Purification Method Using Iodixanol (OptiPrep)-Based Density Gradient Significantly Reduces Cytokine Chemokine Production From Human Islet Preparations, Leading to Prolonged β-Cell Survival During Pretransplantation Culture

Abstract

Purification is one of the most important steps in human islet isolation. Although Ficoll-based density gradients are widely used, OptiPrep-based density gradients are used in few centers. Cytokine/chemokine production from human islet preparations varies widely. Some cytokines/chemokines have been reported to have adverse effects on human islet preparations. Control of cytokine/chemokine production may be a key to improve islet quality and quantity, leading to better transplantation outcomes. The aim of the present study was to investigate the effects on islet preparations of purification methods using various density gradients on viability, cellular composition, and proinflammatory cytokine/chemokine production. After the digestion phase, the extracts were divided into 2 groups for purification using a semiautomated cell processor with Ficoll-based or OptiPrep-based density gradients. Islet preparations cultured for 2 days were assessed regarding islet cell viability (fluorescein diacetate/propidium iodide [FDA/PI]), fractional β-cell viability by FACS, and β-cell content using iCys. Cytokine/chemokine production from islet preparations was also measured by Bio-plex. After purification, the purity, islet equivalents (IEQ), and islet recovery rates were comparable between the 2 groups. Although FDA/PI and fractional β-cell viability showed no significant difference, survival of β cells during culture was significantly higher in the OptiPrep compared with the Ficoll-based density gradient group. There were significantly lower tumor necrosis factor (TNF)-α, interleukin (IL)-1β, interferon (IFN)-γ, IL-6, and MIP-1β productions from the OptiPrep-based density gradient group. OptiPrep-based density gradients reduced cytokine/chemokine production by islet preparations. In addition, OptiPrep-based density gradient purification significantly reduced the loss of β-cell mass during pretransplantation culture.