Perlecan: An Islet Basement Membrane Protein with Protective Anti-Inflammatory Characteristics.

Throughout the isolation process, human islets are subjected to destruction of the islet basement membrane (BM) and reduced oxygen supply. Reconstruction of the BM represents an option to improve islet function and survival post-transplant and may particularly be relevant for islet encapsulation devices and scaffolds. In the present study, we assessed whether Perlecan, used alone or combined with the BM proteins (BMPs) Collagen-IV and Laminin-521, has the ability to protect isolated human islets from hypoxia-induced damage.

The ischaemic preconditioning paradox and its implications for islet isolation from heart-beating and non heart-beating donors.

The impact of ischaemia can severely damage procured donor organs for transplantation. The pancreas, and pancreatic islets in particular, is one of the most sensitive tissues towards hypoxia. The present study was aimed to assess the effect of hypoxic preconditioning (HP) performed ex-vivo in islets isolated from heart-beating donor (HBD) and non heart-beating donor (NHBD) rats.

Basement membrane proteins improve human islet survival in hypoxia: Implications for islet inflammation.

Enzymatic digestion of the pancreas during islet isolation is associated with disintegration of the islet basement membrane (IBM) that can cause reduction of functional and morphological islet integrity. Attempts to re-establish IBM by coating the surface of culture vessels with various IBM proteins (IBMP) have resulted in loss of islet phenotype and function. This study investigated the capability of Collagen-IV, Laminin-521 and Nidogen-1, utilised as single or combined media supplements, to protect human islets cultured in hypoxia.

High Concentrations of Etanercept Reduce Human Islet Function and Integrity.

Background: Most islet transplant groups worldwide routinely use the TNFα inhibitor Etanercept in their peri-transplant protocols. Surprisingly, there have been no published dose-response studies on the effects of Etanercept on human islets. Our study aimed to address this by treating cultured human islets with increasing concentrations of Etanercept.

Proteomic Profiling Reveals the Ambivalent Character of the Mesenchymal Stem Cell Secretome: Assessing the Effect of Preconditioned Media on Isolated Human Islets.

Previous studies in rodents have indicated that function and survival of transplanted islets can be substantially improved by mesenchymal stem cells (MSC). The few human islet studies to date have confirmed these findings but have not determined whether physical contact between MSC and islets is required or whether the benefit to islets results from MSC-secreted proteins. This study aimed to investigate the protective capacity of MSC-preconditioned media for human islets.

Comparison of Clostripain and Neutral Protease as Supplementary Enzymes for Human Islet Isolation.

Although human islet transplantation has been established as valid and safe treatment for patients with type 1 diabetes, the utilization rates of human pancreases for clinical islet transplantation are still limited and substantially determined by the quality and composition of collagenase blends. While function and integrity of collagenase has been extensively investigated, information is still lacking about the most suitable supplementary neutral proteases. The present study compared islet isolation outcome after pancreas digestion by means of collagenase used alone or supplemented with either neutral protease (NP), clostripain (CP), or both proteases.

Does Islet Size Really Influence Graft Function After Clinical Islet Transplantation?

Background: It has been proposed that islet transplants comprised primarily of small rather than large islets may provide better graft function, due to their lower susceptibility to hypoxic damage. Our aim was to determine whether islet size correlated with in vivo graft function in islet transplant recipients with C peptide-negative type 1 diabetes when islets have undergone pretransplant islet culture.

Methods: Human pancreatic islets were isolated, cultured for 24 hours and infused by standardized protocols.

Quantifying the Effects of Different Neutral Proteases on Human Islet Integrity.

Efficient islet release from the pancreas requires the combination of collagenase, neutral protease (cNP), or thermolysin (TL). Recently, it has been shown that clostripain (CP) may also contribute to efficient islet release from the human pancreas. The aim of this study was to evaluate the impact of these proteases on human islet integrity in a prospective approach.

Multicenter Assessment of Animal-free Collagenase AF-1 for Human Islet Isolation.

Animal-free (AF) SERVA Collagenase AF-1 and Neutral Protease (NP) AF GMP Grade have recently become available for human islet isolation. This report describes the initial experiences of 3 different islet transplant centers. Thirty-four human pancreases were digested using 1 vial of the 6 different lots of Collagenase AF-1 (2,000-2,583 PZ-U/vial) supplemented with 4 different lots of NP AF in a range of 50 to 160 DMC-U per pancreas.

Hypoxia-Induced Damage in Human Islets Is Reduced With the Use of Mesenchymal Stem Cell-Preconditioned Medium.

Background: Mesenchymal stem cells (MSCs) are protective for islets when cotransplanted in a hypoxic environment. However, the risk of neoplasia is increased when MSCs are transplanted into immunosuppressed patients. This initial study aimed to investigate whether the production of protective factors from MSC can be stimulated by different culture conditions to benefit human islets cultured in hypoxia.

Effect of Etanercept Concentration on Human Islet Integrity.

Background: Etanercept is widely used as an antiinflammatory drug to improve engraftment after intraportal islet transplantation. In contrast to other immunosuppressive agents, very little is known about detrimental effects of etanercept on islets. The aim of this pilot study was to define the toxic range of etanercept.

Enzyme Development for Human Islet Isolation: Five Decades of Progress or Stagnation?

In comparison to procedures used for the separation of individual cell types from other organs, the process of human pancreatic islet isolation aims to digest the pancreatic exocrine matrix completely without dispersing the individual cells within the endocrine cell cluster. This objective is unique within the field of tissue separation, and outlines the challenge of islet isolation to balance two opposing priorities. Although significant progress has been made in the characterization and production of enzyme blends for islet isolation, there are still numerous areas which require improvement.

Pancreatic Islets: Methods for Isolation and Purification of Juvenile and Adult Pig Islets.

The current situation of organ transplantation is mainly determined by the disbalance between the number of available organs and the number of patients on the waiting list. This obvious dilemma might be solved by the transplantation of porcine organs into human patients. The metabolic similarities which exist between both species made pancreatic islets of Langerhans to that donor tissue which will be most likely transplanted in human recipients.

Comparison of Neutral Proteases and Collagenase Class I as Essential Enzymes for Human Islet Isolation.

Unlabelled: Efficient islet isolation requires synergistic interaction between collagenase class I (CI) and class II (CII). The CI degradation alters the ratio between CI and CII and is responsible for batch-to-batch variations. This study compares the role of neutral protease (NP) plus clostripain (CP) with CI as essential enzymes for human islet isolation.

National Institutes of Health-Sponsored Clinical Islet Transplantation Consortium Phase 3 Trial: Manufacture of a Complex Cellular Product at Eight Processing Facilities.

Eight manufacturing facilities participating in the National Institutes of Health-sponsored Clinical Islet Transplantation (CIT) Consortium jointly developed and implemented a harmonized process for the manufacture of allogeneic purified human pancreatic islet (PHPI) product evaluated in a phase 3 trial in subjects with type 1 diabetes. Manufacturing was controlled by a common master production batch record, standard operating procedures that included acceptance criteria for deceased donor organ pancreata and critical raw materials, PHPI product specifications, certificate of analysis, and test methods. The process was compliant with Current Good Manufacturing Practices and Current Good Tissue Practices.

Evaluation of Perfluorohexyloctane/Polydimethylsiloxane for Pancreas Preservation for Clinical Islet Isolation and Transplantation.

This study aimed to evaluate a 50:50 mix of perfluorohexyloctane/polydimethylsiloxane 5 (F6H8S5) preservation of pancreases in a clinical setting compared with standard solutions for 1) cold ischemia time (CIT) 10 h and 2) an extended CIT 20 h. Procured clinical-grade pancreases were shipped in either F6H8S5 or in standard preservation solutions, that is, University of Wisconsin (UW) or Custodiol. F6H5S5 was preoxygenated for at least 15 min.

High Seeding Density Induces Local Hypoxia and Triggers a Proinflammatory Response in Isolated Human Islets.

Hypoxia is the main threat to morphological and functional integrity of isolated pancreatic islets. Lack of oxygen seems to be of particular importance for functionality of encapsulated islets. The present study was initiated as an experimental model for the environment experienced by human islets in a confined space present during culture, shipment, and in an implanted macrodevice.

Pancreatic L-Glutamine Administration Protects Pig Islets From Cold Ischemic Injury and Increases Resistance Toward Inflammatory Mediators.

The isolation and transplantation of porcine islets represent a future option for the treatment of type 1 diabetic patients. Stringent product release criteria and limited availability of transgenic and specific pathogen-free pigs will essentially require processing of explanted pig pancreata in specialized, possibly remote isolation facilities, whereby pancreata are exposed to cold ischemia due to prolonged tissue transit time. In the present study we investigated whether pancreas oxygenation can be efficiently combined with an antioxidant strategy utilizing intraductal L-glutamine administration.

Anti-caspase-3 preconditioning increases proinsulin secretion and deteriorates posttransplant function of isolated human islets.

Human islet isolation is associated with adverse conditions inducing apoptosis and necrosis. The aim of the present study was to assess whether antiapoptotic preconditioning can improve in vitro and posttransplant function of isolated human islets. A dose-finding study demonstrated that 200 μmol/L of the caspase-3 inhibitor Ac-DEVD-CMK was most efficient to reduce the expression of activated caspase-3 in isolated human islets exposed to severe heat shock.

Liver-directed chemotherapy of cetuximab and bevacizumab in combination with oxaliplatin is more effective to inhibit tumor growth of CC531 colorectal rat liver metastases than systemic chemotherapy.

Colorectal carcinoma is, through to its high rate of liver metastasis (mCRC), the second most cause of cancer death worldwide. Tumor resection represents the only potential cure. In cases of unresectable disease systemic chemotherapy (sCHT) remains the therapy of choice.

Quality of isolated pig islets is improved using perfluorohexyloctane for pancreas storage in a split lobe model.

Pancreas transportation between donor center and islet production facility is frequently associated with prolonged ischemia impairing islet isolation and transplantation outcomes. It is foreseeable that shipment of pig pancreases from distant centralized biosecure breeding facilities to institutes that have a long-term experience in porcine islet isolation is essentially required in future clinical islet xenotransplantation. Previously, we demonstrated that perfluorohexyloctan (F6H8) is significantly more efficient to protect rat and human pancreata from ischemically induced damage compared to perfluorodecalin (PFD).

Oxygen-charged HTK-F6H8 emulsion reduces ischemia-reperfusion injury in kidneys from brain-dead pigs.

Background: Prolonged cold ischemia is frequently associated with a greater risk of delayed graft function and enhanced graft failure. We hypothesized that media, combining a high oxygen-dissolving capacity with specific qualities of organ preservation solutions, would be more efficient in reducing immediate ischemia-reperfusion injury from organs stored long term compared with standard preservation media.

Methods: Kidneys retrieved from brain-dead pigs were flushed using either cold histidine-tryptophan-ketoglutarate (HTK) or oxygen-precharged emulsion composed of 75% HTK and 25% perfluorohexyloctane.