Michael A.J. Zieger

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.

Water and cryoprotectant permeability characteristics of isolated human and canine pancreatic islets.

Cryopreservation allows accumulation of the necessary islet transplantable mass as well as adequate time for tissue typing and infectious disease screening. Cryopreservation protocols may be optimized by modeling the osmotically induced volume excursions that occur during the addition and removal of cryoprotective agents (CPAs). To that end, three transport parameters were measured at 22°C in canine and human islets isolated by collagenase digestion and euroficoll purification: (i) the apparent hydraulic conductivity (Lp), (ii) the permeability coefficient of the CPA (Ps), and (iii) the associated reflection coefficient (σ). The parameters were determined by volumetric analysis of islets upon abrupt exposure to 1, 2, and 3 M dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol (GLY), and propylene glycol (PG). The parameters were calculated using the Kedem-Katchalsky theory to describe islet volume excursion kinetics (assuming islets to be single equivalent osmotic units with the same volume and surface area of the actual islet) and a three-parameter curve fit was performed using the Marquardt-Levenberg method. It was determined that the permeability characteristics of pancreatic islets are species specific, and based upon the measured parameters, the highest Ps values for canine islets were observed following exposure to 2 M EG, and the highest Ps values for human islets were observed following exposure to 2 M PG. The permeability parameters were analyzed adjusting for islet radius using ANCOVA procedures to acquire least square means. For canine islets exposed to 2 M EG these values were determined to be 0.936 μm/min/atm, 2.47 μm/s, and 0.90 (for Lp, Ps, and ϕ, respectively) and for human islets exposed to 2 M PG the values were determined to be 1.56 μm/min/atm, 3.48 μm/s, and 0.85 (for Lp, Ps, and σ, respectively). These parameters were used in a model to calculate osmotically induced islet volumetric response upon addition/dilution of the optimum CPAs, taking into consideration critical volume excursion limits at which irreversible damage occurs.

Osmotic tolerance limits of canine pancreatic islets.

Future improvements in the recovery and function of pancreatic islets following cryopreservation will require a more precise quantification of the stresses that occur at each stage of the cryopreservation protocol. Changes in solution osmolality during the addition and dilution of cryoprotectants and during freezing and thawing induce changes in islet volume that may exceed tolerable limits. The aim of this study was to determine the range of solution osmolalities that results in significant changes in islet function. Islets were isolated from canine pancreases by collagenase digestion and Euro-Ficoll purification. Following 12-h culture at 37°C, islets were counted and dispensed into multiwell plate inserts. Islet function was assessed in each well immediately before and 24 h following a 10-min osmotic challenge with hypo- or hyperosmotic solutions of PBS (0, 75, 150, 300, 600, 1200, or 2300 mOsm/kg) at 22°C. Canine islets reached their osmotic equilibrium within 10 min. Duplicate wells were used for each osmolality treatment for each of six donors (n = 12). No significant differences in basal or glucose-stimulated insulin secretion were found between wells prior to the osmotic challenge (3.35 ± 0.45 and 20.98 ± 3.36 μIU/IE/h, respectively). Following the osmotic challenge and 24-h in vitro tissue culture, a significant increase in basal secretion was observed for islets exposed to 0 and 75 mOsm/kg solutions and a significant decrease for islets exposed to 2300 mOsm/kg solution. Islets exposed to 0 and 2300 mOsm/kg solutions showed significant decreases in the stimulated insulin secretion when compared to controls. Solution osmolalities of 150–1200 mOsm/kg appear to be tolerated by canine islets with no significant deviations in insulin secretion. The corresponding tolerable volume range was 152.6 ± 6.8% to 60 ± 5.1% of the isotonic islet volume. The minimum critical volume was used in a theoretical analysis of the islet volumes that would result from equilibrium freezing in dimethyl sulfoxide (DMSO). The calculations show that 1.5 mol/l DMSO is sufficient to prevent damage to islets due to excessive shrinkage. Further refinement of cryoprotectant addition and dilution protocols, and cooling and warming protocols for canine islets, are now possible.

Improved islet survival and in vitro function using solubilized small intestinal submucosa

In vitro proliferation of isolated pancreaticislets has become an area of great interest given the scarcity of clinicalisletdonors and the islet mass requirements for clinical islet transplantation.Smallintestinal submucosa (SIS), a naturally occurring extracellular matrix, hasbeeninvestigated to promote wound healing, tissue remodeling and cell growth. Thisstudy evaluated recovery and function of isolated canine pancreatic isletsfollowing in vitro tissue culture. Pancreatic islets wereisolated from mongrel dogs using standard surgical procurement followed byintraductal collagenase distension, mechanical dissociation and EuroFicollpurification. Groups of purified islets were cultured in a humidifiedatmosphereof 95% air and 5% CO2 for 48 hours in standard islet cultureconditions of CMRL 1066 tissue culture media (Gibco) which had beensupplementedwith 25μM HEPES, penicillin/streptomycin and either 10% heat inactivatedfetal calf serum (FCS, Gibco) or solubilized SIS solution (Cook Biotech, Inc.,West Lafayette, IN). The mean recovery of islets following the culture periodwas determined by sizing duplicate counts of a known volume and viability wasassessed by static incubation with low glucose (2.8 mM), highglucose (20 mM) and high glucose solution supplemented with 50μm IBMX solution. Remaining islets were embeddedhistologically.From a consecutive series of six culture experiments, a significantly higher (p< 0.05) recovery of islets co-cultured with SIS was observed when comparedtocontrols. Mean islet recovery was 84.5 ± 2.9% (mean ± SEM) fromthe SIS cultured group compared with 64.7 ± 4.5% from the control groupcultured in FCS (p < 0.05, n=6). Islets from the SIS treated group exhibiteda significantly higher (p <, 0.05) insulin response to the high glucosestimulus than islets cultured in the standard FCS cultured solution. Thecalculated stimulation index was 12.3 ± 3.4 for the SIS-treated groupcompared with 5.6 ± 1.8 for the standard cultured group (p < 0.05).The overall mean numbers of islets recovered following invitro culture was also higher in the SIS-treated group. Theproportion of islets with a mean diameter >150 μm increasedfrom 24% to 31% in the SIS-treated group, whereas the same proportion decreasedto 18% from 22% in the control (FCS-treated) group. Histological evaluation offixed tissue samples collected following the culture period identified insulinand glucagon-secreting cells in the SIS and FCS treated groups, however ahigherfrequency of insulin positive cells were detected consistently in the SIStreated group. A proliferation marker (PCNA) identified positive cells withinboth groups as well. This study suggests that co-culture of freshly isolatedcanine islets in medium supplemented with solubilized SIS can improve thepost-culture recovery and in vitro islet function. Futureinvestigations will focus on the cellular interactions of SIS, bothinvitro and in vivo.

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

Abstract 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 × 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 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.

The effects of microencapsulation on pancreatic islet osmotically induced volumetric response.

Microencapsulation of pancreatic islets has been proposed as a means to prevent allograft rejection and to protect islets during cryopreservation. The aim of this study was to investigate: 1) the effects of the cryoprotectants (CPAs) dimethyl sulfoxide (DMSO) and ethylene glycol (EG) on the volume of Ca2+ alginate microcapsules, and 2) the effects of microencapsulation on the volumetric response of human and canine pancreatic islets during CPA equilibration. Stock sodium alginate with a high mannuronic acid content (HM) or a high guluronic acid content (HG) was used to generate empty capsules (mean diameter 200 μm) with an electrostatic generator. The capsules were held in place by a holding pipette system and videotaped during the addition of 2 or 3 M CPA at 22°C. Islets (isolated from human cadaveric donors and mongrel dogs and then cultured overnight at 37°C) were encapsulated in alginate (HM), loaded into a microperfusion chamber, and the change in islet volume was videotaped after exposure to the same CPAs and concentrations. These were compared to the volume responses of nonencapsulated islets. Images were analyzed using a computerized image analysis system and the data were analyzed using ANOVA. HG microcapsules showed a significant (p < 0.05) increase in volume following exposure to EG but not to DMSO. HM microcapsule volume did not change significantly following exposure to either EG or DMSO and was therefore chosen as the substrate for islet encapsulation. Free, nonencapsulated canine and human islets responded to the osmotic challenge of the 2 M DMSO by shrinking to 70.00 ± 1.04% (mean ± SEM) and 70.11 ± 1.05%, and in 2 M EG to 72.89 ± 1.93% and 69.33 ± 1.38%, respectively, of the isotonic volume before returning to the original cell volume. Exposure to 3 M DMSO or EG resulted in a further dehydration to 65.89 ± 0.91.% and 67.67 ± 1.91% for canine and 62.22 ± 0.66.% or 65.89 ± 1.30% for human islets. Minimum volumes were reached within 30–40 s after exposure to the cryoprotectant. Encapsulated human islets reached 86.88 ± 1.47% of their original volume in 2 M and 80.33 ±0.89% in 3 M DMSO, and 87.33 ± 1.86% in 2 M and 82.80 ± 1.57% in 3 M EG. This volume change was significantly less (p < 0.01) than that observed in corresponding free islets. Encapsulated canine islets reached 83.67 ± 2.13% of their original volume in 2 M and 78.22 ± 0.95% in 3 M DMSO, and 85.44 ± 1.92% in 2 M and 78.11 ± 2.01% in 3 M EG. As with human islets, this was significantly different than free islets (p < 0.01). These minimal volumes were reached within 30–50 s. These results demonstrate that there are cryoprotectant and alginate-specific interactions and that microencapsulation modulates the degree of osmotically induced shrinkage of islets. The development or modification of existing cryopreservation protocols to improve postcryopreservation recovery or function must account for these factors.

Lung Preservation: Pulmonary Flush Route Affects Bronchial Mucosal Temperature and Expression of IFN‐γ and Gro in Regional Lymph Nodes

Optimal lung preservation via flush of the pulmonary vasculature minimizes early graft failure post‐lung transplantation. We hypothesized that the route of pulmonary flush has differential effects on thermal gradients in the lung and expression of inflammatory mediators. Swine underwent antegrade flush (AG) via pulmonary artery; AG/RG: antegrade + retrograde flush via pulmonary veins or AG/BA: antegrade + bronchial artery flush via bronchial artery. Temperatures were recorded in bronchial mucosa and peribronchial lymph nodes. RT‐PCR was utilized to detect cytokine gene expression in the nodes. AG/BA flush resulted in greatest cooling of bronchial mucosa and lymph nodes (p < 0.001). The route of flush did not affect expression of RANTES, MCP‐1, IL‐8, IL‐1β, TNF‐α or IL‐6. However, expression of Gro was reduced 4‐h post‐preservation in all groups. Only AG/BA resulted in decreased IFN‐γ transcripts. These data show that, compared to AG or AG/RG, AG/BA flush results in the greatest cooling of lung compartments and down regulates lymph node expression of a cytokine and chemokine that have key roles in inflammation and immunity. These data suggest that pulmonary flush via AG/BA during donor harvest may be optimal to decrease the risk of early graft failure.

A Theoretical Examination of the Biophysical Factors for Development of an Optimized Cryopreservation Procedure for Canine Islets

Pancreatic islet cryopreservation is necessary to facilitate organizational aspects of transplantation, including islet banking, tissue matching, organ sharing, immunomanipulation of islets, and multiple-donor transplantation. It may not be ideal, however, to use a general protocol in which the same cryopreservation strategy is applied to islets isolated from different species. The present study presents a theoretical discussion, based on the use of experimentally measured, species-specific parameters, to propose optimized cryopreservation protocols specific for canine pancreatic islets. This study builds upon previously determined canine islet osmotic and permeability data by measuring hydraulic conductivity and ethylene glycol (EG) permeability at below ambient temperature, allowing calculation of activation energies for hydraulic conductivity and cryoprotectant solute permeability, which were found to be 9.6 and 17.3 kcal/mol, respectively. These data were then used, in conjunction with water/NaCl/EG p...

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.

Hypoosmotic exposure of canine pancreatic digest as a means to purify islet tissue

The development of more effective means to separate pancreatic islets from the unwanted exocrine tissue would greatly advance the field of clinical islet allotransplantation in the treatment of insulin-dependent diabetes mellitus. Recent experiments with hamster islets have demonstrated a selective destruction of dissociated single exocrine cells when exposed to hypotonic conditions. It was the aim of this study to extend these observations to the canine model with collagenase dissociated pancreatic tissue and to evaluate the treatments effect on islet function. Pancreases from five mongrel dogs were digested using an automated protocol of intraductal delivery of collagenase, and gentle dissociation. Duplicate samples of pancreatic digest were removed for insulin and amylase determination prior to and immediately following exposure to 50 mOsm/kg salt solution for a period of 30, 60, or 300 s before returning the digest to isoosmotic conditions. The remaining digest was cultured for a period of 48 h at 37 degrees C before the tissue was recombined, washed, and a third sample removed for insulin and amylase. In vitro viability was then assessed using a static incubation assay with insulin content measured using a double-antibody radioimmunoassay, and amylase was determined using a colorimetric assay system. No difference in the insulin or amylase levels between the experimental groups was observed immediately following the hypotonic exposure; however, a significant decrease in the amylase content was observed following the 48-h culture period in digest that had been hypoosmotically exposed for 60 or 300 s compared with the pretreatment group (2.83 +/- 0.41 IU amylase/mg pancreas vs. 1.29 +/- 0.21 and 0.83 +/- 0.12, mean +/- SEM, p < 0.05). Insulin content was also significantly reduced in the 300-s exposure group compared with nontreated controls (3.2 +/- 0.6 mU insulin/mg pancreas vs. 2.0 +/- 0.2). The insulin/ amylase ratio (I/A), a measure of islet and exocrine content, was 1.1 +/- 0.13 following pancreas dissociation and 1.34 +/- 0.21 for control tissue cultured for 48 h. The I/A ratio increased following hypoosmotic exposure to 1.50 +/- 0.31 for tissue exposed for 30 s, 1.77 +/- 0.19 for 60-s exposure, and 2.54 +/- 0.13 for tissue exposed for 300 s (p < 0.05, vs. pretreatment group). In vitro insulin secretion was equivalent with the exception of the tissue exposed for 300 s, which had an increased basal level of insulin resulting in a significantly decreased stimulation index (3.8 +/- 0.5 vs. 8.1 +/- 1.2 for the purified islet control group, p < 0.05). These results suggest that a brief hypotonic exposure to pancreatic digest can alter the insulin/amylase ratio; however, there is a functional impairment on subsequent islet function after a period of in vitro tissue culture.