Purification Of Human Islets Research Articles

235.5: SEPAX -SEFIA a repurposed closed system for human islet purification and processing in cGMP environment

235.5: SEPAX -SEFIA a repurposed closed system for human islet purification and processing in cGMP environment

Isolation and Purification of Human Pancreatic Islets.

Successful islet isolation is the key to islet transplantation in diabetic patients. However, islet isolation is a technically complex and time-consuming manual process. Optimizing the islet isolation process can improve islet yield and quality, reduce operators, and thus reduce costs.The isolation and purification of human islets include pancreas acquisition and preservation, pancreas digestion, islet purification, islet culture, and islet quality identification. Briefly, after the duodenum was removed, the pancreas was trimmed, the main pancreatic duct was intubated at the distal end of the pancreatic head, collagenase was injected into the pancreatic duct, and the perfused pancreatic tissue was cut and then digested in a Ricordi chamber. A digestion temperature of 37°C was continuously used to assess the number of samples and the integrity of the lysed and released islets. At the end of the digestion process, collect the digested tissue in a 500 mL centrifuge tube prefilled with 25mL of cold (4°C) human serum albumin and centrifuge twice at 150g for 3min. After mixing with UW solution as islet storage solution, put it on ice (shake occasionally to prevent clumping) after 30min. Digested pancreatic tissue was centrifuged at 2200rpm for 5min in a COBE 2991 cell processor to isolate islets from exocrine tissue using a continuous density gradient. The purified islet fractions were washed twice in HBSS supplemented with 10% human serum albumin and finally collected in CMRL1066 medium supplemented with the corresponding liquid. The purity of purified islets was calculated by DTZ staining, the survival rate of islets was calculated by FDA/PI staining, and islet function was determined by in vitro glucose-stimulated insulin secretion test.

Comparison of Purification Solutions With Different Osmolality for Porcine Islet Purification.

The osmolality of the purification solution is one of the most critical variables in human islet purification during islet isolation. We previously reported the effectiveness of a combined continuous density/osmolality gradient for the supplemental purification of human islets. We herein applied a combined continuous density/osmolality gradient for regular purification. The islets were purified with a continuous density gradient without osmolality preparation [continuous density/normal osmolality (CD/NO)] or continuous density/osmolality solution with osmolality preparation by 10× Hank's balanced salt solution (HBSS) [continuous density/continuous osmolality (CD/CO)]. The osmolality of the low-density solution was 400 mOsm/kg in both groups and that of the high-density solution was 410 mOsm/kg in the CD/NO group and 500 mOsm/kg in the CD/CO group. Unexpectedly, we noted no significant differences between the two solutions in terms of the islet yield, rate of viability and purity, score, stimulation index, or the attainability and suitability of posttransplantation normoglycemia. Despite reports that the endocrine and exocrine tissues of pancreata have distinct osmotic sensitivities and that high-osmolality solutions result in greater purification efficiency, the isolation and transplant outcomes did not markedly differ between the two purification solutions with different osmolalities in this study.

An effective purification method using large bottles for human pancreatic islet isolation

The purification process is one of the most difficult procedures in pancreatic islet isolation. It was demonstrated that the standard purification method using a COBE 2991 cell processor with Ficoll density gradient solution harmed islets mechanically by high shear force. We reported that purification using large bottles with a lower viscosity gradient solution could improve the efficacy of porcine islet purification. In this study, we examined whether the new bottle purification method could improve the purification of human islets. Nine human pancreata from brain-dead donors were used. After pancreas digestion, the digested tissue was divided into three groups. Each group was purified by continuous density gradient using ET-Kyoto and iodixanol gradient solution with either the standard COBE method (COBE group) or the top loading (top group) or bottom loading (bottom group) bottle purification methods. Islet yield, purity, recovery rate after purification, and in vitro and in vivo viability were compared. Islet yield per pancreas weight (IE/g) and the recovery rate in the top group were significantly higher than in the COBE and bottom groups. Furthermore, the average size of purified islets in the top group was significantly larger than in the COBE group, which indicated that the bottle method could reduce the shear force to the islets. In vivo viability was also significantly higher in the top group compared with the COBE group. In conclusion, the top-loading bottle method could improve the quality and quantity of human islets after purification.

Evaluation of Osmolality of Density Gradient for Human Islet Purification

For pancreatic islet transplantation, the most common method of islet purification is density gradient centrifugation because of the differences in density between islets and acinar tissue. The density of islets/acinar tissue depends on several conditions, such as osmolality of purification solution. In this study, we evaluated the osmolality of iodixanol-controlled density gradients (400, 450, and 500 mOsm/kg) on the islet purification step. The density of the purification solutions was controlled by changing the volumetric ratio of iodixanol and the purification solutions (iodixanol-Kyoto solutions; IK solutions). The osmolality of density gradients was controlled by addition of 10× Hanks balanced salt solution (HBSS) solution. Density of both islets and acinar tissue increased relative to increase of the osmolality of purification solutions. There were no significant differences among the three groups on islet yield after density-adjusted purification and the rate of postpurification recovery. In vitro and in vivo assays suggest that the quality of islets was similar among the three groups. Our data suggest that efficacy of purification and quality of isolated islets is similar when the osmolality of purification solutions is between 400 and 500 mOsm/kg and density adjustment is applied. Since the density of islet and acinar tissue is changed according to osmolality, the density adjustment is important when using several osmolality solutions.

A Combined Continuous Density/Osmolality Gradient for Supplemental Purification of Human Islets.

For islet transplantation, islet purification minimizes the risks associated with islet infusion through the portal vein. However, islet purification may result in decreased numbers of islets recovered from digested tissue. In this study, we evaluated the effectiveness of performing supplemental purification (SP) after regular purification (RP). We designed the densities of low- and high-density solutions based on the outcome of RP. Moreover, a combined continuous osmolality/continuous density gradient for the SP was used in this study. Low-density/osmolality (1.075-1.110 g/cm3/400-410 mOsm/kg) and high-density/osmolality (1.090-1.125 g/cm3/495-505 mOsm/kg) solutions were produced by changing the volumetric ratio of iodixanol, 10 × HBSS, and RP solutions. The percentage of islet recovery (postpurification IE/prepurification IE × 100) after RP was 77.3 ± 5.6%, and the percentage of islet recovery after addition of SP was 85.3 ± 5.4%. In vitro and in vivo assessments showed that islet viability and function were not altered by the additional purification step. These data suggest that the addition of SP could contribute approximately 8% to islet recovery with viability and potency comparable to that obtained by RP and, therefore, that usage of the combined continuous density and continuous osmolality gradient for SP could efficiently improve islet equivalents in the final preparation.

Prediction of Pancreatic Tissue Densities by an Analytical Test Gradient System Before Purification Maximizes Human Islet Recovery for Islet Autotransplantation/Allotransplantation

Using standard density gradient (SDG) ranges for human islet purification frequently results in islet loss and transplantation of lower islet mass. Measuring the densities of islet and acinar tissue beforehand to customize the gradient range for the actual COBE 2991 cell processor (COBE) purification is likely to maximize the recovery of islets. We developed an analytical test gradient system (ATGS) for predicting pancreatic tissue densities before COBE purification to minimize islet loss during purification. Human islets were isolated from deceased donor (n=30) and chronic pancreatitis pancreata (n=30). Pancreatic tissue densities were measured before purification by the ATGS, and the density gradient range for islet purification in a COBE was customized based on density profiles determined by the ATGS. The efficiency of custom density gradients (CDGs) to recover high islet yield was compared with predefined SDGs. Pancreatic tissue densities from autografts were significantly higher than in allograft preparations. In allograft purifications, a higher proportion of islets were recovered using ATGS-guided CDGs (85.9%±18.0%) compared with the SDG method (69.2%±27.0%; P=0.048). Acinar contamination at 60%, 70%, and 80% cumulative islet yield for allografts was significantly lower in the CDG group. In autograft purifications, more islets were recovered with CDGs (81.9%±28.0%) than SDGs (55.8%±22.8%; P=0.03). CDGs effectively reduced islet loss by minimizing islet sedimentation in the COBE bag. Using ATGS-guided CDGs maximizes the islet recovery for successful transplantations by reducing acinar contamination in allograft preparations and by reducing sedimentation of islets in the COBE bag in autograft preparations.

Human Islet Separation Utilizing a Closed Automated Purification System

A central step within the human islet isolation process is the separation of islets from contaminating exocrine tissue utilizing linear, continuous density gradients manufactured by means of manually controlled standard gradient makers (SGM). The present study was performed to develop a closed, automated purification system (APS) that customizes density gradient profiles aiming to standardize and optimize human islet purification. Digested human pancreata were pooled, split evenly, and incubated in UW solution according to our standard protocol (n = 11). Continuous density gradient centrifugation was performed in parallel in two refrigerated COBE 2991 cell separators loaded with light (1.076 g/ml) and heavy (1.097 g/ml) Ficoll utilizing either an SGM or two computer-controlled pumps connected to Ficoll-containing bags. Quality control included islet equivalent (IE) yield, purity, in vitro function, and islet cytokine expression. Gradient profiles demonstrated that the APS readily customizes linear and nonlinear gradients. In comparison to the SGM, the APS recovered a higher percentage of the expected volume of continuous gradients (90.0 +/- 1.1% vs. 98.2 +/- 2.0%, p < 0.05). Islet yield (120,468 +/- 15,970 vs. 114,570 +/- 15,313 IE, NS) and purity (51.7 +/- 4.8% vs. 54.4 +/- 4.9%, NS) were nearly identical utilizing the SGM or APS. Decreased MCP-1, IL-6, and IL-8 expression indicated that APS-purified islets were possibly exposed to less proinflammatory stress. Compared to standard procedures, similar success and gentle continuous density gradient separation of human islets is feasible utilizing the APS. The APS facilitates the standardization of this complex procedure according to cGMP standards.

Optimization in osmolality and range of density of a continuous ficoll-sodium-diatrizoate gradient for isopycnic purification of isolated human islets

Introduction According to previous estimates from large animals and man, a minimum of approximately 5000 to 6000 engrafted islet equivalents (IEQ)/kg recipient weight is critical to establish insulin independence. Utilizing a single donor, this threshold yield of purified islets can be retrieved from approximately one third of all isolations. The aim of this study was to improve human islet purification by optimization of the osmolality and the density range of the continuous Ficoll-sodium-diatrizoate (FSD) gradient to facilitate consistent purities >80% of human islet preparations without considerable loss of islet yield. Methods Aliquots of human pancreatic digests were placed on continuous density gradients. After centrifugation, sequential aliquots were extracted for amylase and insulin to determine the relative and cumulative density distribution of endocrine and exocrine tissue. We addressed the impact of two factors: (1) osmolalities (300 to 600 mosm/kg) in the gradient of FSD covering a density range of 1.070 to 1.100 g/cm 3; and (2) density (FSD 500/1.070 to 1.100) versus density-osmolarity gradient (DO-FSD 400–530/1.080 to 1.113). Results The density of exocrine and endocrine tissue increased with rising osmolality. Differences in density of both tissues were highest at 450 and lowest at 300 and 600 mOsmol/kg. Purity and recovery were highest at 450 versus 400 or 500 mOsm/kg (NS). Exocrine but not endocrine tissue was more dense in DO-FSD than in FSD gradient ( P < .05). The differences in density were 0.004 versus 0.013 g/cm 3 ( P < .01), resulting in an increased islet purity and recovery. Conclusion The best osmolality for the FSD 1.070 to 1.100 g/cm 3 is at 450 mOsm/kg. Using the DO-FSD may improve human islet purification allowing successful clinical islet transplantation.

Iodixanol density gradient preparation in university of wisconsin solution for porcine islet purification.

Previously published as Graham, J.M. (2002) Purification of Islets of Langerhans from porcine pancreas. TheScientificWorldJOURNAL 2, 1657–1661. ISSN 1537-744X; DOI 10.1100/tsw.2002.847.Generally, prior to the purification of isolated pancreatic islets, the collagenase-digested tissue is incubated in the University of Wisconsin solution (UWS; ~320 mOsm) for osmotic stabilization to preserve or improve the density differences between islets and acinar fragments. The adverse effects arising from the subsequent pelleting and resuspension of the islets in a second, different (often highly hyperosmotic) purification solution are avoided in the protocol described here; preparation of the purification medium is simply achieved by mixing the UWS preincubated islets with a second UWS containing the inert impermeant iodixanol. Flotation of the islets isolated from juvenile porcine pancreases through this mildly hypertonic (~380 mOsm) gradient of iodixanol-UWS achieves a much higher recovery of islets of an improved viability than the customary method using a Ficoll gradient. The method has been extended to human islet purification.

Improved method for the isolation and purification of human islets of Langerhans using Liberase™ enzyme blend

To determine the effects of procedural modifications, 23 human islet isolations were analyzed. Isolations were divided into two groups based on the enzyme used. The influence of Liberase™, with an improved method of mechanical disassociation of pancreas, was compared to an automated method using Sevac collagenase. Pancreases were processed within 10 h of cross clamping. Following ductal injection of the enzyme, tissue was placed in the digestion chamber for disassociation. Purification was accomplished using a COBE 2991 cell processor and continuous gradients of Hypaque EuroFicoll. Isolations in Group I (Sevac) had an average yield of 138,602 ± 128,364 islet equivalents (IE) (2083 ± 1679 IE/g) with a purity of 85 ± 11%. Group II (Liberase) showed an average yield of 389,586 ± 191,161 IE (5,958 ± 3,083 IE/g) with a purity of 90 ± 6.8%. Viability was confirmed by fluorescein diacetate and propidium iodide staining, static incubations, and perifusions. In conclusion, the combination of the enzyme blend, Liberase, and a more gentle system of disassociation has proven to be a more productive method of islet isolation with higher purity than the previously published methods.

LARGE SCALE ISOPYCNIC ISLET PURIFICATION UTILIZING NON-TOXIC, ENDOTOXIN-FREE MEDIA FACILITATES IMMEDIATE SINGLE-DONOR PIG ISLET ALLOGRAFT FUNCTION

120 Early graft function has been difficult to accomplish in clinical islet allotransplantation. It is becoming apparent that reagents used for islet preparation may stimulate the inflammatory response to islet grafts and may thereby interfere with early islet function and engraftment. The purpose of this study therefore was to develop islet purification density gradient media restricted to non-toxic, endotoxin-tested components and to subsequently test whether islets purified on the refined gradients immediately reverse diabetes in the relevant preclinical single-donor pig allograft model. Density gradients were constructed based on iodixanol x-ray contrast media and UW solution which are both approved for clinical use. In the first set of experiments, the osmolality most suitable for islet separation was identified. The effect of four different osmolalities (320; 350; 375; and 400 mosm/kg) on the density profile of islet and acinar tissue was studied by means of continuous test gradients. In the second part of the study, iodixanol/UW gradients were applied for large scale continuous islet density gradient purification on a Cobe 2991 and the percentage recovery of islet equivalents (IE) in fractions with purities ≥ 90% from iodixanol/UW was compared to Ficoll Na-diatrizoate gradients (previous standard). To test whether iodixanol/UW gradients interfere with immediate reversal of hyperglycemia posttransplant, 7134±1830 purified IE/kg body weight were intraportally transplanted into five streptozotocin-diabetic pigs. The percentage of exocrine tissue contaminating 95% yield of islets were 0.45±0.96%, 0.0±0.0%, 0.45±0.69%, and 0.80±1.00% for 320, 350, 375, and 400 mosm/kg iodixanol/UW gradients, respectively. The percentage recovery of pure islet equivalents from 350 mosm/kg iodixanol/UW (n=6) gradients was significantly higher compared to the standard Ficoll Na-diatrizoate (n=4) gradient (86.2±11.8% vs 67.0±9.3%; p=0.013). Four out of five consecutive diabetic single-donor pig islet allograft recipients became normoglycemic and insulin-independent within 24 hrs following transplantation. The data indicate that single donor pig islet allografts purified on non-toxic, endotoxin-free gradients establish insulin independence immediately. Osmolality adjustments should make iodixanol/UW gradients also suitable for human islet purification. Failure to achieve immediate insulin independence in the clinical setting would then point to non-technical obstacles.

Characterization of monoclonal antibodies CBL3 and TT62 with differing antigenic determinants specific for isolated human islets

Identification, separation and purification of human islets from acinar cells following collagenase digestion of the pancreas remains a difficult problem. We have identified and characterized two monoclonal antibodies, CBL3 and TT62, with specific binding activity to human islets but not acinar cells. The antigens these monoclonal antibodies recognize are lipid in nature and thus unaffected by collagenase digestion of the human pancreas. These properties allow isolated human islets to be identified by fluoroscein isothiocyanatelabeled CBL3 and TT62, making these monoclonal antibodies potentially useful reagents in islet identification and purification.