Activation and expression of ERK, JNK, and p38 MAP-kinases in isolated islets of Langerhans: implications for cultured islet survival.

Isolation and purification of islet cells exposes them to ischemic, osmotic and mechanical stresses. The objective of this study was to determine the roles of the MAP-kinases in islets immediately following isolation. During the first 48 h, activity of JNK1 and JNK2 declined markedly. Activity of p38 increased steadily with time in culture while extracellular signal regulated kinase (ERK) activity declined dramatically within 24 h post-isolation. High p38 activation relative to ERK activation immediately following isolation correlated with a decrease in islet survival after 36 h in culture. Absence and/or transiency of ERK signaling in conjunction with sustained activation of p38 pathway could be an important regulator of cell death in islets during and following their isolation by commonly employed procedures.

A prospective comparison of discontinuous EuroFicoll and EuroDextran gradients for islet purification.

Density gradient separation of islets from exocrine tissue is usually performed with Ficoll. However, this reagent adds significantly to the cost of the isolation. The aim of this study was to evaluate the performance of Dextran as a potential low-cost substitute for Ficoll and to evaluate the effects of cold storage of the pancreatic digest prior to purification. Pancreases were procured from mongrel dogs, loaded with collagenase and mechanically dissociated. Washed pancreatic digest was collected and divided into two fractions that were purified using discontinuous gradients on the Cobe 2991 processor using identically prepared EuroFicoll (EF) or EuroDextran (ED) gradients. Alternate groups were suspended in EC and stored on ice, while the other fraction were resuspended in the 1.108-g/mL gradient layer (either EF or ED) and loaded into the COBE. This tissue layer was overlaid with layers of densities 1.096 and 1.037 g/mL and a HBSS cap, and centrifuged for 5 min at 800 x g. Purified islets were collected from the interface between the 1.037 and 1.096 layers and islet recovery, purity, and function were assessed. From a series of eight isolations, 72.9 +/- 8.2% (mean +/- SEM) of the islets were recovered from the EF purified gradients compared with 62.6 +/- 8.3% from ED gradients (p = NS, paired t-test). Gradients of ED that were run following hypothermic storage of the digest in cold EC solution (stored ED) had reduced islet recovery when compared with islet recovery from gradients prepared in EF(stored EF) (51.6 +/- 9.6% for ED stored vs. 72.9 +/- 11.9 for EF stored, p < 0.05). Islet recovery from EF gradients was equivalent between the stored and nonstored groups. The purity of preparations from the stored ED gradients was also reduced (71.3 +/- 4.3%) when compared with islets that were immediately purified after dissociation (82.5 +/- 4.8%, p < 0.05). Static glucose stimulation assay showed equivalence between the islets from ED and EF gradients. The stimulation index (SI) was 9.3 +/- 0.9 for EF islets compared with 7.9 +/- 1.4 for ED islets for digest purified immediately. However, if the digest was hypothermically stored in EC solution, a decrease in functional viability was observed in both the EF and the ED groups (7.7 +/- 1.4 and 5.9 +/- 0.8, respectively). Out of five alloxan-induced diabetic nude mice transplanted under the kidney capsule with 2000 islets isolated from the nonstored groups, three remained euglycemic >50 days posttransplant with either EF or ED islets. These experiments demonstrate effective recovery of equivalent numbers of canine islets using discontinuous gradients of ED or EF immediately following enzymatic digestion. However, following storage of the digest in cold EC solution results in a reduction in both islet recovery and function when gradients of ED are utilized.

Evaluation of a purified enzyme blend for the recovery and function of canine pancreatic islets.

Recently developed technologies enabling the production of a reproducible, purified enzyme blend for optimal human pancreatic islet isolation has renewed interest in clinical islet transplantation. The canine model has been an ideal preclinical model for the development of islet transplantation protocols. As seen in other species, the application of crude collagenase for isolating canine islets resulted in highly variable islet yields, extensive islet fragmentation, and variable islet functionality. We compared the function of commercially available crude collagenases with that of Liberase-CI purified enzyme blend for canine islet isolation. We also compared two manufacturing runs of Liberase-CI enzyme (lots 1 and 2) to demonstrate reproducibility of islet recovery and function. We report on the improved recovery and function of islets isolated using Liberase-CI enzyme. No difference in dog age, mean body weight, or pancreas weight were observed between the experimental groups. We observed a significantly higher postpurification recovery of islet equivalent number (IE) from pancreases processed using two lots of Liberase-CI enzyme (189 +/- 20 x 10(3) IE, n = 4) and lot 2 (234 +/- 39 x 10(3) IE, n = 7) than that obtained from pancreases processed with Sigma Type V (116.8 +/- 27 x 10(3) IE, n = 5), Serva collagenase (49 +/- 11.6 x 10(3) IE, n = 5, p < 0.05) or Boehringer-Mannheim (BM) Type P collagenase (85.4 +/- 25 x 10(3) IE, n = 5, p < 0.05, ANOVA). No significant differences were observed in islet yield recovery from pancreases processed using the two production lots of Liberase-CI enzyme. Islet survival after 48 h in culture at 37 degrees C was significantly higher from islets isolated using Liberase-CI enzyme (88 +/- 3.7% survival) when compared to Sigma Type V (81.8 +/- 3.3%), Serva (71.7 +/- 2.8%), and BM Type P (77 +/- 7.2%) (p < 0.05). Islet functional testing in vitro demonstrated islets isolated using crude collagenase had an increased insulin basal release and a reduced insulin stimulated response when compared with islets isolated using the two lots of Liberase-CI enzyme. The calculated stimulation index was 7.8 +/- 1.7, 3.1 +/- 0.6, and 4.8 +/- 1.1 for Sigma Type V, Serva, and BM Type P isolated islets, respectively, compared to 15.7 +/- 1.6 and 16.2 +/- 1.9 for islets isolated with Liberase-CI enzyme production lots 1 and 2, respectively (p < 0.05). This evaluation demonstrates that a purified enzyme blend can significantly improve islet recovery and function. It also demonstrates the manufacturing reproducibility of Liberase-CI enzyme lots resulting in the isolation of canine islets with the same degree of efficacy. A blend of purified enzymes, specifically formulated for canine islet isolation, can consistently yield large numbers of islets that survive longer in culture and demonstrate an improved insulin response in vitro.

Secretion from islets and single islet cells following cryopreservation.

The ability to cryopreserve pancreatic islets has allowed the development of low-temperature banks that permit pooling of islets from multiple donors and allows time for sterility and viability testing. However, previous studies have shown that during cryopreservation and thawing there is a loss of islet mass and a reduction in islet function. The aim of this study was to measure and compare insulin secretion from cultured nonfrozen and frozen-thawed canine islets and beta-cells. Canine islets were isolated from mongrel dogs using intraductal collagenase distention, mechanical dissociation, and EuroFicoll purification. One group of purified islets was cultured overnight before dissociation into single cells and subsequent analysis. Remaining islets were cultured overnight (22 degrees C) and then cryopreserved in 2 M dimethyl sulfoxide (DMSO) solution using a slow stepwise addition protocol with slow cooling to -40 degrees C before storage in liquid nitrogen (-196 degrees C). Frozen islets were rapidly thawed (200 degrees C/min) and the DMSO removed using a sucrose dilution. From a series of seven consecutive canine islet isolations, islet recovery following postcryopreservation tissue culture was 81.5 +/- 4.8% compared to precryopreservation counts. In vitro islet function was equivalent between cultured nonfrozen and frozen-thawed islets with a calculated stimulation index of 10.4 +/- 1.5 (mean +/- SEM) for the frozen-thawed islets, compared with 12.4 +/- 1.2 for the cultured nonfrozen controls (p = ns, n = 7 paired experiments). Amperometric detection of secretion from single beta-cells in vitro has the sensitivity and temporal resolution to detect single exocytotic events and allows secretion to be monitored from single beta-cells in real time. Secretion from single beta-cells elicited by chemical stimulation was detected using a carbon fiber microelectrode. The frequency of exocytosis events was equivalent between the cultured nonfrozen and frozen-thawed beta-cells with an average of 7.0 +/- 1.32 events per stimulation for the cultured nonfrozen group compared with 6.0 +/- 1.45 events from the frozen then thawed preparations (minimum of 10 cells per run per paired experiment, p = ns) following stimulation with tolbutamide. The average amount of insulin released per individual exocytosis event was equivalent for the cultured nonfrozen and frozen-thawed islets. In addition, beta-cells responded to both tolbutamide and muscarinic stimulation following cryopreservation. It was determined that beta-cells recovered following cryopreservation are capable of secreting insulin at levels and frequencies comparable to those of cultured nonfrozen islet preparations.

Nonviral transfection of intact pancreatic islets.

Ex vivo gene transfer offers a potential means to introduce genes into cells, which may play an important role in preventing graft rejection and inducing graft tolerance. This study examined the efficiency and toxicity of several lipid-based transfection reagents (LipofectAMINE, DOTAP, and DOSPER) in intact pancreatic islets. Isolated islets were transfected with a pCMV-beta-galactosidase plasmid using several DNA/liposome ratios (1:12) of liposomes (3-72 microl) and DNA (3 and 6 microg). Transfection efficiency was quantified by microscopic evaluation of beta-galactosidase gene expression in whole intact islets. Functionality of the transfected islets was measured by insulin response to glucose solutions. All transfection reagents evaluated in this study transfected cells within the islets. As expected, untransfected controls and transfected islets with DNA alone did not express beta-gal. The highest transfection efficiency and functional viability were obtained following a 48-h incubation after exposure to the transfection mixtures as follows: 3 microl DNA and 18 microl DOTAP/ml (1:6 ratio), 6 microg DNA and 12 microl DOSPER/ml (1:2 ratio), or 6 microg DNA and 12 microl Lipofect-AMINE/ml (1:2 ratio). The highest rate of transfected cells per islet was obtained using DOTAP. In vitro functionality was not significantly different between DOTAP and nontreated controls. However, optimal transfection efficiency doses of LipofectAMINE and DOSPER significantly reduced the stimulated insulin response of the transfected islets (p < 0.05, ANOVA). The calculated stimulation index (SI) was 7.8+/-0.6 (mean +/- SEM) for DOTAP-transfected islets compared with 8.4+/-0.5 for nontransfected control islets (p = ns). The SI of DOSPER- and LipofectAMINE-transfected islets was significantly lower (6.1+/-0.5 and 3.4+/-0.5, respectively, p < 0.05). Lipid-based transfection using DOTAP at a DNA/lipid ratio of 1:6 provides an effective means of ex vivo gene delivery without compromising in vitro functionality of the transfected islets.

Submersion culture of the intact fetal lung.

Intact whole lungs from 14 d rat fetuses were explanted to organ culture and maintained fully submerged in medium or positioned at the gas-fluid interface. Striking differences were observed in the morphological development of submersion cultured and interface cultured explants after 7 d in vitro. Submersion cultured lung developed as an expanded hollow organ, whereas the tissue of interface cultures was glandular in appearance and attenuated against the culture substrate. This difference in the development of the cultured lung may be attributed to the influence of surface tension forces at the gas-fluid interface versus the fluid immersed environment. In submersion culture the fetal lung followed a unique pattern of morphogenetic development. Under these culture conditions the lung expanded in three dimensions and retained its discrete lobar structure. The primitive bronchial epithelial tree of the fetal lung at explantation exhibited substantial histoarchitectural changes in vitro. Progressive branching of the epithelium into the surrounding lung mesenchyme gave rise to a pulmonary parenchyma bearing characteristics of the presumptive alveolar region of the late gestation fetal lung. The epithelium consisted predominantly of large, glycogen rich cuboidal cells interspersed with numerous structurally differentiated type II cells, containing lamellar bodies. The pulmonary parenchyma of the submersion cultured explant occupied a narrow margin of the lung wall, surrounding a large central lumen. This unique form of the cultured lung made it possible to examine the pulmonary epithelium by scanning electron microscopy. Data from the present study suggest that submersion culture of the intact fetal lung may provide a useful model system for study of the maturation of epithelial cell surface features during pulmonary development.

Modulation of JNK and p38 stress activated protein kinases in isolated islets of Langerhans: insulin as an autocrine survival signal.

OBJECTIVE: The objective of this study was to determine the effects of islet isolation and cytokine exposure on e-JUN NH2 terminal kinase (JNK) and p38 activation and whether insulin or the p38 inhibitor PD169316 could modify the response. SUMMARY BACKGROUND DATA: Islet transplantation exposes the cells of the graft to a variety of stressful stimuli that could promote beta-cell death and lead to graft failure. METHODS: Islets from canine (n = 12) and cadaveric human (n = 6) pancreata were isolated and purified. Islets were cultured in CMRL 1066 with and without 100 ng/ml insulin. The response to cytokine stimulation with tumor necrosis factor (TNF)alpha and IL-1 beta and the p38 inhibitor PD169316 was also observed. Islet lysates were analyzed by Western blotting for total and phosphorylated JNK and p38 content. Apoptosis was assessed by TdT-mediated dUTP nick end labeling (TUNEL) assay and by a specific cell death enzyme-linked immunosorbant assay (ELISA). RESULTS: In unstimulated islets, JNK activity was highest immediately following isolation, declining over 3 days to a low baseline level. The activity of p38 was lowest immediately after isolation, increasing progressively with time. The addition of insulin resulted in a more rapid decline in JNK activity, as opposed to p38, which showed no decrease in phosphorylation in response to insulin. In the cytokine stimulation studies, IL-1 beta stimulated p38 activation in a dose dependent manner, while JNK was relatively unaffected. PD169316 (100 microg/ml) was able to inhibit p38 activation in response to the isolation procedure as well as cytokine stimulation. Apoptotic activity was highest 24 hours after isolation, and was significantly reduced when islets were maintained in insulin-supplemented medium. CONCLUSIONS: Inhibition of the stress-activated protein kinase (SAPK) pathways may be important for the maintenance of islet cell survival following islet isolation for transplantation. This study supports an autocrine role of insulin in this process.