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Xenotransplantation of porcine islets is a realistic option to restore β-cell function in type 1 diabetic patients. Among other factors, such as islet donor age (fetal, neonatal and adult) and genotype (wild type and genetically modified), choice of the transplantation site, and immune protection of the islets, efficient strategies for islet isolation, culture and engraftment are critical for the success of islet xenotransplantation.Neonatal porcine islets (NPIs) are immature at isolation and need to be matured in vitro or in vivo before they become fully functional. Recent developments include a scalable protocol for isolation of clinically relevant batches of NPIs and a stepwise differentiation protocol for directed maturation of NPIs. In addition, different sources of mesenchymal stem cells were shown to support survival and functional maturation of NPIs in vitro and in various transplantation models in vivo.A plethora of different culture media and supplements have been tested; however, a unique best culture system for NPIs is still missing. New insights, for example from single-cell analyses of islets or from stem cell differentiation toward β cells may help to optimize culture of porcine islets for xenotransplantation in an evidence-based manner.