Islet Harvest in Carbon Monoxide-Saturated Medium for Chronic Pancreatitis Patients Undergoing Islet Autotransplantation.

Stresses encountered during human islet isolation lead to unavoidable ß-cell death after transplantation. This reduces the chance of insulin independence in chronic pancreatitis patients undergoing total pancreatectomy and islet autotransplantation. We tested whether harvesting islets in carbon monoxide-saturated solutions is safe and can enhance islet survival and insulin independence after total pancreatectomy and islet autotransplantation. Chronic pancreatitis patients who consented to the study were randomized into carbon monoxide (islets harvested in a carbon monoxide-saturated medium) or control (islets harvested in a normal medium) groups. Islet yield, viability, oxygen consumption rate, ß-cell death (measured by unmethylated insulin DNA), and serum cytokine levels were measured during the peri-transplantation period. Adverse events, metabolic phenotypes, and islet function were measured prior and at 6 months post-transplantation. No adverse events directly related to the infusion of carbon monoxide islets were observed. Carbon monoxide islets showed significantly higher viability before transplantation. Subjects receiving carbon monoxide islets had less ß-cell death, decreased CCL23, and increased CXCL12 levels at 1 or 3 days post transplantation compared with controls. Three in 10 (30%) of the carbon monoxide subjects and none of the control subjects were insulin independent. This pilot trial showed for the first time that harvesting human islets in carbon monoxide-saturated solutions is safe for total pancreatectomy and islet autotransplantation patients.

Acute Ischemia Induced by High-Density Culture Increases Cytokine Expression and Diminishes the Function and Viability of Highly Purified Human Islets of Langerhans.

BACKGROUND: Encapsulation devices have the potential to enable cell-based insulin replacement therapies (such as human islet or stem cell-derived ß cell transplantation) without immunosuppression. However, reasonably sized encapsulation devices promote ischemia due to high ß cell densities creating prohibitively large diffusional distances for nutrients. It is hypothesized that even acute ischemic exposure will compromise the therapeutic potential of cell-based insulin replacement. In this study, the acute effects of high-density ischemia were investigated in human islets to develop a detailed profile of early ischemia induced changes and targets for intervention. METHODS: Human islets were exposed in a pairwise model simulating high-density encapsulation to normoxic or ischemic culture for 12 hours, after which viability and function were measured. RNA sequencing was conducted to assess transcriptome-wide changes in gene expression. RESULTS: Islet viability after acute ischemic exposure was reduced compared to normoxic culture conditions (P < 0.01). Insulin secretion was also diminished, with ischemic ß cells losing their insulin secretory response to stimulatory glucose levels (P < 0.01). RNA sequencing revealed 657 differentially expressed genes following ischemia, with many that are associated with increased inflammatory and hypoxia-response signaling and decreased nutrient transport and metabolism. CONCLUSIONS: In order for cell-based insulin replacement to be applied as a treatment for type 1 diabetes, oxygen and nutrient delivery to ß cells will need to be maintained. We demonstrate that even brief ischemic exposure such as would be experienced in encapsulation devices damages islet viability and ß cell function and leads to increased inflammatory signaling.

Noninvasive assessment of tissue-engineered graft viability by oxygen-17 magnetic resonance spectroscopy.

Transplantation of macroencapsulated tissue-engineered grafts (TEGs) is being investigated as a treatment for type 1 diabetes, but there is a critical need to measure TEG viability both in vitro and in vivo. Oxygen deficiency is the most critical issue preventing widespread implementation of TEG transplantation and delivery of supplemental oxygen (DSO) has been shown to enhance TEG survival and function in vivo. In this study, we demonstrate the first use of oxygen-17 magnetic resonance spectroscopy (17 O-MRS) to measure the oxygen consumption rate (OCR) of TEGs and show that in addition to providing therapeutic benefits to TEGs, DSO with 17 O2 can also enable measurements of TEG viability. Macroencapsulated TEGs containing ßTC3 murine insulinoma cells were prepared with three fractional viabilities and provided with 17 O2 . Cellular metabolism of 17 O2 into nascent mitochondrial water (H217 O) was monitored by 17 O-MRS and, from the measured data, OCR was calculated. For comparison, OCR was simultaneously measured on a separate, but equivalent sample of cells with a well-established stirred microchamber technique. OCR measured by 17 O-MRS agreed well with measurements made in the stirred microchamber device. These studies confirm that 17 O-MRS can quantify TEG viability noninvasively. Biotechnol. Bioeng. 2017;114: 1118-1121. © 2016 Wiley Periodicals, Inc.

Continuous Quadrupole Magnetic Separation of Islets during Digestion Improves Purified Porcine Islet Viability.

Islet transplantation (ITx) is an emerging and promising therapy for patients with uncontrolled type 1 diabetes. The islet isolation and purification processes require exposure to extended cold ischemia, warm-enzymatic digestion, mechanical agitation, and use of damaging chemicals for density gradient separation (DG), all of which reduce viable islet yield. In this paper, we describe initial proof-of-concept studies exploring quadrupole magnetic separation (QMS) of islets as an alternative to DG to reduce exposure to these harsh conditions. Three porcine pancreata were split into two parts, the splenic lobe (SPL) and the combined connecting/duodenal lobes (CDL), for paired digestions and purifications. Islets in the SPL were preferentially labeled using magnetic microparticles (MMPs) that lodge within the islet microvasculature when infused into the pancreas and were continuously separated from the exocrine tissue by QMS during the collection phase of the digestion process. Unlabeled islets from the CDL were purified by conventional DG. Islets purified by QMS exhibited significantly improved viability (measured by oxygen consumption rate per DNA, p < 0.03) and better morphology relative to control islets. Islet purification by QMS can reduce the detrimental effects of prolonged exposure to toxic enzymes and density gradient solutions and substantially improve islet viability after isolation.

Islet Oxygen Consumption Rate (OCR) Dose Predicts Insulin Independence in Clinical Islet Autotransplantation.

BACKGROUND: Reliable in vitro islet quality assessment assays that can be performed routinely, prospectively, and are able to predict clinical transplant outcomes are needed. In this paper we present data on the utility of an assay based on cellular oxygen consumption rate (OCR) in predicting clinical islet autotransplant (IAT) insulin independence (II). IAT is an attractive model for evaluating characterization assays regarding their utility in predicting II due to an absence of confounding factors such as immune rejection and immunosuppressant toxicity. METHODS: Membrane integrity staining (FDA/PI), OCR normalized to DNA (OCR/DNA), islet equivalent (IE) and OCR (viable IE) normalized to recipient body weight (IE dose and OCR dose), and OCR/DNA normalized to islet size index (ISI) were used to characterize autoislet preparations (n = 35). Correlation between pre-IAT islet product characteristics and II was determined using receiver operating characteristic analysis. RESULTS: Preparations that resulted in II had significantly higher OCR dose and IE dose (p<0.001). These islet characterization methods were highly correlated with II at 6-12 months post-IAT (area-under-the-curve (AUC) = 0.94 for IE dose and 0.96 for OCR dose). FDA/PI (AUC = 0.49) and OCR/DNA (AUC = 0.58) did not correlate with II. OCR/DNA/ISI may have some utility in predicting outcome (AUC = 0.72). CONCLUSIONS: Commonly used assays to determine whether a clinical islet preparation is of high quality prior to transplantation are greatly lacking in sensitivity and specificity. While IE dose is highly predictive, it does not take into account islet cell quality. OCR dose, which takes into consideration both islet cell quality and quantity, may enable a more accurate and prospective evaluation of clinical islet preparations.

Outcomes of a Multimodal Resilience Training Program in an Outpatient Integrative Medicine Clinic.

OBJECTIVE: To investigate the outcomes of resilience training (RT) in an outpatient clinical setting on symptom relief for current or recurrent depression, as well as perceived stress and state and trait anxiety. DESIGN: Observational effectiveness study. SETTINGS/LOCATION: Penny George Institute for Health and Healing, Allina Health, Minneapolis, MN. PARTICIPANTS: A total of 728 men and women age 18 years and older who participated in the RT program between December 1, 2007, and November 31, 2012. Of these individuals, 371 were considered study contributors and completed at least one questionnaire both before (pre-RT) and after (post-RT) completion of the program. The remaining participants were considered study non-contributors and did not complete any questionnaires. INTERVENTIONS: RT is a mindfulness-based program that synergizes elements of mindfulness meditation with nutrition and exercise into a cohesive intervention. OUTCOME MEASURES: Depressive symptoms, as well as state and trait anxiety and perceived stress. RESULTS: Among the 371 RT participant contributors, depressive symptoms, perceived stress, and state and trait anxiety improved significantly from pre-RT to post-RT. Furthermore, among participants with depression at baseline, Center for Epidemiologic Studies Depression Scale-10 scores decreased by a mean of 44.0% (from 17.5 to 9.8), a value below the cutoff for significant depressive symptoms. Baseline perceived stress scores were the most predictive of program success. CONCLUSIONS: This study provides evidence that a multimodal RT program delivered in a real-world clinical setting improves symptoms of depression, anxiety, and stress. Limitations of this effectiveness study include a homogeneous population of mostly white women and a large amount of randomized, imputed, and missing data. Future work should include a randomized controlled trial and potentially studies to separate RT into the three components to determine which may be primarily responsible for the improved outcomes.

Differences in glucose-stimulated insulin secretion in vitro of islets from human, nonhuman primate, and porcine origin.

Porcine islet xenotransplantation is considered a potential cell-based therapy for type 1 diabetes. It is currently being evaluated in diabetic nonhuman primates (NHP) to assess safety and efficacy of the islet product. However, due to a variety of distinct differences between the respective species, including the insulin secretory characteristics of islets, the suitability and predictive value of the preclinical model in the extrapolation to the clinical setting remain a critical issue. Islets isolated from human (n = 3), NHP (n = 2), adult pig (AP, n = 3), and juvenile pig (JP, n = 4) pancreata were perifused with medium at basal glucose (2.5 mm) followed by high glucose (16.7 mm) concentrations. The total glucose-stimulated insulin secretion (GSIS) was calculated from generated insulin secretion profiles. Nonhuman primate islets exhibited GSIS 3-fold higher than AP islets, while AP and JP islets exhibited GSIS 1/3 and 1/30 of human islets, respectively. The insulin content of NHP and AP islets was similar to that of human islets, whereas that of JP islets was 1/5 of human islets. Despite the fact that human, NHP, and AP islets contain similar amounts of insulin, the much higher GSIS for NHP islets than for AP and JP islets suggests the need for increased dosing of islets from JP and AP in pig-to-NHP transplantation. Porcine islet xenotransplantation to humans may require significantly higher dosing given the lower GSIS of AP islets compared to human islets.

Real-time assessment of encapsulated neonatal porcine islets prior to clinical xenotransplantation.

BACKGROUND: Porcine islet transplantation is emerging as an attractive option for the treatment of patients with type 1 diabetes, with the possibility of providing islets of higher and more consistent quality and in larger volumes than available from human pancreata. The use of encapsulated neonatal porcine islets (ENPI) is appealing because it can address islet supply limitations while reducing the need for anti-rejection therapy. Pre-transplant characterization of ENPI viability and potency is an essential component of the production process. We applied the validated assay for oxygen consumption rate normalized for DNA content (OCR/DNA) to characterize ENPI viability. METHODS: ENPI of low viscosity and high m alginate were prepared according to standard methods and characterized at various culture time points up to 5 weeks. RESULTS: The OCR/DNA (nmol/min·mgDNA ± SEM) of ENPI (235 ± 10, n = 9) was comparable to that of free NPI (255 ± 14, n = 13). After encapsulation, NPI OCR/DNA was sustained over a culture period of up to 5 weeks. The average OCR/DNA of ENPI cultured longer than 9 days was higher than that of freshly encapsulated NPI. CONCLUSION: This is the first characterization of ENPI by a validated and more sensitive method for product viability. The NPI encapsulation process does not compromise viability as measured by OCR/DNA, and ENPI can be cultured for up to 5 weeks with maintenance of viability. ENPI meet or exceed current adult porcine islet product release criteria (established at the University of Minnesota) for preclinical xenotransplantation in terms of OCR/DNA.

Paramagnetic microparticles do not elicit islet cytotoxicity with co-culture or host immune reactivity after implantation.

BACKGROUND: Paramagnetic microparticles (MPs) may be useful in pancreatic islet purification, in particular purification of porcine islets as a potential xenotransplantation product. We assessed whether MPs affect islet function or induce an adverse effect following implantation. METHODS: Porcine islets were co-cultured with 0, 500, and 1500 MPs per islet equivalent (IE) for 1 day and with 0 and 1500 MPs/IE for 7 days. Fractional viability was assessed using oxygen consumption rate normalized to DNA content (OCR/DNA) and after 7-day co-culture by perifusion glucose-stimulated insulin secretion (GSIS) and by transplantation under the renal capsule of diabetic nude mice. To assess an inflammatory response or immune reaction, MPs (∼10(7)) were implanted under the renal capsule of C57BL/6 mice. RESULTS: No statistically significant differences were measured in OCR/DNA (mean ± SE) following 1-day co-culture with 0, 500, or 1500 MPs/IE (243.3 ± 4.5, 211.3 ± 8.1, or 230.6 ± 11.3 nmol/min·mgDNA, respectively) or following 7-day co-culture with 0 or 1500 MPs/IE (248.5 ± 1.4 or 252.9 ± 4.7 nmol/min·mgDNA, respectively). GSIS was not affected by the presence of MPs; first- and second-phase insulin area-under-the-curve (mean ± SE) reflected no statistically significant differences after 7-day co-culture between 0 and 1500 MPs/IE (8.36 ± 0.29 and 8.45 ± 0.70 pg/ml·min·ngDNA for first-phase; 69.73 ± 2.18 and 65.70 ± 4.34 pg/ml·min·ngDNA for second-phase, respectively). Islets co-cultured with MPs normalized hyperglycemia in diabetic nude mice, suggesting no adverse effects on in vivo islet function. Implantation of MPs did not elicit tissue injury, inflammatory change or immune reactivity. CONCLUSION: MPs do not adversely affect islet viability or function during co-culture, and MPs are not immune reactive following implantation.