Jennifer P. Kitzmann

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.

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.

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

Kitzmann JP, Law L, Shome A, Muzina M, Elliott RB, Mueller KR, Schuurman H‐J, Papas KK. Real‐time assessment of encapsulated neonatal porcine islets prior to clinical xenotransplantation. Xenotransplantation 2012; 19: 333–336.

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

Suszynski TM, Rizzari MD, Kidder LS, Mueller K, Chapman CS, Kitzmann JP, Pongratz RL, Cline GW, Todd PW, Kennedy DJ, O’Brien TD, Avgoustiniatos ES, Schuurman H‐J, Papas KK. Paramagnetic microparticles do not elicit islet cytotoxicity with co‐culture or host immune reactivity after implantation. Xenotransplantation 2011; 18: 239–244.

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 &bgr; cell transplantation) without immunosuppression. However, reasonably sized encapsulation devices promote ischemia due to high &bgr; 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 &bgr; 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 &bgr; 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 &bgr; 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 βTC3 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.

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.

CO-CULTURE OF PORCINE PANCREATIC ISLETS WITH PARAMAGNETIC MICROPARTICLES DOES NOT RESULT IN REDUCED VIABILITY OR FUNCTION: 1964

T.M. Suszynski1, M.D. Rizzari2, L.S. Kidder3, K. Mueller4, J.P. Kitzmann4, C.S. Chapman4, R.L. Pongratz5, V.A. Kirchner1, G.W. Cline6, E.S. Avgoustiniatos3, K.K. Papas3 1Department Of Surgery, University of Minnesota, Minneapolis/ MN/UNITED STATES OF AMERICA, 2Department Of Surgery, University of Minnesota, Minneapolis/Minnesota/UNITED STATES OF AMERICA, 3Surgery, University of Minnesota, Minneapolis/ UNITED STATES OF AMERICA, 4Department Of Surgery, University of Minnesota, Minneapolis/UNITED STATES OF AMERICA, 5Departments Of Internal Medicine And Cellular And Molecular Physiology, Yale University, New Haven/CT/UNITED STATES OF AMERICA, 6Departments Of Internal Medicine And Cellular And Molecular Physiology, Yale University, New Haven/UNITED STATES OF AMERICA