A scalable human islet 3D-culture platform maintains cell mass and function long-term for transplantation.

Present-day islet culture methods provide short-term maintenance of cell viability and function, limiting access to islet transplantation. Attempts to lengthen culture intervals remain unsuccessful. A new method was developed to permit the long-term culture of islets.

Ubiquitous Luciferase Expression in "Firefly Rats" Does Not Alter the Pancreatic Islet Morphology, Metabolism, and Function.

"Firefly rats" ubiquitously express the luciferase reporter gene under the control of constitutively active promoter in inbred Lewis rats. Due to the minimal immunogenicity of luciferase, wide applications of Firefly rats have been reported in solid organ/cell transplantation studies for imaging, permitting quantitative and non-invasive tracking of the transplanted graft. is a non-coding gene and generally does not affect the expression of other endogenous genes.

Micropyramid-patterned, oxygen-permeable bottomed dish for high density culture of pancreatic islets.

The need for maintaining cell-spheroid viability and function within high-density cultures is unmet for various clinical and experimental applications, including cell therapies. One immediate application is for transplantation of pancreatic islets, a clinically recognized treatment option to cure type 1 diabetes; islets are isolated from a donor for subsequent culture prior to transplantation. However, high seeding conditions cause unsolicited fusion of multiple spheroids, thereby limiting oxygen diffusion to induce hypoxic cell death.

Microwell culture platform maintains viability and mass of human pancreatic islets.

Background: Transplantation of the human pancreatic islets is a promising approach for specific types of diabetes to improve glycemic control. Although effective, there are several issues that limit the clinical expansion of this treatment, including difficulty in maintaining the quality and quantity of isolated human islets prior to transplantation. During the culture, we frequently observe the multiple islets fusing together into large constructs, in which hypoxia-induced cell damage significantly reduces their viability and mass.

Critical Considerations in Bioluminescence Imaging of Transplanted Islets: Dynamic Signal Change in Early Posttransplant Phase and Signal Absorption by Tissues.

Objectives: In pancreatic islet transplantation studies, bioluminescence imaging enables quantitative and noninvasive tracking of graft survival. Amid the recent heightened interest in extrahepatic sites for islet and stem cell-derived beta-like cell transplantations, proper understanding the nature of bioluminescence imaging in these sites is important.

Methods: Islets isolated from Firefly rats ubiquitously expressing luciferase reporter gene in Lewis rats were transplanted into subcutaneous or kidney capsule sites of wild-type Lewis rats or immunodeficient mice.

A Multiparametric Assessment of Human Islets Predicts Transplant Outcomes in Diabetic Mice.

Prior to transplantation into individuals with type 1 diabetes, in vitro assays are used to evaluate the quality, function and survival of isolated human islets. In addition to the assessments of these parameters in islet, they can be evaluated by multiparametric morphological scoring (0-10 points) and grading (A, B, C, D, and F) based on islet characteristics (shape, border, integrity, single cells, and diameter). However, correlation between the multiparametric assessment and transplantation outcome has not been fully elucidated.

Early-Phase Luciferase Signals of Islet Grafts Predicts Successful Subcutaneous Site Transplantation in Rats.

Purpose: The transplantation of pancreatic islets is a promising cell replacement therapy for type 1 diabetes. Subcutaneous islet transplantation is currently under investigation as a means to circumvent problems associated with standard intra-hepatic islet transplantation. As modifications are being developed to improve the efficacy of subcutaneous islet transplantation, it is important to have robust methods to assess engraftment.

Intermittent normobaric oxygen inhalation enhances subcutaneous prevascularization for cell transplantation.

Purpose: Subcutaneous tissue is a promising site for cell transplantation; advantages include minimally invasive procedures and easy post-transplant monitoring. However, limited vascularity is the major known challenge. To address this challenge, a prevascularized graft bed is prepared in recipients.

High Fractions of Large Islets in Human Islet Preparations Detrimentally Affect Posttransplant Outcomes in Streptozotocin-Induced Diabetic Immunodeficient Mice.

Objectives: The aim of this study was to determine whether the size of islets isolated from human donors-measured pretransplant-impacts transplantation outcomes in diabetic mice.

Methods: Human islets (1200 islet equivalents) were transplanted into the kidney capsules of streptozotocin-induced diabetic immunodeficient mice. Data from a total of 174 mice that received islets from 45 isolations were analyzed to evaluate the correlation between pretransplant islet size and posttransplant diabetes reversal.

Semi-Automated Assessment of Human Islet Viability Predicts Transplantation Outcomes in a Diabetic Mouse Model.

In clinical and experimental human pancreatic islet transplantations, establishing pretransplant assessments that accurately predict transplantation outcomes is crucial. Conventional viability assessment that relies on manual counting of viable islets is a routine pretransplant assessment. However, this method does not correlate with transplantation outcomes; to improve the method, we recently introduced a semi-automated method using imaging software to objectively determine area-based viability.

A subcutaneous pancreatic islet transplantation platform using a clinically applicable, biodegradable Vicryl mesh scaffold - an experimental study.

Pancreatic islet transplantation into the liver is an effective treatment for type 1 diabetes but has some critical limitations. The subcutaneous site is a potential alternative transplant site, requiring minimally invasive procedures and allowing frequent graft monitoring; however, hypoxia is a major drawback. Our previous study without scaffolding demonstrated post-transplant graft aggregation in the subcutaneous site, which theoretically exacerbates lethal intra-graft hypoxia.

Inflammatory biomarkers in the blood and pancreatic tissue of organ donors that predict human islet isolation success and function.

The pancreas of brain-dead donors is the primary source of islets for transplantation. However, brain death mediates systemic inflammation, which may affect the quantity and quality of isolated islets. Our aim was to identify inflammatory biomarkers in donor blood and/or pancreatic tissue capable of predicting islet isolation success.

Isolated pancreatic islet yield and quality is inversely related to organ donor age in rats.

Pancreatic islets consist of several endocrine cell types that maintain glucose homeostasis. Type 1 diabetes (T1D) results from autoimmune-mediated destruction of insulin producing beta cells in pancreatic islets. Islet transplantation is a treatment for certain individuals with T1D.

Surfactants Improve Live Cell Imaging of Human Pancreatic Islets.

Objectives: Newport Green is a zinc-specific fluorescent dye developed to monitor cellular zinc transport. In pancreatic islets with zinc-rich β-cells, Newport Green is expected to be useful as an islet-specific indicator for live imaging. However, the low penetration of Newport Green into islets hinders clear detection.

Optimizing Temperature and Oxygen Supports Long-term Culture of Human Islets.

Unlabelled: Islet transplantation is a promising treatment for type-1 diabetes; however, donor shortage is a concern. Even when a pancreas is available, low islet yield limits the success of transplantation. Islet culture enables pooling of multiple low-yield isolations into an effective islet mass, but isolated islets rapidly deteriorate under conventional culture conditions.

Impact of Oxygen on Pancreatic Islet Survival.

Pancreatic islet transplantation is a promising treatment option for individuals with type 1 diabetes; however, maintaining islet function after transplantation remains a large challenge. Multiple factors, including hypoxia associated events, trigger pretransplant and posttransplant loss of islet function. In fact, islets are easily damaged in hypoxic conditions before transplantation including the preparation steps of pancreas procurement, islet isolation, and culture.

Posttransplant oxygen inhalation improves the outcome of subcutaneous islet transplantation: A promising clinical alternative to the conventional intrahepatic site.

Subcutaneous tissue is a promising site for islet transplantation, due to its large area and accessibility, which allows minimally invasive procedures for transplantation, graft monitoring, and removal of malignancies as needed. However, relative to the conventional intrahepatic transplantation site, the subcutaneous site requires a large number of islets to achieve engraftment success and diabetes reversal, due to hypoxia and low vascularity. We report that the efficiency of subcutaneous islet transplantation in a Lewis rat model is significantly improved by treating recipients with inhaled 50% oxygen, in conjunction with prevascularization of the graft bed by agarose-basic fibroblast growth factor.

Oxygen environment and islet size are the primary limiting factors of isolated pancreatic islet survival.

Background: Type 1 diabetes is an autoimmune disease that destroys insulin-producing beta cells in the pancreas. Pancreatic islet transplantation could be an effective treatment option for type 1 diabetes once several issues are resolved, including donor shortage, prevention of islet necrosis and loss in pre- and post-transplantation, and optimization of immunosuppression. This study seeks to determine the cause of necrotic loss of isolated islets to improve transplant efficiency.

Oxygenated thawing and rewarming alleviate rewarming injury of cryopreserved pancreatic islets.

Background/aims: Pancreatic islet transplantation is an effective treatment for Type 1 diabetic patients to eliminate insulin injections; however, a shortage of donor organs hinders the widespread use. Although long-term islet storage, such as cryopreservation, is considered one of the key solutions, transplantation of cryopreserved islets is still not practical due to the extensive loss during the cryopreservation-rewarming process. We have previously reported that culturing islets in a hyperoxic environment is an effective treatment to prevent islet death from the hypoxic injury during culture.

Development of the Nonobese Diabetic Mouse and Contribution of Animal Models for Understanding Type 1 Diabetes.

In 1974, the discovery of a mouse and a rat that spontaneously developed hyperglycemia led to the development of 2 autoimmune diabetes models: nonobese diabetic (NOD) mouse and Bio-Breeding rat. These models have contributed to our understanding of autoimmune diabetes, provided tools to dissect autoimmune islet damage, and facilitated development of early detection, prevention, and treatment of type 1 diabetes. The genetic characterization, monoclonal antibodies, and congenic strains have made NOD mice especially useful.

Mechanisms of islet damage mediated by pancreas cold ischemia/rewarming.

Prolonged pancreas cold ischemia is known to negatively correlate with islet isolation outcomes, hindering successful islet transplantation to treat Type-1 Diabetes. Due to poor islet isolation outcome, pancreata with over 16 h cold ischemia are currently not considered for islet transplantation. Mechanisms involved in pancreas cold ischemia/rewarming mediated islet damage during islet isolation and culture are not well understood.

Involvement of a proapoptotic gene (BBC3) in islet injury mediated by cold preservation and rewarming.

Long-term pancreatic cold ischemia contributes to decreased islet number and viability after isolation and culture, leading to poor islet transplantation outcome in patients with type 1 diabetes. In this study, we examined mechanisms of pancreatic cold preservation and rewarming-induced injury by interrogating the proapoptotic gene BBC3/Bbc3, also known as Puma (p53 upregulated modulator of apoptosis), using three experimental models: 1) bioluminescence imaging of isolated luciferase-transgenic ("Firefly") Lewis rat islets, 2) cold preservation of en bloc-harvested pancreata from Bbc3-knockout (KO) mice, and 3) cold preservation and rewarming of human pancreata and isolated islets. Cold preservation-mediated islet injury occurred during rewarming in "Firefly" islets.