Successful Islet Isolation Research Articles

Analysis on Donor and Isolation-Related Factors of Successful Isolation of Human Islet of Langerhans From Human Cadaveric Donors

We analyzed the preexisting donor factors and isolation variables that affected isolation of human islets of Langerhans. Sixty-nine pancreata from cadaveric donors were analyzed for donor factors of age, gender, body mass index, cause of death as well as graft factors of cold ischemia time, pancreas status, distensibility during intraductal collagenase distension and time of collagenase expansion and digestion. Islet isolations that recovered >100,000 IEQ (n = 53) were compared to those generating less than 100,000 IEQ (n = 16) to analyze the factors affecting islet yield during donor harvest and isolation procedures. The mean islet recovery was 216.0 × 10 3 (IEQ) or 2840 (IEQ) per gram of pancreas. Mean purity was 54%. The success rate of islet isolation was 76%. Mean age was 31 years, and mean cold ischemia time was 6.9 hours. In univariate analysis, the status of the pancreas was the only significant factor for successful isolation, and gender, time of collagenase expansion and digestion were marginal factors. In stepwise multivariate logistic regression analysis of donor and isolation-related factors, donor gender, pancreas status and digestion time were significant factors. During the same period we performed three cases of clinical islet allotransplantation and one autotransplantation. This study confirmed that the same donor factors and variables in the isolation process can affect the ability to obtain successful human islet isolation. Enough experience and pertinent review of donor and isolation factors can make islet isolation consistent, supporting clinical islet transplantation without unnecessary cost.

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.

Analysis of Large-Scale Nonhuman Primate Islet Isolations

Aims It is important to have clinically relevant large animal models, especially nonhuman primates, to improve the efficacy of islet isolation and transplantation prior to clinical trials. The aim of this study was to improve the efficacy of islet isolation by analyzing large-scale nonhuman primate islet isolations. Methods Sixty-one islet isolations were evaluated using nonhuman primates. An automated isolation method was scaled down for islet isolation. Islet yields of prepurification, postpurification, and postculture, purity of islets, viability of islets, and functionality with glucose stimulation test were assessed. Initially, we analyzed relationships between endpoints then analyzed additional factors for successful islet isolation. Those factors included donor characteristics, the two-layer method (TLM) of pancreas preservation, trypsin inhibition during digestion, and digestion and collection time. Results Prepurification islet yields were strongly correlated with postpurification yields and postculture yields. It weakly but significantly correlated with purity, viability, and functionality. The average prepurification yield was 16,267 IE/g with each case divided into either above-average (high-yield group) or below-average groups (low-yield group). In 8 cases, TLM and trypsin inhibition were used and all cases belonged to the high-yield group. There were no significant differences between high- and low-yield groups in terms of donor age, body weight, pancreas weight, and cold ischemic time. The high-yield group had significantly longer digestion times and shorter collection times. Conclusions TLM, trypsin inhibition, complete digestion, and quick collections were key for successful islet isolation. Analysis of nonhuman primate islet isolation techniques provided useful information, which should help to improve clinical islet transplantation.

Technical Improvement of Human Pancreatic Islet Isolation

Introduction A key factor for successful islet isolation is to place the optimal amount of enzyme into the pancreatic ducts prior to starting digestion of pancreatic glands. To improve this procedure, we introduced novel techniques to identify and repair tissue damage resulting in leakage of collagenase solution. Materials and Methods One hundred twelve standardized consecutive islet isolations were for the effects of dye and glue on islet yield, islet function using a perifusion assay, and the possibility of clinical transplantation. One group of pancreata ( n = 26) obtained en bloc together with duodenum were carefully detached with ligation of accessory ducts in an isolation unit (WPD group), whereas the pancreata were dissected from the duodenum in the operating room in the other 86 isolations. In 28 of 86 isolations, whole glands were used (WP group), while only the body and tail area were applied in the remaining 58 isolations (PP group). Results Both dye and glue effectively prevented leakage of collagenase from the gland. Both islet yield and success rate were higher with these tools without adverse effects on islet function or collagenase activity. The success rate of isolations and islet yield were significantly higher in the WPD group ( P = .02 and .001, respectively). Conclusions Dye and glue may be useful tools to improve human islet isolation procedures. In addition, the use of the whole pancreas further improves the outcome.

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.

Can the density of native pancreatic tissue slices predict human islet isolation and purification outcome?

Introduction With currently available technology, the outcomes of human islet isolation and purification are still inconsistent, in part due to a lack of control of the pancreas donor and the procurement conditions. Using a single donor pancreas, the critical islet mass for establishing insulin independence of approximately 5000 engrafted islet equivalents (IEQ)/kg of recipient weight can only be retrieved from about one third of isolations. The purpose of this study was to analyze whether successful islet isolation and purification outcomes might be predicted from the density of native pancreatic tissue. Methods Tissue slices (TS) were obtained from the neck of 9 nondistended human donor pancreata. The density of the TS was determined using gravity sedimentation in continuous density gradients under either iso-osmolar or hyperosmolar conditions. Correlation coefficients were calculated with regard to the density of isolated exocrine and endocrine tissue, donor age, body mass index (BMI), cold ischemia time (CIT), IEQ prepurification and postpurification, IEQ recovery, and purity. Results (1) There was no change in density over time for TS in 300 mOsm/kg (mean, 1.079 ± 0.0019 g/cm 3) (2) In 500 mOsm/kg, there was a significant increase in density from 1.086 ± 0.0021 g/cm 3 to 1.092 ± 0.0021 g/cm 3 over time. (3) Density of isolated exocrine and endocrine became more distinct with lower density of TS (r = −0.776; P < .05). (4) Donor age, BMI, recovery of IEQ from gradients, and number of IEQ after purification did not correlate significantly with TS density. (5) In contrast, a significant inverse correlation existed betwen TS and CIT (r = −0.829; P < .05), and between TS versus IEQ number prior to purification (r = −0.867; P < .05). Conclusion No homogeneous distribution of pancreas tissue density was seen among 9 consecutive human organs. Taken together, the density of native pancreas TS is not a suitable sole predictor for successful islet isolation and purification.

Procurement of the human pancreas for pancreatic islet transplantation.

The full potential of pancreatic islet transplantation (PIT) has not been realized because of the difficulties associated with islet isolation, particularly when associated with a remote islet isolation center. Herein, we describe the principles of pancreatic procurement for PIT, which have allowed us to achieve a successful pancreatic islet isolation rate 67% of the time when using a remote islet isolation center. Between January 16, 2002 and June 30, 2003, 39 pancreata were procured and processed for PIT at a distant islet isolation center. All pancreata were procured by a single surgeon, and special attention was given to careful dissection of the pancreas, maintenance of arterial inflow and the pressure differential between arterial and venous systems during perfusion, rapid organ cooling, and rapid removal of the pancreas from the body. Twenty-six of 39 (67%) procured pancreata yielded more tha 5,000 islet equivalents (IEQ)/kg recipient weight and were transplanted. Median IEQs per isolation was 413,867, whereas median purity and viability were 65% and 100%, respectively. The median time for pancreatic excision was 34 minutes, whereas cold ischemia time was 6 hours and 40 minutes. The principles we have adopted for pancreatic procurement for PIT have resulted in a 67% islet isolation success rate despite the maintenance of more than 5,000 IEQ/kg and the use of a remote islet isolation center.

Influence of pancreas preservation on human islet isolation outcomes: impact of the two-layer method.

Human pancreas preservation for islet transplantation holds additional challenges and considerations compared with whole pancreas transplantation. The purpose of this study was to clarify the limitations of the University of Wisconsin (UW) solution and the potentials of the two-layer method (TLM) for pancreas preservation before human islet isolation. We retrospectively evaluated human islet isolation records between January 2001 and February 2003. One hundred forty-two human pancreata were procured from cadaveric donors and preserved by means of the UW solution (n=112) or TLM (n=30). Human islet isolations were performed using a standard protocol and assessed by islet recovery and in vitro function of islets. Eight to ten hours of cold ischemia in the UW solution is a critical point for successful islet isolations. It is difficult to recover a sufficient number of viable islets for transplantation from human pancreata with more than 10 hours of cold storage in the UW solution. The overall islet recovery in the TLM group was significantly higher than in the UW group. With 10 to 16 hours of cold storage, the success rates of islet isolations remained at 62% in the TLM group but decreased to 22% in the UW group. Transplanted islets in the TLM group worked well in the recipients. There are time limitations for using the UW solution for pancreas preservation before human islet isolation. The TLM is a potential method to prolong the optimal cold storage time for successful islet isolations.

THE TWO-LAYER METHOD AMELIORATES ISLET YIELD AND FUNCTION FROM PANCREATA WITH WARM ISCHEMIC INJURY

P1197 Aims: Shortage of cadaver donors is a problem also for islet transplantation. Recent improvements in isolated islet transplantation have led to a renewed interest in the use of non-heart-beating (NHB) donors. NHB donors could provide a significant source of pancreata for islet transplantation, but have not previously been assessed as a source of islets for transplantation since the additional factor of warm ischemic injury adversely affects the success of islet isolation. In the current study, we tested whether the two-layer method (TL) could improve islet yield and function from damaged pancreata after warm ischemia (WI). Methods: Male Lewis rats were divided into 8 groups. In groups 1 to 4, rats were subjected to 0, 30, 45 and 60 minutes of WI, respectively. WI insult was created by placing animals in 37°C warm bath for designated periods. Subsequently, islets were isolated immediately (subgroup a) or after 3-hour preservation with TL (subgroup b). Before this isolation, pancreatic tissue adenosine triphosphate (ATP) concentration was determined by HPLC. Isolated islets were assessed in terms of islet yield and function (stimulation index). Results: Table summarizes the tissue ATP levels and islet yields in all groups.FigureTL preservation increased the ATP levels in groups 2, 3 and 4. This was more pronounced in groups 2 and 3. Islet yield decreased after WI (groups 2a and 3a). However, TL doubled the yield in group 2 while mealy a small increase was observed in group 3. Increase in ATP levels did not parallel one in islet yield. Stimulation indices in groups 1-a, 2-a and 2-b were 1.66±1.06, 1.14±0.55 and 1.52±0.90, respectively. Conclusions: Islet yield and function results from this experimental study demonstrated that pancreata damaged by warm ischemia for 30 or less minutes were restored with 3-hour TL preservation before isolation. TL may allow the use of NHB donors as an alternative source of islets for transplantation.

KEY FACTORS FOR HUMAN ISLET ISOLATION AND CLINICAL TRANSPLANTATION

P454 Aims: To further increase the success rate of clinical islet transplantation analysis of the impact of donor- and process- related factors could be of great importance. Human pancreata were retrieved from organ donors applying no other exclusion than usually applied for kidney donors. Methods: One hundred twelve standardized consecutive islet isolations, of which 29 were used for clinical islet transplantation, were evaluated. Both univariate and multivariate regression analysis for donor- and process- related variables were correlated to the possibility to proceed to clinical transplantation and to the C-peptide levels of the recipients 2 weeks after transplantation. Results: Multivariate regression analysis identified maximal recorded amylase ≥100 U/l (p=0.03), low BMI (p=0.03), and the use of catecholamine (p=0.04) as crucial donor-related factors related to unsuccessful islet isolation. In addition, short cold ischemia time (p=0.005), applying a dissection technique enabling the use of the whole pancreas (p=0.02), and local procurement team (p=0.03) were identified as isolation-related variables related to successful islet isolation. Furthermore, short hospitalization length (p=0.01), the use of furosemide (p=0.01), low expression of monocyte chemoattractant protein (MCP)-1 in pancreatic tissue (p=0.02), maximal recorded amylase <100 U/l (p=0.03) and short warm ischemia time (p=0.03) were identified as factors associated to high C-peptide values 2 weeks after islet transplantation. Conclusions: The success rate of islet isolation and clinical transplantation is strongly correlated to both donor and pancreas procurement factors. Many of these adverse factors would most likely be possible to reverse by optimal management of the donor.

TECHNICAL IMPROVEMENT OF HUMAN PANCREATIC ISLET ISOLATION

P437 Aims: Despite the breakthrough introduced by ‘Edmonton protocol’, islet transplantation still has a major drawback, i.e., in most cases islets from one donor are insufficient to cure diabetic patients. A key factor for successful islet isolation is to place the optimal amount of enzyme into the pancreatic ducts prior to starting digestion of pancreatic glands. To improve this procedure, we introduced novel techniques to identify and repair tissue damages resulting in leakage of collagenase solution. Furthermore, 3 dissection procedures were also developed and compared to achieve complete digestion of glands. Methods: One hundred twelve standardized consecutive islet isolations were evaluated. Dye solutions (Metyltioninklorid®) and tissue glue (Indermil®) were applied to detect leakage and to repair the damaged parenchyma. The effects of dye and glue were evaluated in terms of islet yield, islet function using the perifusion assay, and the possibility to use the islet preparation for clinical transplantation. One group of pancreata (n=26) were obtained en bloc together with duodenum and carefully detached with ligation of accessory ducts in isolation unit (Whole pancreas with duodenum: WPD group), whereas the pancreata were dissected from the duodenum in the operation room in other 86 isolations. In 28 out of these 86 isolations, the whole glands were used (Whole pancreas: WP group), while only the body and tail area were applied in remaining 58 isolations (Partial pancreas: PP group). Results: Both dye and glue were effective to prevent leakage of collagenase from the gland. Despite having been applied to only damaged pancreata, both islet yield and success rate were higher when these tools were used (Dye: IEQs/g; 4,207±451 vs. 3,469±342 (p=0.02), success rate; 34.8% vs. 23.6% (p=0.27), Glue: IEQs/g; 4,479±809 vs. 3,579±299 (p=0.14), success rate; 50.0% vs. 22.3% (p=0.18)). No adverse effects on islet function or collagenase activity were observed. The success rate of isolations and islet yield were significantly higher in the WPD group (p=0.02 and 0.003, respectively). When WPD group was combined with the use of dye and glue, the success rate significantly increased (50.0% vs. 21.3%, p=0.02). Conclusions: Dye and glue could be applied to human islet isolation as useful tools to improve the islet isolation procedure. In addition, the use of the whole pancreas further improve the outcome.

Analysis of donor factors affecting human islet isolation with current isolation protocol

Background Appropriate donor selection is one of the keys for successful human islet isolation. Previous studies identified several critical donor factors; however, significant improvements in current human islet isolation protocols make reevaluation of donor factors necessary. Study design Review was performed on 31 human islet isolations. Islet isolations were conducted using the standard automated islet isolation method with three protocol revisions that included the two-layer method (TLM) of pancreas preservation prior to islet isolation, usage of purified collagenase mixture Liberase, and continuous density gradient for islet purification. Factors leading to successful isolations (islet yield > 100,000 IE and static incubation stimulation index greater than 2.0) were analyzed. The impacts of various risk factors were also examined. Results Donors in the successful islet isolation group had a significantly lower incidence of elevated peak transaminases and creatinine levels, lower usage of norepinephrine or cardiac arrest, less prolonged hospitalization (> 96 hours), and less prolonged preservation time of donor pancreata (>25 hours). The TLM extended acceptable preservation time of donor pancreata from 8 to 25 hours. When donors had no risk factor, the success rate was 14/16 (87.5%). In sharp contrast, when donors had two or more risk factors, the success rate was 0/7 (0%; P < .001). Conclusion Risk factors for human islet isolation with the current islet isolation protocol were identified. The decision to process pancreata based on review of donor factors should improve the consistency of human islet isolations and transplantation for curing type 1 diabetes.

Endogenous Pancreatic Enzyme Activity Levels Show no Significant Effect on Human Islet Isolation Yield.

Despite advances in human islet isolation, islet yield remains inconsistent and unreliable. In recent studies, it has been suggested that serine proteases, in particular trypsin, have been shown to have a damaging effect on islet yield. This study evaluated enzyme activity levels throughout 42 human islet isolation procedures. Trypsin, chymotrypsin, and elastase activity was determined spectrophotometrically using suitable chromophoric substrates. The results of the islet isolations were rated as successful (n = 19) or unsuccessful (n = 23) based on the islet yield and functionality. The enzyme activity profiles of the isolations were compared. No significant differences in donor-related variables were found in this study. However, in the successful isolations, a significantly greater amount (85.6 ± 1.9%; p = 0.0017) of the pancreas was digested in a significantly shorter digestion time (19.7 ± 0.6 min; p = 0.0054) compared with 74.8 ± 2.5% of digested tissue in 22.6 ± 0.7 min in the poor isolations. This study showed no significant effect of serine protease levels on the outcome of islet isolations, regardless of enzyme inhibitor supplementation. These data suggest that serine protease activity does not sufficiently affect islet yield. However, the data show that the most successful human islet isolations are achieved when the maximum amount of tissue is digested in the shortest amount of time. This suggests that further understanding of the isolation process should focus on the role of the collagenase digestion solution in the dissociation of the endocrine-exocrine tissue connection.

Adjustment of neutral protease activity promotes Successful porcine islet isolation after prolonged cold storage

Adjustment of neutral protease activity promotes Successful porcine islet isolation after prolonged cold storage

The utilization of recombinant collagenase for Successful human islet isolation

The utilization of recombinant collagenase for Successful human islet isolation

The Morphology of Islets within the Porcine Donor Pancreas Determines the Isolation Result: Successful Isolation of Pancreatic Islets can Now be Achieved from Young Market Pigs

Clinical islet allotransplantation has become an increasingly efficient "routine" therapy in recent years. Shortage of human donor organs leads to porcine pancreatic islets as a potential source for islet xenotransplantation. Yet it is still very difficult to isolate sufficient numbers of intact porcine islets, particularly from young market pigs. In the following study islets were successfully isolated from retired breeders [4806 +/- 720 islet equivalents per gram organ (IEQ/g); n = 25; 2-3 years old; RB] and also from young hybrid pigs [2868 +/- 260 IEQ/g; n = 65; 4-6 months old; HY] using LiberasePI and a modified version of Ricordi's digestion-filtration technique. As expected, isolations from RB showed significantly better results (p < 0.002). A retrospective histological analysis of almost all donor pancreases showed that the majority of organs from RB (80%) contained mainly large islets (diameter > 200 microm), in contrast to only 35% of all pancreases from HY. Remarkably, the islet size in situ, regardless whether detected in RB or HY, strongly determined the isolation result. A donor organ with predominantly large islets resulted in significantly higher numbers of IEQs compared with a donor organ with predominantly small islets [RB(Large Islets): 5680 +/- 3,318 IEQ/g (n= 20); RB(Small Islets): 1353 +/- 427 IEQ/g (n = 5); p < 0.02]. In addition, isolation results were strongly influenced by the quality of the LiberasePI batch, and therefore single batch testing is invariably required. Purification was performed using Ficoll or OptiPrep density gradient centrifugation manually or in the COBE cell processor. Although islet purity was highest when OptiPrep was used, final islet yields did not differ between the different purification methods. Our study demonstrates that islet size in situ is an extremely critical parameter for highly successful islet isolation; consequently, we are now performing a morphological screening of each donor organ prior to the isolation process. Under these conditions highly successful isolations can reliably be performed even from young market pigs.

NON-HEART-BEATING ORGAN DONORS

The shortage of organ donors relative to the number of patients on transplant waiting lists has led to a renewed interest in the use of non-heart-beating (NHB) organ donors in many centers. The lack of donors is also a problem for islet transplantation. The disparity between donor organs and potential recipients is further exacerbated by the requirement to transplant a large number of islets to increase the chance of success and the high level of variability in islet isolation yield. Non-heart-beating (NHB) donors have not previously been assessed as a source of islets for transplantation, and it is unknown what affects the additional factor of warm ischemic injury associated with NHB organs may have on the success of islet isolation. This study assesses the yield and function of islets from NHB donors and compares the results with islets obtained from heart-beating brain-dead (HB) donors. There were no differences in the yield of islets per gram of pancreas, 1788 (0-4620) NHB vs. 1580 (26-2544) HB (median, range). The secretory function was also similar in both groups, with stimulation indices of 0.71-3.49 for NHB vs. 0.30-3.57 for HB (overall range). There was no correlation between islet yield and warm ischemia time in the NHB donor group. In conclusion, the study has demonstrated that it is possible to isolate large numbers of islets from NHB donor pancreata and that, where NHB donor programs exist, these could provide a significant addition to the number of potentially transplantable islets.

Evidence for a significant correlation of donor pancreas morphology and the yield of isolated purified human islets.

In clinical islet transplantation to patients with type 1 diabetes mellitus, the number of isolated and purified islet has been identified as a key determinant for functional success of the islet graft. With improved isolation methods based on the original procedure published by Ricordi et al. yield and function of isolated islets were considerably enhanced. However, there is still a large variance in the number, purity, viability and secretory capacity of islets isolated from brain-dead human donor pancreata, significantly hampering utilization of human islet preparations derived from a single donor for one diabetic recipient. The reasons for the limited success in islet isolation and purification have not been clarified in detail yet. Recent studies have indicated, that donor preconditions, and a number of technical factors during organ procurement and the islet isolation process itself are critical to successful islet isolation. This study aimed at identifying distinct morphological and histopathological characteristics of the donor pancreas as determinants for the outcome of human islet isolation and purification.

Fractions from commercial collagenase preparations: use in enzymic isolation of the islets of Langerhans from porcine pancreas.

Transplantation of isolated islets of Langerhans is an intriguing possibility for the treatment of diabetes mellitus. The isolation of islets from pancreata requires the specific dissociation of the tissue. Commercial collagenases from Clostridium histolyticum are widely used for this purpose. Unfortunately, the effectiveness of these commercial enzymes is not predictable and differs considerably between suppliers and even from lot to lot. This is due mainly to differences in their specific collagenase activity and to the presence of other lytic enzymes, as well as to other contaminants. Free flow zone electrophoresis (FFZE) was used to separate the effective protein components from undesired compounds and to prepare a digestive enzyme mixture with controlled composition of lytic activities. Fractionation of crude collagenases by FFZE resulted in partially purified protein fractions that were enriched for collagenase and tryptic activities, and contained only trace amounts of neutral protease. These preparations proved to be highly effective in an in vitro assay for the libration of viable islets from porcine pancreas. To scale up the production of these collagenases with defined enzyme composition, we fractionated two different lots of a commercial collagenase from C. histolyticum (one lot effective in islet isolation, the other not) by using fast protein liquid chromatography (FPLC) on hydroxyapatite. Again, high efficacy of islet release from pancreatic tissue was correlated to high specific tryptic and collagenase activities and low levels of neutral protease. The chromatographic protocol developed in this study converted a non-effective collagenase lot into a preparation that allowed successful islet isolation.

Fractions from commercial collagenase preparations: Use in enzymic isolation of the islets of Langerhans from porcine pancreas

Transplantation of isolated islets of Langerhans is an intriguing possibility for the treatment of diabetes mellitus. The isolation of islets from pancreata requires the specific dissociation of the tissue. Commercial collagenases from Clostridium histolyticum are widely used for this purpose. Unfortunately, the effectiveness of these commercial enzymes is not predictable and differs considerably between suppliers and even from lot to lot. This is due mainly to differences in their specific collagenase activity and to the presence of other lytic enzymes, as well as to other contaminants. Free flow zone electrophoresis (FFZE) was used to separate the effective protein components from undesired compounds and to prepare a digestive enzyme mixture with controlled composition of lytic activities. Fractionation of crude collagenases by FFZE resulted in partially purified protein fractions that were enriched for collagenase and tryptic activities, and contained only trace amounts of neutral protease. These preparations proved to be highly effective in an in vitro assay for the liberation of viable islets from porcine pancreas. To scale up the production of these collagenases with defined enzyme composition, we fractionated two different lots of a commercial collagenase from C. histolyticum (one lot effective in islet isolation, the other not) by using fast protein liquid chromatography (FPLC) on hydroxyapatite. Again, high efficacy of islet release from pancreatic tissue was correlated to high specific tryptic and collagenase activities and low levels of neutral protease. The chromatographic protocol developed in this study converted a non-effective collagenase lot into a preparation that allowed successful islet isolation.