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.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 1.72M | Year: 2009
DESCRIPTION (provided by applicant): VitaCyte LLC is working to improve tissue dissociation enzymes (TDE) required for the isolation of insulin producing pancreatic islet cells from both human and porcine organs. Transplantation of islets into Type 1 Diabetic patients represents a promising therapy as means to replace administration of insulin by external factors. The development of the Edmonton Protocol demonstrated insulin independence in patients transfused with purified pancreatic islets. Insufficient islet yields arising from inconsistencies in TDE manufacturing have plagued continuing attempts to transfer this promising therapy to routine clinical practice. Improved biochemical assays have been used to correlate collagenase molecular forms with tissue dissociation. These data indicate the importance of each molecular form for different tissue types. The goal of this project is to develop a robust and reproducible manufacturing process using recombinant TDE that will improve the quality and commercial availability for both human allotransplantation and porcine xenotransplantation. Specifically, three recombinant constructs of Clostridia histolyticum collagenases: intact Class 2 (C2intact), intact Class 1 with two collagen binding domains (C1intact), and degraded C1 with a single collagen binding domain (C1100kDa) will be generated using standard molecular biology techniques. A large scale bacterial fermentation and expression production protocol will be developed by adopting current industry practices for production scale manufacturing to optimize expression yields. A column chromatography purification protocol will be developed using modifications to existing purification methods currently used in natural collagenase production. This process will be scaled for production of highly pure recombinant enzymes for use in islet isolation procedures. Finally, an assessment of the different molecular forms and their role in islet isolations will be determined in both human and porcine tissue both internally and at leading islet isolation centers. Using a standard modified Ricordi islet isolation procedure, the specific molecular form will be correlated to successful islet yield using design of experiment statistical control methods. PUBLIC HEALTH RELEVANCE: Pancreatic islet transplantation represents a promising therapy for Type 1 Diabetic patients as a replacement for traditional insulin therapies. Hurdles to moving this therapy to routine clinical practice lies in achieving greater islet yields for either human allotransplantation or porcine xenotransplantation. VitaCyte is working to develop a robust and reproducible manufacturing process using recombinant tissue dissociation enzymes to improve islet isolation procedure yields.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 276.03K | Year: 2007
DESCRIPTION (provided by applicant): The quality of tissue dissociation enzymes (TDEs) containing collagenase was critical to the success of the Edmonton Protocol, an islet transplantation procedure that led to successful treatment of adult type 1 diabetic patients who were disabled by refractory hypoglycemia. The quality and yield of human islets recovered from pancreata were improved when Liberase(tm) HI Purified Enzyme Blend (HI) was used in place of crude collagenase to dissociate the tissue. HI has become the primary TDE reagent for human islet isolation because of its defined compostion (contains 3 purifed proteases: two classes of collagenase -class I and class II- purified from Clostridium histolyticum culture supernatants and thermolysin, a bacterial neutral protease) and low amounts of endotoxin contamination. The number of islet transplants performed over the last 6 years (~550) since publication of the Edmonton Protocol now exceeds the number of transplants performed the prior 26 years (n=483). A strong concern of the islet transplant community is inconsistent islet yields that many believe reflects the variable quality of the HI product (see http://icr.coh.org/workshops.asp). Collagenase is the critical reagent since it is the only enzyme that can degrade collagen fibers present in the extracellular matrix. Once the fibers are cut, other neutral proteases accelerate the degradation of the collagen leading to release of islets from the tissue. Inconsistent islet yields may in part be due to the protease degradation of collagenase during the protein purification or tissue dissociation process. Structure-function studies of these enzymes showed that once degraded, they are ineffective in cutting collagen. This issue will not be resolved until a superior purifed collagenase enzyme is used in purified TDE products. This project addresses this issue by developing a protease resistant, recombinant (rec) class I & class II enzymes using sequences of C. histolyticum genes found in public databases. Once this reagent is developed, then systematic studies can be performed to assess factors that affect the quality and yield of islets. The basic elements of the project are as follows: CI or CII collagenase purified from C. histolyticum culture supernatants will be digested with neutral proteases present in the tissue dissociation mixture, the peptides purified by reverse phase HPLC, then the sensitive residues determined using mass spectrometry. Critical amino acid residues attacked by proteases will be changed by using PCR mutagenesis techniques and the mutated genes used to express the rec enzymes. After confirming the resistance of the mutated collagenases to protease degradation, enzyme production will be scaled up, & the mutant enzymes purified and assessed for their effectiveness to isolate human and porcine islets. Collagenase is a critical reagent for isolating islets from pancreatic tissue. The success of islet transplantation as a cellular therapy to treat adult type diabetic patients relies on isolating a sufficient number of high quality islets from human pancreata. The inconsistent quality of purified collagenase used in islet isolation procedures today often leads to suboptimal islet yields. Development of recombinant collagenase enzymes that are resistant to protease degradation will minimize the "collagenase problem" and begin to build a foundation for obtaining a deeper understanding of other factors that affect islet yield.
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.
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. Source