Continuous Quadrupole Magnetic Separation of Islets during Digestion Improves Purified Porcine Islet Viability

Abstract

Islet transplantation (ITx) is an emerging and promising therapy for patients with uncontrolled type 1 diabetes. The islet isolation and purification processes require exposure to extended cold ischemia, warm-enzymatic digestion, mechanical agitation, and use of damaging chemicals for density gradient separation (DG), all of which reduce viable islet yield. In this paper, we describe initial proof-of-concept studies exploring quadrupole magnetic separation (QMS) of islets as an alternative to DG to reduce exposure to these harsh conditions. Three porcine pancreata were split into two parts, the splenic lobe (SPL) and the combined connecting/duodenal lobes (CDL), for paired digestions and purifications. Islets in the SPL were preferentially labeled using magnetic microparticles (MMPs) that lodge within the islet microvasculature when infused into the pancreas and were continuously separated from the exocrine tissue by QMS during the collection phase of the digestion process. Unlabeled islets from the CDL were purified by conventional DG. Islets purified by QMS exhibited significantly improved viability (measured by oxygen consumption rate per DNA, p < 0.03) and better morphology relative to control islets. Islet purification by QMS can reduce the detrimental effects of prolonged exposure to toxic enzymes and density gradient solutions and substantially improve islet viability after isolation.

Culturing with modified EGM2 medium enhances porcine neonatal islet‐like cell clusters resistance to apoptosis in islet xenotransplantation

“…In this study, the effect of EGM2 medium on NICC in vitro survival was examined. As islet OCR measurement is a sensitive assay of their viability, our findings of greater mitochondrial capacity and better viability detected by OCR assessment in EGM2 medium cultured NICC than that observed in NICC cultured with Ham's F‐10 medium further confirmed the enhanced NICC capacity to survive during culture shown by both the AO/EB staining and FACS analyses in this study. Compared to that cultured in conventional Ham's F‐10 medium, NICC cultured in modified EGM2 medium exhibited increased number, enhanced viability and in vitro insulin secretion, upregulated expression of endocrine hormone genes, and more impressed, enhanced capability to resist apoptosis resulted from hypoxia and/or nutrient deficiency.…”

Carbohydrate antigen microarray analysis of serum IgG and IgM antibodies before and after adult porcine islet xenotransplantation in cynomolgus macaques

“…Adult porcine islets were isolated as previously described [ 19 , 20 ]. Briefly, donor pancreases were retrieved from exsanguinated pigs, dissected, distended intraductally with collagenase and neutral protease, and dissociated using the automated method at 28° to 32°C [ 21 ].…”