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. In this study, we introduce a clinically applicable subcutaneous islet transplantation platform using a biodegradable Vicryl mesh scaffold to prevent aggregation in a diabetic rat model. Islets were sandwiched between layers of clinically proven Vicryl mesh within thrombin-fibrin gel. In vitro, the mesh prevented islet aggregation and intra-islet hypoxia, which significantly improved islet viability. In vivo rat syngeneic islet transplantations into a prevascularized subcutaneous pocket demonstrated that the mesh significantly enhanced engraftment, as measured by assays for graft survival and function. Histological examination at 6 weeks showed well-vascularized grafts sandwiched in a flat shape between the mesh layers. The biodegradable mesh was fully absorbed by three months, which alleviated chronic foreign body reaction and fibrosis, and supported long-term graft maintenance. This simple graft shape modification approach is an effective and clinically applicable strategy for improved subcutaneous islet transplantation.

Determination of Islet Viability Using a Zinc-Specific Fluorescent Dye and a Semiautomated Assessment Method.

Islet transplantation is an effective therapy that allows the achievement of insulin independence in patients with type 1 diabetes (T1D). To ensure successful transplantation, islet viability and function are of great importance. Viability assessments most often use fluorescein diacetate (FDA)/propidium iodide (PI) staining. However, results using this method often do not correlate well with graft function. Because FDA nonspecifically penetrates all cells present in the islet preparation, including islets and contaminating acinar cells, its use often complicates viability assessments of the overall cell population. Furthermore, the manual method for determining viability percentages is highly subjective. Shortcomings of the conventional islet viability assay can be potentially improved by staining cells with Newport Green (NG). NG, is a zinc-specific fluorescent dye that specifically reacts with zinc-rich ß cells. Two kinds of NG dyes, NG-DCF and NG-PDX, are currently available. We examined the zinc specificity of these NG dyes and compared NG staining with traditional FDA staining to explore the potential of NG dyes to improve islet viability assessment. Of the two NGs tested, NG-DCF showed the higher specificity toward a ß-cell line as well as human islets. NG-DCF accurately identified the islet area, even in low-purity islets, while neither FDA nor NG-PDX did. Although NG-DCF staining required a longer incubation time, the addition of poloxamer F127 and incubation at 37°C allowed viability assessment to take place within 30 min. Unlike FDA/PI staining, NG-DCF/PI staining allowed for islet-specific assessment. We also introduced a semiautomated measurement to determine NG-DCF/PI staining results, which enabled us to obtain objective and reproducible results. NG-DCF/PI staining is easy and reliable, and this method permits highly objective islet-specific viability assessments.