Adenine nucleotide levels in a closed enzymatic digestion system for porcine islet isolation

Obtaining viable islets is a crucial step for successful islet transplantation. Adenosine triphosphate (ATP) is a marker of cell viability. However, little is known about any changes in the energy status of the tissues that are being digested during the digestion phase. We herein examined whether the ATP content in serially digested pancreatic tissue samples could be specific objective parameters that signal the optimal point to stop the digestion process. We obtained partial pancreata (body to tail) from 4- to 5-year-old pigs from a slaughterhouse. The tissue samples were preserved in M-Kyoto solution for less than 3 h. They were digested using an automated enzymatic and mechanical dissociation system at 37°C for 90 min following intraductal injection of Liberase HI. Samples were collected from the digestive circuit every 5 or 10 min to determine the ATP level, total adenine nucleotide (TAN) level, islet count (count/g), and yield of islet equivalent (IEQ) in the serial digestive fluids. The ATP and TAN levels, IEQ and islet count were increased and then decreased during digestion process. The profile of these parameters differed from case to case. However, when ATP changing ratio (respective value/precedent value) was compared with IEQ changing ratio, a greater than threefold increase in the ATP changing ratio followed by an increase in the islet count changing ratio within 5 min was consistently observed, indicating the optimal time to stop the digestion. The ATP levels of the handpicked islets in the digested samples were lower in the overdigested phase in comparison to those in the earlier digested phase. These results indicate that the ATP level in digested fluid could be an effective indicator to estimate the viability of cells as well as determine the optimal time to terminate the digestion process in order to obtain viable islets.     

Comparison of automated and manual methods for islet isolation

The authors used the principal features of a collagenase perfusion technique and an automated dissociation technique to determine if islets could be isolated from the large mammal pancreas and to compare the effects of the two methods on isolated islets. The pancreases of 16 dogs were cannulated and perfused with collagenase at 4 degrees C, then warmed to 37 degrees C. Group 1 (eight) pancreases were perfused at 37 degrees C until digested, then dissociated manually by teasing and trituration. Group 2 (eight) pancreases were transferred to a closed chamber for continued collagenase digestion and dissociation at 37 degrees C. Islets were purified using identical Ficoll gradients. Aliquots were stained with dithizone and evaluated for number, size and purity. Total islet volume was calculated. Group 2 pancreases were thoroughly digested leaving only a few residual ducts, but undigested fragments persisted in group 1 pancreases. Islet size was similar in both groups. There was a greater islet volume before and after Ficoll purification in group 2, but the difference was not significant. Purity was greater than 90% in both groups. Perifusion with 28 mM glucose elicited a biphasic insulin release from islets in both groups. The data show that the combined protocol enables mass isolation of purified islets from the canine pancreas. Compared with the manual technique, the automated protocol for pancreas dissociation tends to improve the yield of islets without compromising islet size and viability. It provides the advantages of a closed system with increased control over the extent of collagenase digestion.     

Improved human islet isolation by a tube method for collagenase infusion.

BACKGROUND: Collagenase infusion into the pancreatic duct is an essential step in human islet isolation. We developed a new method for ductal canulation and collagenese infusion. METHODS: A total of 53 pancreata were divided into two groups: group 1 (n=23), the new tube method, and group 2 (n=30), the standard angiocatheter method. In group 1, a polyethylene tube was inserted into the duct and pushed to the tail. The tail was first expanded, followed by expansion of the body and then the head, by pulling out the tube. RESULTS: Total islet number and number/g pancreas (mean+/-SE) were significantly higher in group 1 (481,123+/-43,218 and 8,010+/-722) (mean+/-SE) than in group 2 (300,974+/-35,122 and 5,090+/-515, P<0.01). Total islet equivalent number and islet equivalent number per gram pancreas were also significantly higher in group 1 (319,176+/-39,354 and 5,455+/-652) than in group 2 (202,022+/-23,331 and 3,722+/-468, P<0.04). Islet purity and fragmentation showed no differences between the groups. CONCLUSIONS: The tube method improved islet yields. We recommended this method for human islet isolation.     

A large-scale automated method for hepatocyte isolation: effects on proliferation in culture

Large-scale sterile methods for isolating hepatocytes are desirable for the development of bioartificial liver support systems. In this study the traditional centrifuge method was compared with the use of a Baylor Rapid Autologous Transfusion (BRAT) machine for isolating large quantities of porcine hepatocytes. After isolating hepatocytes, the methods were evaluated in terms of cell viability and yield per liver, proliferation over 7 days, and the effects on the cell cycle using the trypan blue exclusion test, conventional phase-contrast light microscopy, the lactate to pyruvate ratio, the leakage of lactate dehydrogenase (LD) and aspartate aminotransferase (AST), lidocaine clearance, albumin production, and flow cytometry. With the centrifuge method the mean cell viability was 92.5%, while with the BRAT method the viability was 95.9%. The minimal cell yields with the BRAT procedure were 7.3 x 10(9) for 250-ml centrifuge bowls and 2.8 x 10(9) for 165-ml bowls, which compares well with that found by other authors. Because the same initial procedures were employed in both methods the total hepatocyte yield per liver was comparable. Flow cytometry confirmed that the proliferation of hepatocytes was facilitated by oxygenation during the isolation procedure. The recovery of hepatocytes in culture following isolation was similar after either method. Daily microscopic investigation indicated that cytoplasmic vacuolization and granularities were present after either procedure and these disappeared following 3-4 days of culturing. Flow cytometry indicated that the hepatocyte cell cycle was similar after either method; at 7 days the profile indicated that the cells were still proliferating. Trends in the lactate to pyruvate ratio and the leakage of LD and AST indicated that the functional polarity of hepatocytes was regained after approximately 3 days. Lidocaine clearance at 4 days indicated that the cytochrome P450 system was active, while significant albumin production was apparent at day 5. The benefit of using BRAT technology in hepatocyte isolation lies in guaranteed sterility, convenience, speed, and the ability to oxygenate media and cell suspensions during the procedure.     

Validation of the scoring system for standardization of the pancreatic donor for islet isolation as used in a new islet isolation center

BACKGROUND: The aim of this study was to evaluate the effectiveness of the Edmonton Donor Scoring System for use in our much less active islet center. Because the ability to recognize an appropriate donor may help to achieve consistent and predictable success of pancreatic islet isolation, it should lead to increased effectiveness and lower cost. MATERIAL AND METHODS: Charts of 36 consecutive pancreas donors were reviewed to assess the donor points (DP). DP ranged from 0 to 100 based on donor age, body mass index, cause of death, social and medical history, hospital stay, vasopressor dosages, laboratory tests, cold ischemia time and procurement team, as well as pancreas size, consistency, fat content, damage, and quality of procurement and packing. RESULTS: Successful isolation was achieved in 39% of donors (14 of 36), a value similar to that achieved in Edmonton (40%). We used the optimal cutoff value (DP = 79) proposed by the Edmonton group. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 66%, 75%, 57%, 82% and 72%, respectively. Successful islet isolation from poor or marginal donors (DP < 49.5 and 50 to 59.5) was 0% and 28.6% respectively; it was 63% and 100% in optimal donors (DP = 80 to 89.5 and 90 to 100). We concluded that islet isolation success correlated with the previously proposed donor scoring system. CONCLUSIONS: The Donor Scoring System can be successfully implemented regardless of the level of activity of an experienced isolation center. This system permits identification of a suitable donor prior to organ processing. It may guide a center's donor selection strategy based on its goals and its budget.     

Development of a novel digestion chamber for human and porcine islet isolation

BACKGROUND: The current technique of human pancreas digestion for islet isolation relies on selective distribution of collagenase delivered via the pancreatic duct to produce digestion and removal of peri-acinar fibrous tissue. However, the collagenase has relatively little effect on the interlobular fibrous tissue, which must therefore be broken down by mechanical means within the digestion chamber so as to release the contained acini and islets. The current way of achieving this in the Ricordi chamber is to place five or six stainless steel balls within the chamber and shake vigorously. The shaking presumably breaks down the interlobular fibrous tissue by a combination of shear force induced by the movement of tissue through the shaking process, assisted by numerous blows from the steel balls. Intuitively, one would expect some islets would be destroyed rather than released by such a battering. METHODS: In an attempt to improve the efficiency of islet isolation we have designed a new digestion/filtration chamber that consists of a glass cylinder, sealed with Teflon plates holding in mesh filters at each end, secured in place by a central threaded tie-rod and external knurled nuts. A ring-shaped piston within the cylinder can be pushed up and down the travel by two rods passing out through sealed ports in the Teflon disk at one end and connected to an external handle. The handle is used to gently push the piston up and down the travel of the cylinder, which pushes the fluid and tissue through the central lumen of the ring-piston. A series of hooks attached to the central tie-rod catch the fibrous strands of the passing tissue; the shearing forces produced cause disruption by a process thought to be similar to teasing the tissue apart with fine forceps. RESULTS: A series of initial experiments with human pancreas showed the prototype to be too large, causing temperature control problems, and a redesigned smaller chamber was produced, maintaining the crucial design features. Experience processing five human pancreata has now demonstrated that in three of five pancreata the new chamber produced a good yield (>200,000 I.E.) of remarkably well separated and intact islets, the entire dispersion process being under 1 hour. However, in two isolations the collagenase digestion was poor, with few free islets. A copy of the new chamber (reserved for porcine work only) has been produced, as well as a copy of the Ricordi chamber. We have confirmed that the new chamber can isolate porcine islets in large numbers (>5000 islets/g pancreas [n = 2], but note that pig islets are small). CONCLUSION: These preliminary studies are sufficiently encouraging to justify further direct comparison with the Ricordi chamber for the purpose of animal and human islet isolation.     

Study of the human keratinocyte isolation methods and in vitro culture techniques in a single laboratory

The survey was conducted to analyze different human keratinocyte isolation methods and in vitro culture techniques used in current practice. The aim was to find which would work best in our laboratory to obtain epidermal sheets suitable for grafting. The techniques for epidermal removal and keratinocyte isolation involve dispase II or trypsin solutions at different concentrations and times. The number of cells and cellular viability were measured from different origins and size samples. The main approaches developed for in vitro culture of keratinocytes include the use of serum-containing media and of a feeder-layer of lethally irradiated mouse fibroblasts, serum-free medium in the absence of a feeder-layer and a commercially prepared serum-free medium. Other techniques include the use of different substrates such as laminin, fibronectin, collagen IV or fibrin glue to obtain more complete differentiation of epithelial sheets. The growth rate and cellular attachment have been studied using these media and support systems. In summary, for the isolation of keratinocytes, the best results were obtained with 0.17% trypsin solution overnight. The best attachment and growth rates of cell cultures have been obtained when using serum-containing medium and a feeder-layer of lethally irradiated mouse fibroblasts. Received: 15 January 1997 / Accepted: 30 April 1998     

Methods of human islet culture for transplantation

The ability to maintain isolated human islet preparations in tissue culture has recently been adopted by most islet transplant centers, and improves the safety as well as the practicality of islet transplantation. Maintaining islet viability and recovery, however, remains challenging in a clinical setting, due to stringent conditions required for culture. Islet culture is further complicated by the fact that islets do not form a monolayer. This review aims to clarify media, supplementation, and conditions that have been shown to be relevant to human islets, as well as to offer avenues of future research. Factors examined that may influence islet survival include base medium, glucose concentration, vitamin, inorganic ion, lipid, hormone, growth factor, amino acid, and binding protein composition and concentration, as well as culture temperature and seeding density. In addition, this article reviews novel techniques, such as coculture and matrices, that have been employed in an attempt to improve islet survival and functional viability.     

Key factors for human islet isolation and clinical transplantation

BACKGROUND: To further improve the outcome of clinical islet transplantation analysis of the impact of donor- and process-related factors could be of great importance. MATERIALS AND METHODS: Thirty-eight consecutive clinical islet transplantations were performed with consecutive islet isolations. Univariate analysis for donor- and isolation-related variables were correlated with recipient C-peptide levels at 2 and 4 weeks after transplantation. "Warm ischemia time" was defined as the time from start of University of Wisconsin solution perfusion in the donor until the pancreas was removed to the back table. RESULTS: Short "warm ischemia time" (WIT), low expression of tissue factor (TF) in pancreatic tissue, and high creatinine levels in the donor were variables related to high C-peptide values after islet transplantation. Furthermore, hospitalization length longer than 4 days was associated with low C-peptide levels. The number of islet equivalents (IEQ) did not correlate with the clinical outcome, possibly due to the fact that IEQ number was included in the release criteria for clinical islet transplantation CONCLUSIONS: Successful clinical islet transplantation is strongly correlated with donor and pancreas procurement factors rather than isolation process-related variables. "WIT" may induce TF expression in the pancreatic tissues. TF has been identified as the main trigger of the instant blood-mediated-inflammatory reaction in clinical islet transplantation. Therefore, assay of TF expression in pancreatic tissues could be applied as useful screening tool to identify "good" pancreata for clinical transplantation.     

Quantitative assessment of collagenase blends for human islet isolation

BACKGROUND: The variability in collagenase blends has been speculated as the single most important determinant of the success or failure in isolated islet yields in clinical islet transplantation. Examination of the formulation and potency of the widely used Liberase HI enzyme blend will uncover possible sources of imprecision. METHODS: High performance liquid chromatography (HPLC) and kinetic measurements of collagenase and protease activity were used to assess potency. Between four and nine clinical lots were assessed for various parameters such as relative formulation of collagenase isoforms, and recovered collagenase and protease potencies postreconstitution. RESULTS: Six vials from a single typical lot had a mean enzyme content of 489+/-62.5 mg (mean+/-SEM; range 398-610 mg). The mean recovered collagenase activity was 2235+/-310 Wünsch units (WU)/vial (range 1794-2968 WU/vial). The percent coefficients of variation for collagenase and protease activity in these vials were 17.4%, and 13.4%, respectively. The increase in the presence of the collagenase Ib (CIb) isoform detected by HPLC analysis was related to the chronological order of the date of manufacture. The CIb isoform was found to have a reduced specific activity compared to intact collagenase I (CI) (3.8+/-1.2 WU/mg vs. 2.1+/-0.7 WU/mg, P < 0.05). The presence of CIb was related to reduced islet yields in twelve human isolations studied. CONCLUSIONS: Variation in potency was observed between, and within lots of Liberase HI in this study. Differences in relative collagenase isoform composition may also affect the stability and potency characteristics of these blends.     

Assessment of a novel two-component enzyme preparation for human islet isolation and transplantation

BACKGROUND: Biologic characteristics of enzyme products used for islet isolation are critical for the success of islet transplantation. In particular, lot-to-lot variability significantly affects the yields of the isolation procedure. In this study, we have evaluated a new enzyme preparation in which highly purified collagenase can be blended with separately supplied neutral protease in a predetermined ratio. METHODS: We compared the results of human-islet isolations performed either with Collagenase NB1 supplemented with Neutral Protease NB (group I, n=9) or with Liberase (group II, n=9). RESULTS: Endotoxin contents of the enzyme preparations were lower in group I. Total islet yields were similar in both groups, but islet equivalents per gram of pancreas was higher in group I (4,020+/-1,240 vs. 2,360+/-1,350; P<0.05). Islet morphology was improved in group I with significantly higher proportion of free and intact islets (71+/-9% vs. 52+/-14%; P<0.01). In vitro function was improved and apoptosis rate was lower in group I. CONCLUSIONS: This new enzyme blend was as efficient as Liberase in terms of islet yields and showed improvements in islet morphology, viability, and in vitro function. The possibility to control lot-to-lot variability and the low endotoxin contents make Collagenase NB1 a promising product for human-islet isolation.     

Design of a simple, in vitro method for evaluating the efficiency of crude Clostridium histolyticum collagenase and its components for porcine islet isolation

Abstract: The wide variability of different batches of crude collagenase is one of the major factors influencing the isolation of porcine islets of Langerhans. In order to enable the production of reproducible collagenase batches, however, it is first necessary to determine which of the many components of crude collagenase are required for porcine islet isolation. For this to be done, experiments must control for the inter-pancreatic variation seen with porcine pancreata, and this requires methodology that enables many different crude collagenases or collagenase components to be tested on any one pancreas. The aim of this study was to assess four different in vitro methods for evaluating different batches of crude collagenase. Each of the methods involved placing blocks of either distended or undistended pancreas in centrifuge tubes, adding crude collagenase in various concentrations dissolved in Hanks' and incubating the tubes in a waterbath at 35°C. The remainder of the pancreas was simultaneously processed using the semi-automated method. Every 10 min, samples were taken from both the semi-automated circuit and the relevant centrifuge tubes and assessed for number of cleaved islets, cleavage index, degree of fragmentation and quality of exocrine digestion. A correlation was made between each in vitro method and the "gold standard" semi-automated method. We conclude that it is possible to use an in vitro method to evaluate crude collagenase that correlates well with the digestion occurring in the Ricordi chamber and that enables the testing of many batches or components of crude collagenase on any one porcine pancreas.     

Two-layer method in short-term pancreas preservation for successful islet isolation

BACKGROUND: We sought to determine whether the two-layer method (TLM) offers advantages over UW storage solution for locally procured pancreata with cold ischemia time of <8 hours for successful islet isolation. METHODS: From October 2003 through February 2005, 22 human pancreata were procured locally from cadaveric donors and preserved using UW solution (n = 11) or TLM (n = 11). RESULTS: Donor characteristics were similar in the two groups, with no statistical difference. Cold ischemia time was 4.5 +/- 0.6 (2.5 to 8) hours in the UW and 5.1 +/- 0.5 (3 to 8) hours in TLM group (P > .05). Organs preserved with TLM were exposed to PFC for 4 +/- 0.5 (2 to 7.5) hours. After TLM preservation, 8 of 11 (72%) pancreata yielded >300,000 IEQ pancreatic islets, which met all criteria for clinical transplantation; after UW cold storage, only 3 of 11 isolations were equally successful (27%) (P < .05). Mean IEQ was higher in the TLM than in the UW group: 349,000 +/- 37,000 vs 277,800 +/- 34,000; IEQ/g was also higher at 5100 +/- 760 vs 3000 +/- 570, respectively (P < .05). Islet quality, characterized by purity, viability, and insulin SI, did not differ statistically in the two groups: 67 +/- 4 vs 74 +/- 4%, 87 +/- 2 vs 83 +/- 4%, and 4 +/- 0.7 vs 4.8 +/- 1, respectively (P > .05). CONCLUSIONS: The Two Layer Method for locally procured human pancreata with cold ischemia time lower than 8 hours offers significant advantage over UW cold storage increasing the pancreatic islet isolation yield and the isolation success rate.     

A closed system for islet isolation and purification using the COBE2991 cell processor may reduce the need of clean room facilities

During the isolation of human islets of Langerhans the digest has repeated direct contact with the ambient atmosphere. In order to fulfill GMP requirements in clinical applications, the entire cell preparation must be performed in clean room facilities. We hypothesized that the use of a closed system, which avoids the direct exposure of tissue to the atmosphere, would significantly ease the preparation procedure. To avoid the direct atmosphere exposure we tested a modification of the isolation and purification process by performing all islet preparation steps in a closed system. In this study we compared the isolation outcome of the traditional open preparation technique with the new closed system. Pancreata from 6-month-old hybrid pigs were procured in the local slaughterhouse. After digestion/filtration the digest was cooled, collected, and concentrated in centrifugation containers and purified thereafter in the COBE2991 by top loading (control). In the control group 502 +/- 253 IEQ per gram pancreas were purified. The total preparation time amounted to 12 h. In the closed system the digest was cooled and directly pumped into the COBE2991 for centrifugation followed by supernatant expelling. Bag filling, centrifugation, and expelling were repeated several times. Islets in pellet form were then purified by adding a gradient (bottom loading). Using this closed system 1098 +/- 489 IEQ per gram pancreas were purified with a total cell viability of 67 +/- 10% and a beta-cell viability of 41 +/- 13%. The total preparation time reduced to 6 h. After 24 h of cell culture the viability of beta-cells was still 56 +/- 10% and was only reduced after the addition of proapoptotic IL-1 and TNF-alpha to 40 +/- 4%, indicating that freshly isolated islets are not apoptotic. In conclusion, the closed system preparation is much faster, more effective, and less expensive than the traditional islet preparation. The closed system may be applicable for human islets preparations to restrict the need of clean room facilities for islet preparations to a minimum and may open the way for islet preparations without clean room demand.     

Long-term preservation of the pig pancreas by a one-layer method for successful islet isolation

Background

Pancreas preservation by two-layer method (TLM) was recently established for clinical islet transplantation. The extensive use of TLM would require enormous efforts to solve logistical and technical problems. Omitting University of Wisconsin solution (UW) as second layer would facilitate the regular application of oxygenated perfluorocarbon; (PFC). To clarify whether long-term pancreas preservation is feasible by this simplified procedure, pancreases from retired breeder pigs were subjected to 7-hour preservation utilizing PFC alone in a one-layer method (OLM, n = 8) or in combination with UW (TLM, n = 10).

Methods

Resected pancreata were intraductally flushed with cold UW. Subsequently, pancreata were promptly processed (n = 6) as previously described or stored by TLM or OLM.

Results

Compared to unstored (429200 ± 86700 IEQ) and OLM-stored pancreases (338600 ± 42100 IEQ), (P = ns vs unstored) postpurification islet yield decreased after TLM storage (238000 ± 26600 IEQ, P < .05). No significant differences were found regarding purity (>90%), adenosine triphosphate (ATP) content, and viability as determined by formazan production and trypan-blue exclusion (>95%). Glucose stimulation index of freshly isolated islets (2.5 ± 0.4) was significantly decreased after TLM storage (1.8 ± 0.2, P < .05) but not after OLM storage (2.3 ± 0.6). Islet transplantation in diabetic nude mice demonstrated sustained graft function in all experimental groups.

Conclusions

This study demonstrates that viable pig islets can be successfully isolated after prolonged ischemia utilizing PFC alone for oxygenation of cold-stored pig pancreases. The easy handling of OLM could facilitate the regular application of PFC as pancreas preservation solution.     

小鼠胰岛的分离及胰岛移植

目的研究小鼠胰岛分离和移植的方法。方法在Gotoh等所介绍的小鼠胰岛分离方法的基础上作了一些改进,由原来经胆总管注入胶原酶消化液改为由胆囊注入,并在消化液和Ficoll分离液中加入了胰酶抑制剂和BSA。结果使分离纯化后的胰岛产量由原来方法的41.7±13.2个提高到了266.5±32.1个(P<0.01),活性在95%以上,除了少量导管外几乎不含腺泡组织。结论改进后的方法可以在肉眼条件下注入消化液,而不需要解剖显微镜,既方便了操作又提高了成功率;避免了整个消化和分离过程中胰酶对胰岛的消化作用,提高了得率,且具有很好的重复性。     

Resuscitation of the ischemically damaged human pancreas by the two-layer method prior to islet isolation

A two-layer cold storage method (TLM) allows sufficient oxygen delivery to pancreata during preservation and resuscitates the viability of ischemically damaged pancreata. This study determined the effect of additional preservation of ischemically damaged human pancreata by the TLM before islet isolation. Human pancreata were procured from cadaveric organ donors and preserved by the TLM for 3.2 +/- 0.5 hours (mean +/- SEM) at 4 degrees C after 11.1 +/- 0.9 hours of cold storage in University of Wisconsin solution (UW) (TLM group), or by cold UW alone for 11.0 +/- 0.3 hours (UW group). Islet isolations of all pancreata were performed using the Edmonton protocol. Islet recovery and in vitro function of isolated islets were significantly increased in the TLM group compared with the UW group. In the metabolic assessment of human pancreata, ATP levels were significantly increased after the TLM preservation. This study showed that additional short-term preservation by the TLM resuscitates the viability of ischemically damaged human pancreata before islet isolation, leading to improvements in islet recovery and in vitro function of isolated islets.     

A reliable method for isolation of viable porcine islet cells

HYPOTHESIS: Mechanical injury and oxidative stress caused by reoxygenation of isolated porcine islet cells result in their unresponsiveness to glucose stimulation. DESIGN: Adult pigs (weighing 25-30 kg) were anesthetized, and following intra-arterial infusion of ice-cold University of Wisconsin solution, a complete pancreatectomy was performed. The pancreatic duct was cannulated for infusion of digestion medium containing collagenase type P, 1.5 mg/mL; deoxyribonuclease I, 10 000 U; and a water-soluble analogue of vitamin E (Trolox), 1 mmol/L. After 20-minute incubations on ice, and at 37 degrees C, the pancreas was hand shaken for 1 minute, followed by filtration and separation on an automatic cell separator (COBE 2991). Islet cells, identified by dithizone staining, were perifused at 37 degrees C. RESULTS: The mean +/- SEM yield of intact purified islet cells (50-200 microm in diameter), and mostly present in clusters, was 2398 +/- 143 cells per gram (n = 12). Glucose stimulation caused a significant increase in biphasic insulin secretion in the perifusion experiments. CONCLUSION: We have developed a simple, reproducible, and reliable procedure for isolating intact and viable porcine islet cells suitable for xenotransplantation.