Background Islet transplantation is emerging as a treatment option for selected patients with type 1 diabetes. The limited human islet supply from cadavers and poor islet yield and quality remain substantial impediments to progress in the field. Use of porcine islets holds great promise for large-scale application of islet transplantation. Consistent isolation of porcine islets is dependent on advances in pancreas procurement and preservation, and islet isolation requiring detailed knowledge of the porcine pancreatic anatomy. The primary aim of this study was to describe the vascular and ductal anatomy of the porcine pancreas in order to guide and improve organ preservation and enzyme perfusion. Methods Pancreata were removed by en bloc viscerectomy from 65 female Landrace pigs. Results 15% of organs exhibited inconsistent vascular branching from the celiac trunk. All organs had uniform patterns of branching at the superior mesenteric artery. The superior and inferior mesenteric veins (IMV) merged to become the portal vein in all but one case in which the IMV drained into the splenic vein. 97% of pancreata had three lobes: duodenal (DL), connecting (CL), and splenic (SL); 39% demonstrated ductal communication between the CL and the other two lobes; 50% had ductal communication only between the CL and DL; and 11% presented other types of ductal delineation. Conclusions Accounting for the variations in vascular and ductal anatomy, as detailed in this study, will facilitate development of protocols for preservation, optimal enzyme administration, and pancreas distention and digestion, and ultimately lead to substantial improvements in isolation outcomes.

In this concept article, we outline a variety of new approaches that have been conceived to address some of the remaining challenges for developing improved methods of biopreservation. This recognizes a true renaissance and variety of complimentary, high-potential approaches leveraging inspiration by nature, nanotechnology, the thermodynamics of pressure, and several other key fields. Development of an organ and tissue supply chain that can meet the healthcare demands of the 21st century means overcoming twin challenges of (1) having enough of these lifesaving resources and (2) having the means to store and transport them for a variety of applications. Each has distinct but overlapping logistical limitations affecting transplantation, regenerative medicine, and drug discovery, with challenges shared among major areas of biomedicine including tissue engineering, trauma care, transfusion medicine, and biomedical research. There are several approaches to biopreservation, the optimum choice of which is dictated by the nature and complexity of the tissue and the required length of storage. Short-term hypothermic storage at temperatures a few degrees above the freezing point has provided the basis for nearly all methods of preserving tissues and solid organs that, to date, have proved refractory to cryopreservation techniques successfully developed for single-cell systems. In essence, these short-term techniques have been based on designing solutions for cellular protection against the effects of warm and cold ischemia and basically rely upon the protective effects of reduced temperatures brought about by Arrhenius kinetics of chemical reactions. However, further optimization of such preservation strategies is now seen to be restricted. Long-term preservation calls for much lower temperatures and requires the tissue to withstand the rigors of heat and mass transfer during protocols designed to optimize cooling and warming in the presence of cryoprotective agents. It is now accepted that with current methods of cryopreservation, uncontrolled ice formation in structured tissues and organs at subzero temperatures is the single most critical factor that severely restricts the extent to which tissues can survive procedures involving freezing and thawing. In recent years, this major problem has been effectively circumvented in some tissues by using ice-free cryopreservation techniques based upon vitrification. Nevertheless, despite these promising advances there remain several recognized hurdles to be overcome before deep-subzero cryopreservation, either by classic freezing and thawing or by vitrification, can provide the much-needed means for biobanking complex tissues and organs for extended periods of weeks, months, or even years. In many cases, the approaches outlined here, including new underexplored paradigms of high-subzero preservation, are novel and inspired by mechanisms of freeze tolerance, or freeze avoidance, in nature. Others apply new bioengineering techniques such as nanotechnology, isochoric pressure preserv...

19F MRI is valuable for in vivo imaging due to the only trace amounts of fluorine in biological systems. Because of the low sensitivity of MRI however, designing new fluorochemicals remains a significant challenge for achieving sufficient 19F signal. Here, we describe a new class of high-signal, water-soluble fluorochemicals as 19F MRI imaging agents. A polyamide backbone is used for tuning the proteolytic stability to avoid retention within the body, which is a limitation of current state-of-the-art perfluorochemicals. We show that unstructured peptides containing alternating N-ε-trifluoroacetyllysine and lysine provide a degenerate 19F NMR signal. 19F MRI phantom images provide sufficient contrast at micromolar concentrations, showing promise for eventual clinical applications. Finally, the degenerate high signal characteristics were retained when conjugated to a large protein, indicating potential for in vivo targeting applications, including molecular imaging and cell tracking.

Oxygen homeostasis is important in the regulation of biological function. Disease progression can be monitored by measuring oxygen levels, thus producing information for the design of therapeutic treatments. Non-invasive measurements of tissue oxygenation require the development of tools with minimal adverse effects and facile detection of features of interest. Fluorine magnetic resonance imaging (19F-MRI) exploits the intrinsic properties of perfluorocarbon (PFC) liquids for anatomical imaging, cell tracking, and oxygen sensing. However, the highly hydrophobic and lipophobic properties of perfluorocarbons require the formation of emulsions for biological studies. Though, stabilizing these emulsions has been challenging. To enhance the stability and biological loading of perfluorocarbons, one option is to incorporate perfluorocarbon liquids into the internal space of biocompatible mesoporous silica nanoparticles. Here, we developed perfluorocarbon-loaded ultraporous mesostructured silica nanoparticles (PERFUMNs) as 19F-MRI detectable oxygen sensing probes. Ultraporous mesostructured nanoparticles (UMNs) have large internal cavities (average = 1.76 cm3 g−1), facilitating an average 17% loading efficiency of PFCs, meeting the threshold fluorine concentrations needed for imaging studies. Perfluoro-15-crown-5-ether PERFUMNs have the highest equivalent nuclei per PFC molecule, and a spin-lattice (T1) relaxation-based oxygen sensitivity of 0.0032 mmHg−1 s−1 at 16.4 T (657 MHz). The option of loading PFCs after synthesizing UMNs, rather than the more traditional in situ core-shell syntheses, allows for use of a broad range of PFC liquids from a single material. The biocompatible and tunable chemistry of UMNs combined with the intrinsic properties of PFCs makes PERFUMNs a MRI sensor with potential for anatomical imaging, cell tracking, and metabolic spectroscopy with improved stability.

Background 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 remains a critical issue. Methods Islets isolated from human (n=3), NHP (n=2), adult pig (AP, n=3) and juvenile pig (JP, n=3) pancreata were perifused with medium at basal glucose (2.5mM) followed by high glucose (16.7mM) concentrations. The total glucose-stimulated insulin secretion (GSIS) was calculated from generated insulin secretion profiles. Results NHP islets exhibited GSIS 3-fold higher than human islets, while AP and JP islets exhibited GSIS 1/3 and 1/16 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 1/3 of human islets. Conclusion Despite the fact that human, NHP, and AP islets contain similar amounts of insulin, the much higher GSIS for NHP islets than for human, AP and JP islets suggests the need for increased dosing of islets from JP and AP in pig-to-NHP transplantation which may be substantially higher than that required for humans. Finally, porcine islet xenotransplantation to humans may require significantly higher dosing given the lower GSIS of AP islets compared to human islets.

Background-Islet transplantation is a promising treatment for type 1 diabetes. Due to a shortage of suitable human pancreata, high cost, and the large dose of islets presently required for long-term diabetes reversal; it is important to maximize viable islet yield. Traditional methods of pancreas preservation have been identified as suboptimal due to insufficient oxygenation. Enhanced oxygen delivery is a key area of improvement. In this paper, we explored improved oxygen delivery by persufflation (PSF), ie, vascular gas perfusion.

Prolonged anoxia has deleterious effects on islets. Gas-permeable cell culture devices can be used to minimize anoxia during islet culture and especially during shipment when elimination of gas-liquid interfaces is required to prevent the formation of damaging gas bubbles. Gas-permeable bags may have several drawbacks, such as propensity for puncture and contamination, difficult islet retrieval, and significantly lower oxygen permeability than silicone rubber membranes (SRM). We hypothesized that oxygen permeability of bags may be insufficient for islet oxygenation. We measured oxygen transmission rates through the membrane walls of three different types of commercially available bags and through SRM currently used for islet shipment. We found that the bag membranes have oxygen transmission rates per unit area about 100-fold lower than SRM. We solved the oxygen diffusion-reaction equation for 150-microm diameter islets seeded at 3000 islet equivalents per cm2, a density adequate to culture and ship an entire human or porcine islet preparation in a single gas-permeable device, predicting that about 40% of the islet volume would be anoxic at 22 degrees C and about 70% would be anoxic at 37 degrees C. Islets of larger size or islets accumulated during shipment would be even more anoxic. The model predicted no anoxia in islets similarly seeded in devices with SRM bottoms. We concluded that commercially available bags may not prevent anoxia during islet culture or shipment; devices with SRM bottoms are more suitable alternatives.

Islet transplantation is emerging as a promising treatment for patients with type 1 diabetes. It is important to maximize viable islet yield for each organ due to scarcity of suitable human donor pancreata, high cost, and the high dose of islets required for insulin independence. However, organ transport for 8 hours using the two-layer method (TLM) frequently results in lower islet yields. Since efficient oxygenation of the core of larger organs (eg, pig, human) in TLM has recently come under question, we investigated oxygen persufflation as an alternative way to supply the pancreas with oxygen during preservation. Porcine pancreata were procured from non-heart-beating donors and preserved by either TLM or persufflation for 24 hours and fixed. Biopsies were collected from several regions of the pancreas, sectioned, stained with hematoxylin and eosin, and evaluated by a histologist. Persufflated tissues exhibited distended capillaries due to gas perfusion and significantly less autolysis/cell death than regions not exposed to persufflation or tissues exposed to TLM. The histology presented here suggests that after 24 hours of preservation, persufflation dramatically improves tissue health when compared with TLM. These results indicate the potential for persufflation to improve viable islet yields and extend the duration of preservation, allowing more donor organs to be utilized.ISLET TRANSPLANTATION is emerging as a promising treatment for patients with type I diabetes. 1,2 The minimally invasive nature of the procedure as well as the islets' intrinsic ability to tightly control blood glucose levels on demand via insulin release offer a combination of advantages that cannot be matched by other presently available treatments, such as selfmonitoring or whole-organ pancreas transplantation. Despite recent advances building upon the Edmonton Protocol 3 published in 2000, most centers still require multiple transplants to achieve long-term diabetes reversal. 4 The large dose of islets required, the high cost of the Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A lack of proper oxygenation during preservation places hypoxic or anoxic stress on the tissue and may predispose islets to death during the isolation process.5 -8 It has been postulated that improving pancreas preservation via enhanced oxygenation may (1) extend the effective duration of preservation, (2) expand the donor pool up to 10-fold, (3) reduce the immunogenicity of transplanted islets, and (4) reduce the number of donors needed per patient. 9 Presently, the two-layer method (TLM) is the state of the ar...