonton group that human islet transplantation can be successfully used to manage adult type 1 diabetes patients with refractory hypoglycemia has led to increased funding of clinical trials andfurther research to extend the scope of this therapy by using porcine islets in place of human islets. Significant advances have been made in improving immunosuppression treatment regimens so that results obtained from treating adult diabetic patients with human islet transplants are similar to those obtained after pancreas transplantation. The major hurdle to move this therapy from clinical research to routine clinical practice is to improve the yield and quality of islets recovered from human or porcinepancreas. Presently, there are no standardized methods that can accurately assess the number or quality of islets that are used in the islet transplantation procedures so that results between laboratories can be objectively evaluated. This grant is focusedon developing a robust, islet image analysis software to objectively analyze the number and quality of porcine islets recovered from the pancreas. The two major aims of the project are first to develop an improved image analysis software program that willprovide a standardized measurement of the number and mass of porcine islets in a cell preparation. And second, enhance the capabilities of the software program by correlating the image signatures of each porcine islet to an artificial category. Porcine islets of similar size will be handpicked and sorted into three categories based on the shape, border, integrity, or uniformity of dithizone staining. The first software enhancement will find those features in the images that can be used to distinguish the different categories of islets. The second enhancement will assess the feasibility of using machine learning methods to correlate these features with data recovered from the images but also other discrete or continuous variables that are used to characterize the porcine islet preparations. If successful, the ability to use a rapid and objective image analysis methodology will improve the assessment of the number and quality of islets within and between laboratories; correlate image features with success of transplantation as measured by graft survival and insulin independence; and improve the islet isolation methods to achieve favorable islet image scores that are determined by retrospective analysis. The ability of a commercial firm focused on improving islet yields by focusing on tissue dissociation with a leading academic laboratory that has sophisticated expertise in developing software algorithms from microscopic images provides a fresh approach to a difficult medical that needs to be resolved to realizethe full potential of islet transplantation to treat adult type 1 diabetic patients. PUBLIC HEALTH RELEVANCE: An objective, reliable and accurate method for the assessment of islet quantity and quality is paramount to the standardization and subsequent success of islet transplantation as a treatment for type 1 diabetes. Conventional manual methods for determining islet yields using an optical microscope with a calibrated eyepiece reticule are subjective, time consuming and often overestimate islet mass due to sampling errors and erroneous assumptions in the conversion of islet numbers to islet equivalents. The research proposed will utilize recent advances in digital image analysis, including machine learning and pattern recognition, to develop a software algorithm for the rapid characterization of islets destined for transplantation procedures.

Vitacyte, Llc | Date: 2016-03-08

Highly purified and rigorously characterized enzymes that dissociate cellular matrices, sold as individual enzyme products and as enzyme formulations, for dissociating cells from tissue, detaching cells from tissue culture vessels and for enzyme treatment of specific clinical conditions, all for medical research purposes.

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