Comparison of the collagen VI content within the islet-exocrine interface of the head, body, and tail regions of the human pancreas.

Efficient islet isolation depends on the use of collagenase to digest the extracellular matrix within the islet-exocrine interface, the molecular structure of which is poorly understood. Recently it has been reported that transplantable yields of islets can be isolated from the tail segment of the pancreas alone. This study aimed to quantify and compare the amount of collagenase-resistant collagen VI within the islet-exocrine interface of the head, body, and tail of the human pancreas. Human adult pancreata (n = 5) were retrieved from heart-beating donors (age range, 40-62 years; cold ischemia times <10 hours). Tissue blocks from the head, body, and tail region of each pancreas were fixed in formalin and processed for immuno-labelling of collagen VI, which was quantified in the islet-exocrine interface using a Zeiss KS-400 image analysis system. Data were expressed as area of collagen at the interface relative to the islet area. Statistical analysis was done using paired t test. The mean islet areas in the head, body, and tail regions were not significantly different. Collagen VI was uniformly present within the islet-exocrine interface of all regions of the pancreas and was 0.326 +/- 0.064, 0.324 +/- 0.060, and 0.334 +/- 0.052 microm(2)/islet area (P = .441) in the head, body, and tail, respectively. The content of collagen VI within the islet-exocrine interface was uniform throughout all parts of the adult pancreas. Targeting this collagen subtype with novel collagenase blends may result in consistently improved islet yields and enable a wider number of available donor pancreata to be used.

The risks of total pancreatectomy and splenic islet autotransplantation.

The intraportal site is the most common site for islet transplantation. Many other sites have been tried experimentally, including the spleen, which has successfully lead to insulin independence in a number of animal models. Nevertheless, there are no detailed reports of total pancreatectomy and splenic islet autotransplantation in humans. Five patients underwent total pancreatectomy and splenic islet autotransplantation for chronic pancreatitis. Four patients had a pylorus-preserving total pancreatectomy and one patient a duodenal-preserving pancreatectomy. In three cases islets were embolized into both the portal vein and spleen. Two patients received splenic islet transplants alone. Islets were transplanted by retrograde venous infusion via the short gastric veins (n = 3), splenic vein stump (n = 1), and the left gastroepiploic vein (n = 1). The total volumes of transplanted pancreatic digest in those receiving combined intraportal and splenic autografts (n = 3) were 15.8, 13.0, and 13.5 ml. The volumes in those receiving a splenic-alone autograft (n = 2) were 12.0 and 5 ml. The mean rise in portal pressure was 18 cm of water. Complications related to the splenic autograft included a wedge splenic infarct, an emergency splenectomy, and a portal vein thrombosis in one patient having a combined intraportal and splenic autograft. Two patients developed insulin independence. two patients were still insulin independent at 1-year follow-up, and all had normal HbA1c levels (mean 5.6, range 5.2-6.3). Splenic islet autotransplantation, after total pancreatectomy, does lead to insulin independence. However, in our experience the combined procedure has a high morbidity because of splenic infarction and venous thrombosis.

A comparison of cross sectional surface area densities between adult and juvenile porcine islets of Langerhans.

The object of this study was to evaluate differences in islet diameters, their distribution and both cross sectional surface areas and densities of insulin containing islets between adult and juvenile porcine pancreata using a computerised image analysis system (Improvision). Five adult (A) (2-3 yrs) and 5 juvenile (J) (< 12 mths) Large White pancreata were assessed. Biopsies were taken from 5 different regions (posterior lobe, duodenal lobe, along with the head, body and tail regions of the splenic lobe) of the pancreas and tissue sections stained for insulin. In both A and J pancreata islet numbers increased with decreasing islet diameter, showing a skewed distribution. There was no statistical significance between the cross sectional surface area within A (mean 5.04 x 10(3) microm2) or J (mean 5.99 x 10(3) microm2) pancreata. Assuming islets are spherical, extrapolation from pir2 showed that the mean diameter for A was 80 microm and 87 microm in J. These compared with A 77 microm and J 86 microm diameters using conventional microscopic techniques. The percentage islet volume density relative to exocrine tissue, derived from the principle of Delesse (Area density = volume density), did not significantly differ between each of the 5 areas studied, either in A or J. The percentage islet volume densities did show a significant difference between A (mean 1.83%) (P = 0.001) but not between J pancreata (mean 2.13 %). In conclusion poor islet yields can be attributed to differences in islet volume density of islets within porcine pancreata. These results also suggest that the posterior and duodenal lobes should be used along with the splenic lobe in order to improve porcine islet yields. Furthermore, the current practise of reporting porcine islet yields and the isolation index relative to 150 microm (IEQs) needs to be redefined, based on the assumption that the average size of an adult porcine islet is 80 microm.

A preliminary study of the activation of endogenous pancreatic exocrine enzymes during automated porcine islet isolation.

The activation of endogenous pancreatic enzymes during automated pancreas digestion may be detrimental to islet isolation. In this report we assessed the activation of trypsin, chymotrypsin, elastase, carboxypeptidases A and B, phospholipase A2, and lipase using a porcine model. Four islet isolations were examined. Duplicate aliquots were taken from the automated circuit at 5-min time intervals up to the completion of pancreas digestion (approx 60 min). One aliquot was activated in vitro with exogenous trypsin in order to convert the enzymes into their active non-"proform," with the exception of trypsinogen, which was activated with exogenous enterokinase. This was done to assess the percentage activation of each individual enzyme (total potentially activatable enzyme release). The extent of activation between isolations was extremely variable. During the closed (recirculating) circuit phase of pancreas digestion there were both gradual and rapid increases in the levels of enzymes released. Peak activity of enzyme activation varied from 13 to 30 min; similarly, total potentially activatable peaks occurred between 13 and 38 min. Lipase and carboxypeptidase B showed greater than 70% activation, chymotrypsin, carboxypeptidase A, and phospholipase A2 between 50% and 70% activation, and trypsin and elastase less than 20%. There were up to 30-fold differences between the four islet preparations. In summary, it is unlikely that poor islet yields are soley explained by variations between collagenases; the variable activation of endogenous pancreatic exocrine enzymes is also likely to be influential to porcine islet yields.

An investigation into the distribution of different collagen types within adult and juvenile porcine pancreata.

One of the difficulties of porcine islet isolation is their fragility during collagenase digestion. The object of this study was to determine the distribution of 4 different collagen types within adult and juvenile porcine pancreata. Five different areas from each pancreas were analysed by light microscopy. The distribution of collagen types I, IV, V and VI were measured within the interlobar, intralobular (acinar), peri-insular and intra-islet regions. Their was an abundance of collagen type VI compared to I, IV and V in both the interlobar and intralobular septa in both juveniles (P<0.001) and adults (P<0.001). The periinsular collagen content also showed diversity. This was mainly attributable to the distribution of collagen type I (weak) and type VI (intense) in both adults and juveniles. In general, the peri-insular capsule was fragmentary and contained less than 50% of the total islet circumference. The latter finding taken together with the distributions of different collagen types can partly explain some of the variability of porcine islet isolation.

Different treatment regimens to reduce microbial contamination of purified porcine islets of Langerhans.

BACKGROUND: Different treatment regimens for minimizing microbial contamination of purified porcine islets were analyzed. METHODS: Purified islets, prepared from abattoir-excised pancreata (n=26), were cultured in Hams F-12 with or without antibiotics after islet isolation. Aliquots of pancreas transport media were all contaminated with microbes (100%). Of those islets cultured with no antibiotics, 70% were contaminated, compared with 42% of the islets cultured in penicillin and streptomycin (P/S) (P>0.05). A further 20 consecutive pancreata were then randomized to saline washing (N=10) before islet isolation. Islets were then cultured in either Hams F-12 with P/S (groups C and E) or Hams F-12 with gentamicin, penicillin, and amphotericin B (G/P/A) (groups D and F). RESULTS: None of those purified islets prepared from washed pancreata and then undergoing culture in Hams F-12 with G/P/A (group F) were contaminated. This compared with 30% contamination when islets were cultured in P/S (group E). Neither antibiotic regimen compromised stimulated insulin release (P=0.13). CONCLUSIONS: In conclusion pancreas washing in saline and culture in G/P/A was shown to eradicate detectable microbial contamination of purified porcine islets isolated from abattoir-excised pancreata.

Influence of different collagenase solvents and timing of their delivery on porcine islet isolation.

Methods to minimize the effect of cold ischaemia on porcine islet isolation were investigated. Forty pancreata were randomized to intraductal collagenase delivery in University of Wisconsin solution (UW) or Hanks balanced salt solution (HBSS) (control) both before and after 65 min of cold pancreas storage. Collagenase was also administered in a specially designed cold storage solution (University of Leicester solution, ULEIC), before cold storage. Median islet yield was significantly greater if the pancreas was distended with collagenase in either UW (21524 islet equivalents (IEQ) per g) or ULEIC (19814 IEQ/g) before cold storage, compared with that after distension with HBSS (6924 IEQ/g) following cold storage (P < 0.05). Islet fragmentation, islet purification and glucose-stimulated insulin release were not significantly different after collagenase delivery in either UW or ULEIC compared with those after administration in HBSS. It is concluded that porcine islet yields can be improved significantly by intraductal collagenase administration in either UW or ULEIC immediately after excision of the pancreas.

The effect of UW solution and its components on the collagenase digestion of human and porcine pancreas.

University of Wisconsin (UW) solution is used extensively as a cold storage solution during the procurement and transport of the pancreas prior to islet isolation. However, it has been observed that UW inhibits the collagenase digestion phase of human but not porcine islet isolation, resulting in poor islet yields and islets of poor viability. The aim of this study was, therefore, to confirm this species difference and to determine which components of UW are responsible for the inhibition in the human. In the initial experiment, blocks of human and porcine pancreas (n = 7) were incubated in test tubes containing collagenase at a concentration of 4 mg/mL at 37 degrees C dissolved in 4 mL of either Hanks' solution or UW. Every 5 min the tubes were manually shaken and the degree of tissue dissociation scored on a scale of + and 3+. Our results confirm the inhibition of collagenase digestion in the human but not the pig. Using the same methodology, we then investigated the components of UW that were causing the observed inhibition in the human pancreas (n = 7). This time the collagenase was dissolved in individual or combinations of UW components. Using Hank's as a control, the results were then expressed as a median ratio. The components found to be most inhibitory were magnesium, the Na+/K+ ratio, hydroxyethyl starch (HES), and adenosine. Allopurinol in combination with either lactobionate or glutathione was markedly inhibitory (i.e., median ratio 1.8 and 1.9, respectively). The most inhibitory solution tested was combination of the three components raffinose, glutathione, and lactobionate (median ratio 2.1). This combination was almost as inhibitory as UW itself (median ratio 2.7). These findings are essential for the development of effective cold-storage solutions for the human pancreas that do not inhibit the subsequent collagenase digestion phase of islet isolation.

Storage of pancreatic digest before islet purification. The influence of colloids and the sodium to potassium ratio in University of Wisconsin-based preservation solutions.

The density-dependent purification of islets from several species of mammalian pancreata is improved by prior storage of the dispersed, collagenase-digested pancreas in suitable storage solutions, such as University of Wisconsin (UW) solution. The optimal composition of such solutions, however, is not fully established, although previous investigations have suggested separately that cellular impermeants and colloids are important components. To investigate this issue further, dispersed tissues from 7 porcine and 7 human pancreata were stored in UW or in solutions containing the impermeants lactobionate and raffinose, with either no added colloid or in the presence of the colloids hydroxyethyl starch, dextran 40, dextran 250, or Ficoll 400; hydroxyethyl starch-containing solutions in which the principal cation was sodium, rather than potassium, were also studied. Subsequent purification of islets on continuous linear density gradients of BSA was then assessed by insulin/amylase assay of gradient fractions. Islet purity was slightly reduced using solutions containing impermeants but lacking a colloid, compared with using UW. In the combined presence of impermeants and a colloid, however, islet purity was similar to that obtained with UW, and for porcine pancreata, solutions containing Ficoll 400 or dextran 40 were slightly superior to UW. Purity was not, however, influenced by the sodium to potassium ratio of storage media. In conclusion, impermeants and colloids are both essential components of solutions used to preserve pancreatic tissue before islet purification, findings which may be relevant when designing media for use during other phases of islet isolation, e.g., during collagenase digestion/density gradient purification.