Human Islet Isolation Research Articles

Using HTK for Prolonged Pancreas Preservation Prior to Human Islet Isolation

Histidine-tryptophan-ketoglutarate (HTK) has been established as an alternative to University-of-Wisconsin solution (UWS) for abdominal organ preservation, but data about HTK efficiency to preserve pancreata during prolonged cold ischemia time (CIT) are conflicting. In human islet transplantation, HTK provided similar isolation outcomes after short CIT. The present study aimed to investigate whether islets can be successfully isolated from HTK-preserved pancreata after prolonged CIT compared with UWS. Sixty-four human pancreata retrieved from donors meeting criteria for kidney donation were perfused utilizing either HTK or UWS and preserved for more or less than 10 h prior to islet isolation. Along with parameters related to isolation and islet quality assessment, the dry-to-wet weight ratio was evaluated. Donor- and procurement-related factors did not vary between HTK- and UWS-perfused pancreata. The dry-to-wet weight ratio was lower in HTK-preserved pancreata indicated tissue edema (21.0% ± 3.5% versus 24.8% ± 2.0%, P = 0.007). Isolation-related variables differed between experimental groups after prolonged CIT with respect to purified packed tissue volume (9.1 ± 5.0 versus 17.2 ± 8.1 μL/g, P = 0.004) and islet yield (1910 ± 980 versus 3150 ± 1420 IE/g, P = 0.012). Islet purity and survival after culture were similar after HTK or UWS perfusion. The preservation solution did not affect in vitro function and transplantability of isolated islets. Compared with UWS, HTK has similar efficiency to preserve human pancreata for subsequent islet isolation during <10 h CIT but seems to be limited for prolonged cold storage.

Calcineurin Signaling Regulates Human Islet β-Cell Survival

The calcium-regulated phosphatase calcineurin intersects with both calcium and cAMP-mediated signaling pathways in the pancreatic β-cell. Pharmacologic calcineurin inhibition, necessary to prevent rejection in the setting of organ transplantation, is associated with post-transplant β-cell failure. We sought to determine the effect of calcineurin inhibition on β-cell replication and survival in rodents and in isolated human islets. Further, we assessed whether the GLP-1 receptor agonist and cAMP stimulus, exendin-4 (Ex-4), could rescue β-cell replication and survival following calcineurin inhibition. Following treatment with the calcineurin inhibitor tacrolimus, human β-cell apoptosis was significantly increased. Although we detected no human β-cell replication, tacrolimus significantly decreased rodent β-cell replication. Ex-4 nearly normalized both human β-cell survival and rodent β-cell replication when co-administered with tacrolimus. We found that tacrolimus decreased Akt phosphorylation, suggesting that calcineurin could regulate replication and survival via the PI3K/Akt pathway. We identify insulin receptor substrate-2 (Irs2), a known cAMP-responsive element-binding protein target and upstream regulator of the PI3K/Akt pathway, as a novel calcineurin target in β-cells. Irs2 mRNA and protein are decreased by calcineurin inhibition in both rodent and human islets. The effect of calcineurin on Irs2 expression is mediated at least in part through the nuclear factor of activated T-cells (NFAT), as NFAT occupied the Irs2 promoter in a calcineurin-sensitive manner. Ex-4 restored Irs2 expression in tacrolimus-treated rodent and human islets nearly to baseline. These findings reveal calcineurin as a regulator of human β-cell survival in part through regulation of Irs2, with implications for the pathogenesis and treatment of diabetes following organ transplantation.

Quantitative Assessment of β-Cell Apoptosis and Cell Composition of Isolated, Undisrupted Human Islets by Laser Scanning Cytometry

Assays for assessing human islet cell quality, which provide results before transplantation, would be beneficial to improve the outcomes for islet transplantation therapy. Parameters such as percent β-cell apoptosis and cell composition are found to vary markedly between different islet preparations and may serve as markers of islet quality. We have developed fluorescence-based assays using laser scanning cytometry for assessing β-cell apoptosis and islet cell composition on serial sections of intact isolated islets. Isolated human islets were fixed in formalin and embedded in paraffin. Serial sections were immunostained for the pancreatic hormones and acinar and ductal cell markers. DNA fragmentation was used to label apoptotic cells. Stained cells were quantified using an iCys laser scanning cytometer. Islet preparations from 102 human pancreatic islet isolations were analyzed. For the whole set of islet preparations, we found a mean islet cell composition of 54.5%±1.2% insulin-positive, 33.9%±1.2% glucagon, 12.1%±0.7% somatostatin, and 1.5%±0.2% pancreatic polypeptide-positive cells. The apoptotic β cells were 2.85%±0.4% with a range of 0.27% to 18.3%. The percentage of apoptotic β cells correlated well (P<0.0001, n=59) with results obtained in vivo by transplantation of the corresponding islets in diabetic NODscid mice. The analysis of whole, nondissociated islets for cell composition and β-cell apoptosis using laser scanning cytometry gives reliable and reproducible results and could be performed both before islet transplantation and on preserved cell blocks at any time in future. Thus, they can be a powerful tool for islet quality assessment.

One Hundred Human Pancreatic Islet Isolations at Baylor Research Institute

The effectiveness of pancreatic islet isolation must be maximized to make islet cell transplantation (ICT) a standard therapy. We have performed 100 human islet isolations at Baylor Research Institute including islet isolations for research, for clinical allogeneic transplantation, and for autologous islet transplantation. In this study, we analyzed the results of these isolations. First, we assessed 79 islet isolations using brain-dead donors to determine variables associated with successful islet isolation. Univariate logistic regression analysis revealed that seven variables influenced the success of islet isolation for allogeneic ICT: cause of death, mechanism of death, techniques for pancreas procurement and preservation, heavy fatty infiltration, collagenase type, dilution time, and islet purification method. Multivariate regression analysis revealed that only the current isolation protocol, the Baylor Islet Isolation Method (BIIM)—with its four required elements of pancreas procurement by the team, pancreatic ductal injection, the two-layer method with perfluorocarbon, and density-adjusted density gradient purification—had a significant positive impact on successful islet isolation (P = 0.02). Second, we compared allogeneic and autologous ICT using the BIIM. There were no significant differences in islet yields between allogeneic and autologous ICT using the BIIM; total islet yield after purification was 628 ± 84 × 103 IE in allogeneic ICT vs. 576 ± 49 × 103 IE in autologous ICT (P = 0.59). This retrospective study revealed that the BIIM provided favorable outcomes for both autologous and allogeneic ICT.

Effectiveness of a Web-Based Automated Cell Distribution System

In recent years, industries have turned to the field of operations research to help improve the efficiency of production and distribution processes. Largely absent is the application of this methodology to biological materials, such as the complex and costly procedure of human pancreas procurement and islet isolation. Pancreatic islets are used for basic science research and in a promising form of cell replacement therapy for a subset of patients afflicted with severe type 1 diabetes mellitus. Having an accurate and reliable system for cell distribution is therefore crucial. The Islet Cell Resource Center Consortium was formed in 2001 as the first and largest cooperative group of islet production and distribution facilities in the world. We previously reported on the development of a Matching Algorithm for Islet Distribution (MAID), an automated web-based tool used to optimize the distribution of human pancreatic islets by matching investigator requests to islet characteristics. This article presents an assessment of that algorithm and compares it to the manual distribution process used prior to MAID. A comparison was done using an investigator's ratio of the number of islets received divided by the number requested pre- and post-MAID. Although the supply of islets increased between the pre- versus post-MAID period, the median received-to-requested ratio remained around 60% due to an increase in demand post-MAID. A significantly smaller variation in the received-to-requested ratio was achieved in the post- versus pre-MAID period. In particular, the undesirable outcome of providing users with more islets than requested, ranging up to four times their request, was greatly reduced through the algorithm. In conclusion, this analysis demonstrates, for the first time, the effectiveness of using an automated web-based cell distribution system to facilitate efficient and consistent delivery of human pancreatic islets by enhancing the islet matching process.

The Effect of Truncated Collagenase Class I Isomers on Human Islet Isolation Outcome

The Effect of Truncated Collagenase Class I Isomers on Human Islet Isolation Outcome

Comparison of Human Islet Isolation Outcomes Using a New Mammalian Tissue-Free Enzyme Versus Collagenase NB-1

After the discontinuation of the manufacturing Liberase HI because of a small potential for prion disease transmission, Roche Diagnostics (Indianapolis, IN) developed a new enzyme product (Liberase MTF [mammalian tissue free]), which is similar to Liberase HI with the exception that no mammalian tissue is used in the manufacture of the collagenase component. We report our experience using the MTF enzyme in clinical islet isolations compared with Serva NB-1 with modified enzyme delivery method. Islets were isolated from 41 pancreata using MTF enzyme (n=17) or NB-1 enzyme (n=24). NB-1 enzymes were delivered using a modified (nonsimultaneous) enzyme delivery method whereas isolations using MTF used the standard method of simultaneous collagenase and thermolysin perfusion. Islets were purified on a COBE 2991 Cell Blood Processor and subsequently cultured. The average islet mass after purification was 392+/-36 x 10 islet equivalent (IE) for MTF versus 371+/-40 x 10 IE for Serva NB-1 (P=0.63). Post-IE/cm of tissue was 110+/-9 x 10 IE/cm and 91+/-11 x 10 IE/cm for MTF and NB-1, respectively (P=0.07). The isolation success rate (>400,000 IE) for MTF was 53% compared with 33% for Serva (P=0.33). We conclude that MTF may be successfully used for high-yield human islet isolation and clinical transplantation and provides similar quality islets to those derived using NB-1.

Improved Method of Human Islet Isolation for Young Donors

Background Although islet transplantation using young donors is more effective than older donors, islet isolation from young donor is notoriously difficult. This may relate to islet ontogeny and collagen composition in the young pancreas. Therefore, we examined whether a high concentration of collagenase could improve the separation of islets from exocrine tissues resulting in an high islet yield. Methods We used six human pancreata from brain-dead donors of less than 30 years old. Islet isolation was performed based on the Edmonton protocol with modifications. All pancreata were digested with Collagenase NB1 Premium Grade (Serva). The pancreas was expanded by injecting either 200 mL of cold collagenase solution (2.5 mg/mL, standard group, n = 3) or 100 mL of solution (5 mg/mL, new group, n = 3) in a controlled manner under low pressure for 5 minutes. Then the pressure was raised for another 5 minutes. The following procedure and evaluation were performed based on the Edmonton protocol. Results Phase II time in the new group was significantly shorter than the standard group. The ratio of embedded islets in the new group was significantly lower than the standard group. The postpurification islet equivalents per pancreas weight (IEQ/g) and the recovery rate in the new group were higher than the standard group, but not significantly. There was no significant difference in the postpurification purity, viability, and final tissue volume. Conclusion Our simple modification with an initially concentrated collagenase preparation using a syringe significantly improved the ratio of embedded islets, resulting in a higher yield from young donors.

Islet Isolation From Human Pancreas With Extended Cold Ischemia Time

The general consensus among transplant centers is that a cold ischemia time (CIT) beyond 8 hours results in reduced yields and quality of human islets. We sought to optimize the isolation process and enzymes for pancreata with extended CIT. We processed 16 extended CIT pancreata (13.2 ± 0.7 hours). Donors averaged 50.8 ± 2.6 (standard error of the mean) years old with a body mass index of 28.6 ± 1.5. Glands were shipped in cold organ preservation solution without oxygenated perfluorocarbon. Isolations were performed under a protocol optimized for digestion with the new cGMP collagenase from Roche. Purification used continuous Euroficoll/University of Wisconsin gradients. Islets were cultured in two types of Prodo cGMP islet culture media and/or in Miami 1A media. Glucose-stimulated insulin secretion assays were performed after 8 to 16 days of culture. Prepurification yield averaged 415 ± 41 KIEQ postpurification, 359 ± 29 KIEQ (purification loss 13.5%); and postculture 317 ± 27 KIEQ (culture loss 11.7%). Our process liberated an average of 4278 IEQ/g of pancreas (97 ± 5 g). Most islets were recovered in the purest fraction (purity 79.7% ± 1.9%). Culture loss in our enhanced culture media was 11.7%. After 2 to 3 days in culture, viability was 92% ± 1%. Islets exhibited compactness and dithizone staining. Glucose-stimulated insulin secretion assays performed after 3 to 23 days in our PIM(R) media resulted in a stimulation index of 6.8 ± 1.7 (G50 to G350). We concluded that our human islet isolation process permitted the recovery of large numbers of high-quality human islets from extended CIT pancreata and that our cGMP islet culture media was superior to the current standard CMRL-based media.

A New Method to Value Efficiency of Enzyme Blends for Pancreatic Tissue Digestion

Islet transplantation, since the 1990s, has been an example of human cell therapy. Nevertheless, the islet isolation procedure is not completely standardized; in fact, >50% of islet procedures do not eventuate in transplantation due both to the variability of a donor's pancreas and to the unpredictable efficiency of an enzymatic blend. The enzymes used in pancreas isolation to digest several substrates are extracted from Clostridium histolyticum. In particular, they have strong collagenolytic activity compared with vertebrate collagenases. However, several impediments persist in human islet isolation success, probably owing to the variable composition and concentration of collagenases employed during the digestion phase. For islet isolation processes, neutral proteases play important roles. However, they should be considered to be double-edged swords, contributing to tissue dissociation but, sometimes, decreasing islet yield through fragmentation, breakdown, and inactivation. Protease activities cannot be preciously adjusted in a narrow range, there is no approach to determine the optimal dosage and composition of enzymes for extraction of human islets from the pancreas. At this time, available data on commercial enzymatic activity are not sufficient to predict their efficiency for pancreas digestion; consequently, it is difficult to select enzyme batches. For these reasons, we sought to generate an innovative evaluation assay to select enzymes useful for isolation procedures of pancreatic islets.

Assessment of Human Islet Isolation With Four Different Collagenases

Background The isolation of islets from the human pancreas critically depends on the efficiency of the digestive enzymes. Liberase HI had been used as a standard preparation until the issues concerning bovine spongiform encephalopathy. Thus, we must now use other collagenases for clinical islet transplantation, four of which we have evaluated herein. Methods The digestion of each of 17 pancreata from brain-dead donors was performed using the following collagenases: Liberase HI (HI; Roche, n = 9); Liberase MTF C/T (MTF; Roche, n = 4); Collagenase NB1 Premium Grade (NB1; Serva, n = 7); or Clzyme Collagenase HA (CI, VitaCyte, n = 4). Islet isolations were based on the Edmonton protocol for HI, whereas our modified islet isolation method was used for the three new enzymes (MTF, NB1, and CI). Results There were no significant differences in donor age, body mass index, pancreas size, and cold ischemic time among the four groups. The phase I time in the NB1 group was significantly shorter than in the CI group ( P = .0014). The prepurification IEQ/g in the HI group was significantly lower than the others ( P = .0003 vs MTF, .0007 vs NB1, and .0009 vs CI, respectively). The postpurification IEQ/g in the MTF group was significantly higher than in the HI group ( P = .006). The viability in the NB1 group was significantly greater than the HI group ( P = .003). Conclusion Three new enzymes (MTF, NB1, and CI) may enable us to obtain higher islet yields than with HI.

Tissue Dissociation Enzyme Neutral Protease Assessment

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 isothyocyanate–conjugated 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.

Comparison of Modified Celsior Solution and M-Kyoto Solution for Pancreas Preservation in Human Islet Isolation

Since the successful demonstration of the Edmonton protocol, islet transplantation has advanced significantly on several fronts, including improved pancreas preservation systems. In this study, we evaluated two different types of organ preservation solutions for human islet isolation. Modified Celsior (Celsior solution with hydroxyethyl starch and nafamostat mesilate; HNC) solution and modified Kyoto (MK) solution were compared for pancreas preservation prior to islet isolation. Islet yield after purification was significantly higher in the MK group than in the HNC group (MK = 6186 ± 985 IE/g; HNC = 3091 ± 344 IE/g). The HNC group had a longer phase I period (digestion time), a higher volume of undigested tissue, and a higher percentage of embedded islets, suggesting that the solution may inhibit collagenase. However, there was no significant difference in ATP content in the pancreata or in the attainability of posttransplant normoglycemia in diabetic nude mice between the two groups, suggesting that the quality of islets was similar among the two groups. In conclusion, MK solution is better for pancreas preservation before islet isolation than HNC solution due to the higher percentage of islets that can be isolated from the donor pancreas. MK solution should be the solution of choice among the commercially available solutions for pancreatic islet isolation leading to transplantation.

Successful Human Islet Isolation and Transplantation Indicating the Importance of Class 1 Collagenase and Collagen Degradation Activity Assay

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. 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. 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). 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.

Multicenter Analysis of Novel and Established Variables Associated with Successful Human Islet Isolation Outcomes

Islet transplantation is a promising therapy used to achieve glycometabolic control in a select subgroup of individuals with type I diabetes. However, features that characterize human islet isolation success prior to transplantation are not standardized and lack validation. We conducted a retrospective analysis of 806 isolation records from 14 pancreas‐processing laboratories, considering variables from relevant studies in the last 15 years. The outcome was defined as postpurification islet equivalent count, dichotomized into yields ≥315 000 or ≤220 000. Univariate analysis showed that donor cause of death and use of hormonal medications negatively influenced outcome. Conversely, pancreata from heavier donors and those containing elevated levels of surface fat positively influence outcome, as did heavier pancreata and donors with normal amylase levels. Multivariable logistic regression analysis identified the positive impact on outcome of surgically intact pancreata and donors with normal liver function, and confirmed that younger donors, increased body mass index, shorter cold ischemia times, no administration of fluid/electrolyte medications, absence of organ edema, use of University of Wisconsin preservation solution and a fatty pancreas improves outcome. In conclusion, this multicenter analysis highlights the importance of carefully reviewing all donor, pancreas and processing parameters prior to isolation and transplantation.

Large-Scale Comparison of Liberase HI and Collagenase NB1 Utilized for Human Islet Isolation

For more than a decade Liberase HI was commonly used as the standard enzyme blend for clinical human islet isolation until enforced replacement by collagenase NB1 (NB1). This change resulted initially in a reduction in islet isolation outcome and transplant activities worldwide. This retrospective study was initiated to compare the efficiency of NB1 premium grade with Liberase in 197 human islet isolations. All pancreata were processed between January 2006 and June 2008 utilizing the same procedures for isolation and quality assessment except the administration of preselected lots of either Liberase (n = 101) or NB1 (n = 96). Utilizing Liberase, significantly more digested tissue and purified islet yield was produced compared to NB1. In contrast, the use of NB1 was associated with significantly higher purity and glucose stimulation index during dynamic perifusion. The expression of proinflammatory markers was almost identical except tissue factor expression, which was higher after utilization of Liberase. No difference was found in the percentage of pancreata fulfilling the criteria for clinical islet transplantation. The results suggest that Liberase is more efficient for pancreas dissociation than collagenase NB1 but seems to be more harmful to exocrine cells and islet tissue.

Comparison of modified Celsior solution and M-Kyoto solution for pancreas preservation in human islet isolation

Comparison of modified Celsior solution and M-Kyoto solution for pancreas preservation in human islet isolation

The effect of different concentrations of Liberase HI used in a non automated method for human adult pancreatic islet isolation

Liberase HI has many advantages over traditional collagenases. Liberase performs the rapid dissociation of human pancreata and allows for the preservation of the morphological and functional integrity of isolated islets. The aim of this study was to investigate the effect of different concentrations of Liberase HI (1.5, 2.5 and 3.5 mg/ml) on the yield, viability and function of islets isolated by a non-automated method. Based on our results, the Liberase HI concentration 1.5 mg/ml is recommended for this method of isolation. .

Mammalian Tissue-Free Liberase: A New GMP-Graded Enzyme Blend for Human Islet Isolation

Islet transplantation has found its niche in diabetes treatment. It has contributed to a better quality of life and better glycemic control of patients with diabetes suffering from severe hypoglycemia that are not eligible for vascularized pancreas transplantation. Islet isolation is a technically challenging procedure. The different studies within this doctoral thesis aim to improve and standardize different steps in the isolation procedure. They are in particular looking to improve human pancreas preservation during cold storage, to optimize islet release from the exocrine tissue and to assess whether the isolated islet yield can be predicted from a biopsy. We found that pancreas preservation with pre-oxygenated perfluorodecalin (two-layer method) did not improve the ischemic tolerance of the human pancreas as compared to cold storage with the University of Wisconsin (UW) solution. Furthermore, in pancreas with long cold ischemia time (CIT) (>10 hours), Histidine-Tryptophan-Ketoglutarate (HTK) had a limited preservation capacity as compared with the UW solution with respect to isolation outcome. We also found that during enzymatic pancreas digestion, Vitacyte HA was able to provide a similar islet yield and quality as Serva NB1 with less collagenase activity and shorter digestion time. We further describe the first experience with a new GMP manufactured enzyme called Liberase MTF-S for successful human islet isolation. Finally, we found that the isolated islet yield could not be predicted from a biopsy taken from the head of the pancreas concerning solely morphological parameters of the islets tissue. The improvement of pancreas preservation will allow for marginal organs with prolonged cold ischemia time to expand the donor pool. Better knowledge of how the pancreatic extracellular matrix is digested by collagenase will lead to a fast and predictable islet release from the exocrine tissue. By standardizing the isolation procedure and improving organ selection we will increase the success rate in human islet isolation, thereby making islet transplantation available for more patients.

The effect of extracellular matrix components on the preservation of human islet function in vitro

Human islet isolation leads to the loss of the ECM basement membrane which contributes to eventual apoptosis in vitro. The reestablishment of this environment is vital in understanding the mechanism of islet interaction with its surroundings in order to arrive at conditions favourable to islet culture in vitro. In this study, we investigated the effects of the main ECM components collagen I and IV, fibronectin, and laminin on human islet adhesion, survival, and functionality. Results have provided insight into integrin-mediated effects and behaviour. Collagen I/IV and fibronectin induced adhesion, while fibronectin was the only ECM protein capable of maintaining islet structural integrity and insulin content distribution. Furthermore, islet phenotype was eventually lost, but insulin gene expression was highest in islets cultured on collagen I and IV. However, insulin release was highest on fibronectin, along with a decrease in SUR1 expression, while glucose metabolism, along with GLUT2 and GCK expression, was highest on collagen I and IV surfaces. These findings provide a basis for the future establishment of a modified three-dimensional construct for the culture of human pancreatic islets in vitro.