Human Islet Isolation Research Articles

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

BackgroundEfficient 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.MethodsHuman islets were isolated using 4 different enzyme mixtures composed of CII plus either intact (CI-115) or degraded CI (CI-100). Blends were administered either with or without NP/CP. Purified islets were cultured for 3 to 4 days before islet quality assessment.ResultsWhereas using intact CI-115 without NP/CP did not significantly reduce islet yield (3429 ± 631 vs 3087 ± 970 islet equivalent/g, nonsignificant), administration of degraded CI-100 without NP/CP decreased islet yield from 3501 ± 580 to 1312 ± 244 islet equivalent/g (P < 0.01), doubled the amount of undigested tissue from 11.8 ± 1.6 to 24.4 ± 1.2% (P < 0.01) and triplicated the percentage of trapped islets from 7.7 ± 2.8 to 22.5 ± 3.6% (P < 0.05). Islet yield did not vary between supplemented CI-115 and CI-100, but was increased using CI-115 when NP/CP was omitted (P < 0.05). A trend toward higher viability and increased secretory insulin response was noted in both CI-100 and CI-115 when NP/CP was not added.ConclusionsThis study suggests that NP/CP can compensate reduced CI activity. Future attempts to optimize enzyme blends should consider the possibility to increase the proportion of collagenase CI to reduce the need for potentially harmful NPs.

Research-Focused Isolation of Human Islets From Donors With and Without Diabetes at the Alberta Diabetes Institute IsletCore.

Recent years have seen an increased focus on human islet biology, and exciting findings in the stem cell and genomic arenas highlight the need to define the key features of mature human islets and β-cells. Donor and organ procurement parameters impact human islet yield, although for research purposes islet yield may be secondary in importance to islet function. We examined the feasibility of a research-only human islet isolation, distribution, and biobanking program and whether key criteria such as cold ischemia time (CIT) and metabolic status may be relaxed and still allow successful research-focused isolations, including from donors with type 1 diabetes and type 2 diabetes. Through 142 isolations over approximately 5 years, we confirm that CIT and glycated hemoglobin each have a weak negative impacts on isolation purity and yield, and extending CIT beyond the typical clinical isolation cutoff of 12 hours (to ≥ 18 h) had only a modest impact on islet function. Age and glycated hemoglobin/type 2 diabetes status negatively impacted secretory function; however, these and other biological (sex, body mass index) and procurement/isolation variables (CIT, time in culture) appear to make only a small contribution to the heterogeneity of human islet function. This work demonstrates the feasibility of extending acceptable CIT for research-focused human islet isolation and highlights the biological variation in function of human islets from donors with and without diabetes.

In Vitro Assessment of Human Islet Vulnerability to Instant Blood-Mediated Inflammatory Reaction (IBMIR) and Its Use to Demonstrate a Beneficial Effect of Tissue Culture.

Culture of human pancreatic islets is now routinely carried out prior to clinical islet allotransplantation, using conditions that have been developed empirically. One of the major causes of early islet destruction after transplantation is the process termed instant blood-mediated inflammatory reaction (IBMIR). The aim of this study was to develop in vitro methods to investigate IBMIR and apply them to the culture conditions used routinely in our human islet isolation laboratory. Freshly isolated or precultured (24 h, 48 h) human islets were incubated in either ABO-compatible allogeneic human blood or Hank's buffered salt solution (HBSS) for 1 h at 37°C. Tissue factor (TF) expression and leukocyte migration were assessed by light microscopy. TF was also quantified by ELISA. To assess β-cell function, glucose-stimulated insulin secretion (GSIS) assay was carried out. The extent of islet β-cell damage was quantified using a proinsulin assay. Islets cultured for 24 h had higher GSIS when compared to freshly isolated or 48-h precultured islets. Freshly isolated islets had significantly higher TF content than 24-h and 48-h precultured islets. Incubation of freshly isolated human islets in allogeneic human blood released 6.5-fold higher level of proinsulin in comparison to freshly isolated human islets in HBSS. The high level of proinsulin released was significantly attenuated when precultured islets (24 h or 48 h) were exposed to fresh blood. Histological examination of fresh islets in blood clot showed that some islets were fragmented, showing signs of extraislet insulin leakage and extensive neutrophil infiltration and necrosis. These features were markedly reduced when the islets were cultured for 24 h. These results suggest that our standard 24-h islet culture is markedly beneficial in attenuating IBMIR, as evidenced by increased GSIS, lower content of TF, decrease islet fragmentation, and proinsulin release.

Cloning a neutral protease of Clostridium histolyticum, determining its substrate specificity, and designing a specific substrate.

Islet transplantation is a prospective treatment for restoring normoglycemia in patients with type 1 diabetes. Islet isolation from pancreases by decomposition with proteolytic enzymes is necessary for transplantation. Two collagenases, collagenase class I (ColG) and collagenase class II (ColH), from Clostridium histolyticum have been used for islet isolation. Neutral proteases have been added to the collagenases for human islet isolation. A neutral protease from C. histolyticum (NP) and thermolysin from Bacillus thermoproteolyicus has been used for the purpose. Thermolysin is an extensively studied enzyme, but NP is not well known. We therefore cloned the gene encoding NP and constructed a Bacillus subtilis overexpression strain. The expressed enzyme was purified, and its substrate specificity was examined. We observed that the substrate specificity of NP was higher than that of thermolysin, and that the protein digestion activities of NP, as determined by colorimetric methods, were lower than those of thermolysin. It seems that decomposition using NP does not negatively affect islets during islet preparation from pancreases. Furthermore, we designed a novel substrate that allows the measurement of NP activity specifically in the enzyme mixture for islet preparation and the culture broth of C. histolyticum. The activity of NP can also be monitored during islet isolation. We hope the purified enzyme and this specific substrate contribute to the optimization of islet isolation from pancreases and that it leads to the success of islet transplantation and the improvement of the quality of life (QOL) for diabetic patients.

Clostripain, the Missing Link in the Enzyme Blend for Efficient Human Islet Isolation.

Effective digestive enzymes are crucial for successful islet isolation. Supplemental proteases are essential as they synergize with collagenase for effective pancreas digestion. The presence of tryptic-like activity has been implicated in efficient enzyme blends and the present study aimed to evaluate if addition of clostripain, an enzyme with tryptic-like activity, could improve efficacy of the islet isolation procedure. Clostripain was added to the enzyme blend just before pancreas perfusion. Islets were isolated per standard method and numerous isolation parameters, islet quality control, and the number of isolations fulfilling standard transplantation criteria were evaluated. Two control organs per clostripain organ were chosen by blindly matching against body mass index, cold ischemia time, hemoglobin A1c, donor sex, and donor age. There were no differences in pancreas weight, dissection time, digestion time, harvest time, percent digested pancreas, or total pellet volume before islet purification between control or clostripain pancreases. Glucose-stimulated insulin release results were similar between groups. Total isolation islet equivalents, purified tissue volume and islet equivalents/g pancreas as well as fulfillment of transplantation criteria favored clostripain processed pancreases. The addition of clostripain to the enzyme blend soundly improved islet yields and transplantation rates. It gently aided pancreas digestion and maintained proper islet functionality. The addition of clostripain to the enzyme blend has now been implemented into standard isolation protocols at the isolation centers in Uppsala and in Oslo.

Human pancreatic islet transplantation: an update and description of the establishment of a pancreatic islet isolation laboratory.

Type 1 diabetes mellitus (T1DM) is associated with chronic complications that lead to high morbidity and mortality rates in young adults of productive age. Intensive insulin therapy has been able to reduce the likelihood of the development of chronic diabetes complications. However, this treatment is still associated with an increased incidence of hypoglycemia. In patients with "brittle T1DM", who have severe hypoglycemia without adrenergic symptoms (hypoglycemia unawareness), islet transplantation may be a therapeutic option to restore both insulin secretion and hypoglycemic perception. The Edmonton group demonstrated that most patients who received islet infusions from more than one donor and were treated with steroid-free immunosuppressive drugs displayed a considerable decline in the initial insulin independence rates at eight years following the transplantation, but showed permanent C-peptide secretion, which facilitated glycemic control and protected patients against hypoglycemic episodes. Recently, data published by the Collaborative Islet Transplant Registry (CITR) has revealed that approximately 50% of the patients who undergo islet transplantation are insulin independent after a 3-year follow-up. Therefore, islet transplantation is able to successfully decrease plasma glucose and HbA1c levels, the occurrence of severe hypoglycemia, and improve patient quality of life. The goal of this paper was to review the human islet isolation and transplantation processes, and to describe the establishment of a human islet isolation laboratory at the Endocrine Division of the Hospital de Clínicas de Porto Alegre - Rio Grande do Sul, Brazil.

Effects of Donor-, Pancreas-, and Isolation-Related Variables on Human Islet Isolation Outcome: A Systematic Review

Different factors have been reported to influence islet isolation outcome, but their importance varies between studies and are hampered by the small sample sizes in most studies. The purpose of this study was to perform a systematic review to assess the impact of donor-, pancreas-, and isolation-related variables on successful human islet isolation outcome. PubMed, Embase, and Web of Science were searched electronically in April 2009. All studies reporting on donor-, pancreas-, and isolation-related factors relating to prepurification and postpurification islet isolation yield and proportion of successful islet isolations were selected. Seventy-four retrospective studies had sufficient data and were included in the analyses. Higher pre- and postpurification islet yields and a higher proportion of successful islet isolations were obtained when pancreata were preserved with the two-layer method rather than University of Wisconsin solution in donors with shorter cold ischemia times (CITs) [1 h longer CIT resulted in an average decline of prepurification and postpurification yields and proportion of successful isolations of 59 islet equivalents (IEQs)/g, 54 IEQs/g, and 21%, respectively]. Higher prepurification yields and higher percentage of successful islet isolations were found in younger donors with higher body mass index. Lower yields were found in donation after brain death donors compared to donation after cardiac death donors. Higher postpurification yields were found for isolation with Serva collagenase. This review identified donor-, pancreas-, and isolation-related factors that influence islet isolation yield. Standardized reports of these factors in all future studies may improve the power and identify additional factors and thereby contribute to improving islet isolation yield.

Different digestion enzymes used for human pancreatic islet isolation: A mixed treatment comparison (MTC) meta-analysis

Collagenases are critical reagents determining yield and quality of isolated human pancreatic islets and may affect islet transplantation outcome. Some islet transplantation centers have compared 2 or more collagenase blends; however, the results regarding differences in quantity and quality of islets are conflicting. Thus, for the first time, a mixed treatment comparison (MTC) meta-analysis was carried out to compile data about the effect of different collagenases used for human pancreas digestion on islet yield, purity, viability and stimulation index (SI). Pubmed, Embase and Cochrane libraries were searched. Of 755 articles retrieved, a total of 15 articles fulfilled the eligibility criteria and were included in the MTC meta-analysis. Our results revealed that Vitacyte and Liberase MTF were associated with a small increase in islet yield (islet equivalent number/g pancreas) when compared with Sevac enzyme [standardized mean difference (95% credible interval – CrI) = −2.19 (−4.25 to −0.21) and −2.28 (−4.49 to −0.23), respectively]. However, all other enzyme comparisons did not show any significant difference regarding islet yield. Purity and viability percentages were not significantly different among any of the analyzed digestion enzymes. Interestingly, Vitacyte and Serva NB1 were associated with increased SI when compared with Liberase MTF enzyme [unstandardized weighted mean difference (95% CrI) = −1.69 (−2.87 to −0.51) and −1.07 (−1.79 to −0.39), respectively]. In conclusion, our MTC meta-analysis suggests that the digestion enzymes currently being used for islet isolation works with similar efficiency regarding islet yield, purity and viability; however, Vitacyte and Serva NB1 enzymes seem to be associated with an improved SI as compared with Liberase MTF.

Circulating microRNAs: Understanding the Limits for Quantitative Measurement by Real‐Time PCR

Ever since the discovery of small, non-coding RNAs in circulation, microRNAs have gained significant attention as biomarkers or, in some instances, regulators of disease and/or associated complications. Until now, most articles published use real-time quantitative PCR (qPCR) for assessment of

Filtration is a time-efficient option to Histopaque, providing good-quality islets in mouse islet isolation

Pancreatic islet transplantation is a promising therapy for Type I Diabetes. For many years the method used worldwide for islet purification in both rodent and human islet isolation has been Ficoll-based density gradients, such as Histopaque. However, it is difficult to purify islets in laboratories with staff limitations when large scale isolations are required. We hypothesized that filtration could be a more simple and fast alternative to obtain good quality islets. Four separate islet isolations were performed per method, comparing filtration and Histopaque purification with handpicking as the gold standard method for islet purity. Different parameters of quality were assessed: yield in number of islets per pancreas, purity by dithizone staining, viability by Fluorescein Diacetate/Propidium Iodide vital staining and in vitro functionality assessed by Glucose Stimulated Insulin Secretion. Time efficiency and cost were also analyzed. The overall quality of the islets obtained both by Histopaque and filtration was good. Filtration saved almost 90% of the time consumed by Histopaque purification, and was also cheaper. However, one-third of the islets were lost. Since human and rodent islets share similar size but different density, filtration appears as a purification method with potential interest in translation to clinic.

Human Islet Isolation Processing Times Shortened By One Hour

Human Islet Isolation Processing Times Shortened By One Hour : Minimized Incubation Time Between Tissue Harvest and Islet Purification

Quantification of Islet Loss and Graft Functionality During Immune Rejection by 3-Tesla MRI in a Rat Model

Because metabolic markers are not suitable for early diagnosis of islet graft dysfunction, magnetic resonance imaging (MRI) has been used to study islets that were labeled pretransplantation with superparamagnetic iron oxide nanoparticles. However, the relation between graft functionality assessed by glycemia, and MRI signal remains unclear. We transplanted hyperglycemic rats intraportally with 2500 ferucarbotran-labeled syngeneic (n=10) or allogeneic (n=12) islet equivalents or normoglycemic rats with 5000 xenogeneic human islet equivalents. Images were acquired on a clinical 3-Tesla MRI scanner. When rejection occurred on days 4 and 8 in xenogeneic and allogeneic recipients, 60% (57-68) and 55% (46-73) of the initial signal remained compared to 93% (71-104) and 82% (59-90) in syngeneic controls (P=0.006 and 0.03). With a cutoff value of 84% on day 4 for the diagnosis of allogeneic rejection, sensitivity of 91% and specificity of 70% were obtained. Based on MRI signal on day 4, treatment with antilymphocytic serum from day 4 allowed graft rescue in 75% of recipients. In this model, MRI of pretransplantation superparamagnetic iron oxide nanoparticle-labeled islet grafts allows timely diagnosis of immune rejection.

Effect of osteocalcin on human beta cell proliferation and function and its role on insulin secretion

Effect of osteocalcin on human beta cell proliferation and function and its role on insulin secretion

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. Ac-DEVD-CMK-pretreated or sham-treated islets were transplanted into immunocompetent or immunodeficient diabetic mice and subjected to static glucose incubation to measure insulin and proinsulin secretion. Antiapoptotic pretreatment significantly deteriorated graft function resulting in elevated nonfasting serum glucose when compared to sham-treated islets transplanted into diabetic nude mice (p < 0.01) and into immunocompetent mice (p < 0.05). Ac-DEVD-CMK pretreatment did not significantly change basal and glucose-stimulated insulin release compared to sham-treated human islets but increased the proinsulin release at high glucose concentrations (20 mM) thus reducing the insulin-to-proinsulin ratio in preconditioned islets (p < 0.05). This study demonstrates that the caspase-3 inhibitor Ac-DEVD-CMK interferes with proinsulin conversion in preconditioned islets reducing their potency to cure diabetic mice. The mechanism behind this phenomenon is unclear so far but may be related to the ketone CMK linked to the Ac-DEVD molecule. Further studies are required to identify biocompatible caspase inhibitors suitable for islet preconditioning.

Tracing the Fate of Extracellular Matrix Proteins During Human Islet Isolation and Culture

Introduction: The precise action of different collagenase blends on the digestion of the peri-islet extracellular matrix (ECM) during human islet isolation is largely unknown. It is also unclear how the remaining ECM of purified islets fares during culture. The presence of certain ECM proteins in purified islets may improve survival and promote islet engraftment. The aims of this pilot study were to characterize 1) the digestion of pancreatic ECM proteins during islet isolation, and 2) their expression in purified islets in culture prior to transplantation. Methods: With appropriate consent and ethical approval, human pancreases were retrieved from multiorgan donors (n = 3, age 44-59, CIT 5-7.5h, BMI 29-31). 0.5cm3 biopsies were taken from the head of the pancreas prior to islet isolation. 1ml samples were taken from the circulation fluid during the digestion phase of islet isolation, and an aliquot of islets taken following purification and after 24h and 48h in culture. Immunofluorescence labelling and Western blotting were used to identify specific ECM proteins. Results: Collagen I, IV, V, VI and Laminin expression were widespread throughout the pancreas, both around and within the islets. Western blotting of samples from the digestion circuit indicate variations in the release of digested fragments of these ECM proteins, most notably Collagen I. ECM proteins were present in purified islets, with Collagen VI expression being the most extensive. The expression of ECM proteins decreased over time in culture, with Laminin expression lost completely from islets by 48h. Conclusion: The total loss of Laminin and the decline of other ECM proteins suggests that the glycoprotein framework of the islet basement membrane is destroyed, raising concerns that islets may fragment following isolation, which could partially account for the reduction in islet yield commonly seen during culture. The reduction in Collagen I would suggest the loss of endothelial cells important for angiogenesis during engraftment. Furthermore, the degradation of Collagen IV may reveal cryptic regions with anti-angiogenic properties.

Pancreas Digestion Increases Hypoxia and Stress Markers During Human Islet Isolation

Introduction: The human islet isolation procedure is known to be metabolically stressful for the pancreatic tissue and may be accompanied by hypoxia. The aim of this study was to identify and compare the hypoxia-induced deleterious changes at sequential steps of the isolation process. Methods: With appropriate consent and ethical approval, human pancreases were retrieved from multiorgan donors. 1ml of tissue samples were collected from the islet isolation circuit, during i) collagenase digestion, ii) digest collection, iii) islet purification, and iv) from the final islet preparation (n=18 islet isolations). Measurements of HIF-1α translocation in the nuclear extract (ELISA), superoxide dismutase activity (enzymatic assay), and caspase 3 activation (ELISA) in the cytoplasmic extract were determined. Results from samples taken at different time points were compared with the first sample taken during digestion (t=0; 10 minutes after closing the chamber). Results: HIF-1α translocation into the nucleus was significantly increased (1.5±0.2 vs. 1.0±0.0; p< 0.001) throughout the first 30 minutes of the digestion and collection processes. Surprisingly, the level of HIF1α in the nuclei remained high after digestion even with the temperature reduced to 4°C (1.7±0.4). Superoxide dismutase activity was variable throughout the isolation process, with an increase during digestion (122.7±13.0%), a decrease during collection (41.5±11.3%), and an increase again during purification (131.6±20.0%). Caspase 3 activation was stable during digestion and purification, but was increased in final islet preparations (2.4±0.9 vs. 0.9±0.4 t=0) when the islet yield was low (< 2500 IEQ/g pancreas), but decreased with higher yields (>2500 IEQ/g pancreas), (0.2±0.4 vs. 1.2±1.1 t=0). Conclusion: From an early time point in human islet isolation, hypoxia and oxidative stress may influence the outcome of the islet isolation by activating the apoptosis pathways. By modifying islet isolation solutions with antioxidants and oxygen supply, we may be able to prevent this and improve the outcome of islet isolation.

Human Islet Isolation for Clinical Transplantation: University of Minnesota Experience with Different Islet Isolation Protocols and Isolation Enzymes

Introduction: Clinical islet transplantations are offering hope of a greater quality of life to patients suffering from type I diabetes with the potential of insulin independence from highly successful transplants. Although human islet isolation techniques have been constantly improving, it is still difficult to consistently isolate adequate quantities of intact islets from donor pancreases. Islet yield critically depends on the donor characteristics, efficiency of the enzymes used for pancreas dissociation and isolation protocols used. In this study, we retrospectively evaluated the impact of donor, enzyme and isolation protocol on final islet yield and overall isolation outcome in 69 clinical pancreata (2007-2012). Methods: Human islets were isolated from donor pancreata (n=69) with two different islet isolation protocols (A&B) with the following three enzyme combinations: (I) SERVA collagenase and neutral protease (n=13), (II) VitaCyte-collagenase and VitaCyte-thermolysin (n=19), and (III) New enzyme mixture (NEM) containing intact C1 collagenase (VitaCyte-collagenase combination with SERVA-neutral protease) (n=37). Protocol A (P-A) was followed from 2007-2009 (n=35 isolations) after which protocol B (P-B) was used until 2012 (n=34). The basic method of islet isolation and donor acceptance criteria in both protocols were the same; however, there were minor variations in the solutions utilized between protocols. Isolated islets were subjected to quality assessment and clinical transplantations were performed in successful cases. Statistical analysis was done using multivariate regression model, T-test and ANOVA. Results: The isolation results were separated into protocols A&B as well as 3 enzymes used. There were no significant difference in age, BMI, and pancreas weight between P-A and P-B. However, the cold ischemia time was lower in P-A compared to P-B (p=0.01). Significant differences were observed in digestion time, digested islet count, digested IEQ and IEQ/gram pancreas between the groups. When islet isolations were separated according to the enzyme use we observed that high islet yield was obtained from the NEM. The percentage of isolations leading to clinical transplantation using enzymes I, II and III are 23.1, 36.8 and 67.6 respectively (p=0.01). Protocol-B resulted in a greater number of preparations suitable for clinical transplantation (67.6% Vs 34.3% with P-A, p=0.01). Even from the failed group in NEM, the average islet yield was high (3,475 ± 1,261 IEQ/gram) compared to other two enzymes. Collagenase unit/g pancreas used was not different across the groups. Conclusion: The source of isolation enzyme appears to be a critical factor affecting islet yield and may, therefore, significantly affect the clinical outcome. We conclude that human islet isolations performed with the new enzyme mixture are more productive than any other enzymes at improving the islet transplantation rate.Table: [Clinical islet isolation]

A Proteomic Approach for Investigating Pancreatic Extracellular Matrix Digestion During Human Islet Isolation

A Proteomic Approach for Investigating Pancreatic Extracellular Matrix Digestion During Human Islet Isolation

A New Approach to Contain Costs for the Establishment of a Clinical Islet Transplant (ITX) Program

Background: Despite improvements in pancreatic islet auto- and allotransplantation the diffusion of ITX programs has been limited. This is in part due to the high cost of building a specialized islet isolation facility and its operation. Facilities are built in areas requiring demolition and a complete refitting of large spaces to accommodate the isolation room, air filtration system, all ancillary services (pre-post clean area, scrubbing, changing, etc.) and new certifications that can cost $1-2 million. In addition these facilities require dedicated personnel for maintenance and operation (cleaning, monitoring, setting up etc.) 24/7. In our Institution, we developed a novel strategy for cost-containment of a clinical ITX program. Methods and Results: We designed the islet isolation facility in the operating room (OR) area at our Institution. The facility consists of ˜250 sq ft. that includes an ante-room (computer/monitor, storage, back-up refrigerator/freezer, CO2 tanks) and the fully equipped isolation facility. Floor, walls and ceiling were built of non porous surfaces for cleaning. Equipment included: 6 ft safety cabinets (2), COBE 2991 cell separator (2), mechanical shaker, organ perfusion apparatus, centrifuges (2), microscope with remotely connected camera and room screen, cabinets/storage, refrigerator/freezer, incubators, pass-through window, hands free intercom and door operation, room/equipment monitoring system (24/7) with alarm report for temperature, humidity, CO2, pressure. The cost of this conversion and equipping was approximately $200K, with an additional $100K to classify the room at 10.000 (ISO-7) particle level standards with HEPA filters. Air handler and power supply connected to emergency generator, sterilization equipment, changing and scrubbing area, storage, room monitoring system, ice maker, and certain certifications as well as personnel for cleaning, setting up and monitoring were already available in the OR reducing the cost of dedicated FTEs to 50%. Same location of cell preparation and recipient reduced risk of contamination and improved coordination between teams. We performed 8 human islet isolations and 3 clinical auto-ITX with no complication. Positive pressure was maintained at all times and microbiologic cultures were negative in all samples. Conclusions: Integration of an islet isolation facility in the OR area presents many functional advantages and a significant cost containment.

Collagenase does Not Persist in Human Islets following Isolation

Optimal human islet isolation requires the delivery of bacterial collagenase to the pancreatic islet-exocrine interface. However, we have previously demonstrated the presence of collagenase within human islets immediately following intraductal collagenase administration. This potentially has significant implications for patient safety. The present study aimed to determine if collagenase becomes internalized into islets during the isolation procedure and if it remains within the islet postisolation. Islet samples were taken at various stages throughout 14 clinical human islet isolations: during digest collection, following University of Wisconsin solution incubation, immediately postisolation, and after 24 h of culture. Samples were embedded in agar, cryosectioned, and then assessed by immunolabeling for collagenase and insulin. Immunoreactivity for collagenase was not observed in isolated islets in any preparation. Collagenase labeling was detected in one sample taken at the digest collection phase in one islet preparation only. No collagenase-specific labeling was seen in islets sampled at any of the other time points in any of the 14 islet preparations. Collagenase that enters islets during intraductal administration is washed out of the islets during the collection phase of the isolation process and thus does not remain in islets after isolation. This observation alleviates some of the important safety concerns that collagenase remains within islet grafts.