title: High Seeding Density Induces Local Hypoxia and Triggers a Proinflammatory Response in Isolated Human Islets.
abstract: Hypoxia is the main threat to morphological and functional integrity of isolated pancreatic islets. Lack of oxygen seems to be of particular importance for functionality of encapsulated islets. The present study was initiated as an experimental model for the environment experienced by human islets in a confined space present during culture, shipment, and in an implanted macrodevice. Quadruplicate aliquots of isolated human islets (n = 12) were cultured for 24 h at 37°C under normoxic conditions using 24-well plates equipped with 8-µm pore size filter inserts and filled with islet aliquots adjusted to obtain a seeding density of 75, 150, 300, or 600 IEQ/cm(2). After culture viability, glucose-stimulated insulin release, DNA content as well as Bax and Bcl-2 gene expression were measured. Culture supernatants were collected to determine production of VEGF and MCP-1. Viability correlated inversely with IEQ seeding density (r = -0.71, p < 0.001), while the correlation of VEGF and MCP-1 secretion with seeding density was positive (r = 0.78, p < 0.001; r = 0.54, p < 0.001). Decreased viability corresponded with a significant increase in the Bax/Bcl-2 mRNA ratio at 300 and 600 IEQ/cm(2) and with a sigificantly reduced glucose-stimulated insulin secretion and insulin content compared to 75 or 150 IEQ/cm(2) (p < 0.01). The present study demonstrates that the seeding density is inversely correlated with islet viability and in vitro function. This is associated with a significant increase in VEGF and MCP-1 release suggesting a hypoxic and proinflammatory islet microenvironment.
authors.forename: Daniel
authors.initials: D
authors.lastname: Brandhorst
authors.affiliation: Nuffield Department of Surgical Sciences, University of Oxford, Headington, Oxford, UK.
authors.forename: Heide
authors.initials: H
authors.lastname: Brandhorst
authors.forename: Niamh
authors.initials: N
authors.lastname: Mullooly
authors.forename: Samuel
authors.initials: S
authors.lastname: Acreman
authors.forename: Paul R V
authors.initials: PR
authors.lastname: Johnson
mesh_terms: D000328:Adult; D018932:Chemokine CCL2 / Q000378:metabolism; D005260:Female; D006801:Humans; D000860:Hypoxia / Q000378:metabolism / Q000503:physiopathology*; D007249:Inflammation / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D007515:Islets of Langerhans / Q000276:immunology* / Q000378:metabolism / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D009924:Organ Culture Techniques; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D018932:Chemokine CCL2; D019253:Proto-Oncogene Proteins c-bcl-2; D042461:Vascular Endothelial Growth Factor A; D051028:bcl-2-Associated X Protein