Cost and Scalability Analysis of Porcine Islet Isolation for Islet Transplantation: Comparison of Juvenile, Neonatal and Adult Pigs.

The limited availability of human islets has led to the examination of porcine islets as a source of clinically suitable tissue for transplantation in patients with diabetes mellitus. Islets from porcine donors are commonly used in both in vitro and in vivo experiments studying diabetes mellitus. However, there are significant differences in quality and quantity of islet yield depending on donor pig age, as well as substantial differences in the costs of pancreas procurement in adult versus neonatal and juvenile pigs. In this study, we compared the total cost per islet of juvenile pig pancreata with that of neonatal and adult pigs. Although adult porcine pancreata yield, on average, more than five times the amount of islets than do juvenile and neonatal pancreata, we found that the high price of adult pigs led to the cost per islet being more than twice that of juvenile and neonatal islets (US $0.09 vs $0.04 and $0.02, respectively). In addition, neonatal and juvenile islets are advantageous in their scalability and retention of viability after culture. Our findings indicate that isolating neonatal and juvenile porcine islets is more cost-effective and scalable than isolating adult porcine islets.

In vitro maturation of viable islets from partially digested young pig pancreas.

Isolation of islets from market-sized pigs is costly, with considerable islet losses from fragmentation occurring during isolation and tissue culture. Fetal and neonatal pigs yield insulin unresponsive islet-like cell clusters that become glucose-responsive after extended periods of time. Both issues impact clinical applicability and commercial scale-up. We have focused our efforts on a cost-effective scalable method of isolating viable insulin-responsive islets. Young Yorkshire pigs (mean age 20 days, range 4-30 days) underwent rapid pancreatectomy (<5 min) and partial digestion using low-dose collagenase, followed by in vitro culture at 37°C and 5% CO2 for up to 14 days. Islet viability was assessed using FDA/PI or Newport Green, and function was assessed using a glucose-stimulated insulin release (GSIR) assay. Islet yield was performed using enumeration of dithizone-stained aliquots. The young porcine (YP) islet yield at dissociation was 12.6 ± 2.1 × 10(3) IEQ (mean ± SEM) per organ and increased to 33.3 ± 6.4 × 10(3) IEQ after 7 days of in vitro culture. Viability was 97.3 ± 7% at dissociation and remained over 90% viable after 11 days in tissue culture (n = ns). Glucose responsiveness increased throughout maturation in culture. The stimulation index (SI) of the islets increased from 1.7 ± 2 on culture day 3 to 2.58 ± 0.5 on culture day 7. These results suggest that this method is both efficient and scalable for isolating and maturing insulin-responsive porcine islets in culture.

Assessment of glycemic control after islet transplantation using the continuous glucose monitor in insulin-independent versus insulin-requiring type 1 diabetes subjects.

BACKGROUND: The aim of this study was to assess and compare glycemic control using the continuous glucose monitor (CGMS, Medtronic Minimed, Northridge, CA) in type 1 diabetes mellitus (T1DM) subjects who are insulin-independent versus those who require insulin after islet transplantation alone (ITA). METHODS: Glycemic control was assessed using 72-h CGMS in eight T1DM subjects who were insulin-independent after ITA (ITA-II), eight T1DM subjects who were C-peptide-positive but insulin-requiring after ITA (ITA-IR), and eight non-transplanted (NT) T1DM subjects. RESULTS: Standard deviation of glucose values was not significantly different between ITA-II and ITA-IR subjects (ITA-II, 1.2 +/- 0.1 mM; ITA-IR, 2.0 +/- 0.3 mM; P = 0.072). Both ITA groups were more stable than NT subjects (NT, 3.3 +/- 0.3 mM; P = 0.001 vs. ITA). Mean high glucose values were significantly lower in ITA subjects compared with NT subjects (ITA-II, 10.5 +/- 0.6 mM; ITA-IR, 13.0 +/- 1.0 mM; NT, 16.1 +/- 1.1 mM; P = 0.002). Mean average glucose values were not significantly different among all groups (ITA-I, 6.7 +/- 0.2 mM; ITA-IR, 7.8 +/- 0.3 mM; NT, 7.7 +/- 0.6 mM; P = 0.198). Mean low glucose values were significantly higher in both ITA groups compared with NT subjects (ITA-II, 4.5 +/- 0.2 mM; ITA-IR, 4.3 +/- 0.3 mM; NT, 3.0 +/- 0.2 mM; P = 0.003). Duration of hypoglycemic excursions (<3.0 mM) was markedly reduced in both ITA groups (ITA-II, 0%; ITA-IR, 2.4 +/- 0.2%; NT, 11.8 +/- 4.2%). Glycated hemoglobin was not significantly different between ITA groups (ITA-II, 6.4 +/- 0.2%; ITA-IR, 6.5 +/- 0.3%) and was significantly higher in NT subjects (8.3 +/- 0.2%; P < 0.001 vs. ITA). CONCLUSIONS: CGMS monitoring demonstrates that glycemic lability and hypoglycemia are significantly reduced in C-peptide-positive islet transplant recipients, whether or not supplementary, exogenous insulin is used, compared with non-transplanted T1DM subjects.

Estimation of pancreas weight from donor variables.

Previous studies have identified several donor factors affecting the outcome of islet isolation. Pancreas weight has not been considered as a donor selection criterion, because a value cannot be obtained prior to organ procurement. However, a larger pancreas will likely contain a higher number of islets. Therefore, the prediction of pancreas weight would be helpful in donor selection, benefiting cost and efficiency of the islet isolation laboratory. The purpose of this study was to investigate normal pancreas weight in cadaveric donors and identify pancreas weight predictors from demographic data of cadaveric organ donors. We retrospectively analyzed data on pancreas weight from 354 cadaveric donors with respect to gender, age, body weight, body height, body mass index (BMI), and body surface area (BSA). In men, pancreas weight correlated more closely with body weight than with age, height, or BMI. BSA was as strong a correlate of pancreas weight as body weight. In women, pancreas weight had a similar pattern of relationships, with generally lower correlation coefficients. On the basis of the observation of gender-specific pancreas weight difference in elderly donors, stepwise multiple linear regression analyses were conducted separately for younger (< or =40 years) and elderly (> or =41 years) donors. In younger donors, body weight and age were the major predictors of pancreas weight [pancreas weight (g) = 4.355 + 0.742 x body weight (kg) + 0.837 x age (years) (R2 = 0.564, p < 0.001)]. In contrast, pancreas weight of elderly donors was best predicted by BSA and gender [pancreas weight (g) = -17.624 + 60.036 x BSA (m2) - 7.152 x gender (R2 = 0.372, p < 0.001; "gender": 1 = female, 0 = male)]. Pancreas weight was found to be positively associated with pre- and postpurification islet yields. These formulae should contribute to the estimation of pancreas weight, and thus improve donor selection for islet isolation and 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.

Beta-score: an assessment of beta-cell function after islet transplantation.

OBJECTIVE: Success after islet transplantation can be defined in terms of insulin independence, C-peptide secretion, or glycemic control. These measures are interdependent and all need to be considered in evaluating beta-cell function after islet transplantation. For the current study, a composite beta-score was developed that provides an integrated measure of beta-cell function success after islet transplantation. RESEARCH DESIGN AND METHODS: The proposed scoring system gave 2 points each for normal fasting glucose, HbA(1c), stimulated C-peptide, and absence of insulin or oral hypoglycemic agent use. No points were awarded if the fasting glucose was in the diabetic range, the HbA(1c) was >6.9%, C-peptide secretion was absent on stimulation, or daily insulin use was in excess of 0.24 units/kg. One point was given for intermediate values. The score ranged from 0 to 8 and was correlated with the glucose value 90 min after a standard mixed meal challenge (n = 218) in 57 subjects before and after islet transplantation. The score was also used to follow subjects for up to 5 years after islet transplantation. RESULTS: The beta-score correlated well with the plasma glucose level 90 min after a mixed meal challenge (r = -0.849, P < 0.001). On follow-up, the beta-score rose after the first transplant and was maintained up to 5 years, demonstrating continuing function of the transplanted beta-cells. CONCLUSIONS: The beta-score provides a simple clinical scoring system that encompasses glycemic control, diabetes therapy, and endogenous insulin secretion that correlates well with physiological measures of beta-cell function. On this basis, it is suitable as an overall measure of beta-cell transplant function. The beta-score gives an integrated measure of beta-cell function as a continuum that may be more useful than simply assessing the presence or absence of insulin independence.

Islet graft assessment in the Edmonton Protocol: implications for predicting long-term clinical outcome.

The success of the Edmonton Protocol for islet transplantation has provided new hope in the treatment of type 1 diabetes. This study reports on the assessment of 83 human islet grafts transplanted using the Edmonton Protocol since 1999. Cellular composition, as assessed by immunohistochemistry, showed a lower islet purity (approximately 40%) than has been reported in previous studies using dithizone staining to quantitate islet equivalents. Furthermore, grafts were found to contain substantial populations of exocrine and ductal tissue. Total cellular insulin transplanted was 8,097.6 +/- 3,164.4 microg/patient, and was significantly lower in bottom gradient layer grafts than top gradient layer or whole/combined grafts (P < 0.0005). A static incubation test for islet function gave a stimulation index of 3-4, although this measure did not correlate with posttransplant metabolic outcome. Furthermore, we confirmed a previously reported trend in which donor age affects islet yield and purity. It is important to note that a significant positive correlation was observed between the number of islet progenitor (ductal-epithelial) cells transplanted and long-term metabolic success as assessed an by intravenous glucose tolerance test at approximately 2 years posttransplant. In summary, careful assessment of islet graft composition is needed in a clinical transplantation program to accurately estimate islet purity and assess the contribution of other cell types present, such as islet progenitor cells.

Assessment of Different Transfection Parameters in Efficiency Optimization.

Achieving optimal transfection efficiency is the most critical step in overcoming the primary obstacle to success in nonviral-mediated gene therapy. Several transfection parameters were being examined including the effects of different types of transfection media, glucose concentration, reporter DNA concentration, and incubation time in lipotransfectant. Efficiency of transfection obtained was highest for Opti-MEM I (29 ± 2.28%; p = 0.001) followed by M199 (24 ± 1.54%; p = 0.009), both of which performed significantly better than DMEM (14 ± 0.28%) as a transfection medium. The rate of transfection was affected by glucose levels in only DMEM with higher efficiency achieved using low glucose containing DMEM (17 ± 0.38%) than its counterpart. Furthermore, transfection rate and cell viability were severely hampered by lengthened exposure to transfection complexes, leading to an overall mean efficiency of 5 ± 0.87%. However, doubling the DNA content in the transfection mixture did not significantly change the mean rate of transfection in M199 medium (24 ± 1.54% to 27 ± 1.54%; p = 0.273). The overall range of mean efficiency acquired with our protocol under different transfection conditions was between 14% and 29%. Hopefully results from this study will further potential success in nonviral-mediated gene transfer.