The development of new density gradient media for purifying human islets and islet-quality assessments

Abstract

Successful islet transplantation is dependent on the quality and quantity of islets infused. Islets are purified on density gradients, but procedures currently used have limited capacity for pancreatic digests, islet yield, and viability. We aimed to improve islet purification with a modified gradient medium. Biocoll was diluted in University of Wisconsin solution to create linear density gradients of 1.065 to 1.095 g/mL. Properties of islets purified from 22 human pancreas digests with modified medium were compared with 15 preparations using standard medium. The modification increased the capacity of gradients for pancreatic digests from 20 to 60 mL, islet yield increased from 218,000 to 435,318 per isolation, and viability increased from 65.4% to 92.1%. Islet fractions contained greater than 95% of recovered insulin. Islets showed good physiologic responses to secretagogues and restored normoglycemia in streptozotocin-induced diabetic severe combined immunodeficiency disease mice. The new medium enhances yield, purity, and viability of human islet preparations for clinical islet transplantation.

The novel chemokine receptor, G-protein-coupled receptor 75, is expressed by islets and is coupled to stimulation of insulin secretion and improved glucose homeostasis

“…Human islets were isolated from nondiabetic donors at the King's College Hospital Islet Transplantation Unit (Kings College Hospital, London, UK) with appropriate ethics approval [18]. Four separate human islet preparations were used, with a donor age range of 22 to 60 years and BMI of 20 to 37 kg/m 2 .…”

Insulin-producing cells derived from human pancreatic non-endocrine cell cultures reverse streptozotocin-induced hyperglycaemia in mice

“…Cold ischaemia time was 5.2±1.5 h (mean±SD). The pancreases were subjected to liberase digestion and density gradient fractionation as previously described [17]. Gradient fractions enriched with exocrine cells were collected, washed twice with Hanks' balanced salt solution and cultured in suspension as clusters [18] in CMRL1066 medium (Invitrogen, Inchinnan, UK), supplemented with 10% FCS and 0.95 mmol/l freshly prepared streptozotocin (STZ; Sigma) at 37°C in a humid 5% carbon dioxide incubator for 5 days, with medium and STZ changed daily to remove contaminating beta cells and dead cells.…”

Activin Receptor-Like Kinase 5 Inhibition Reverses Impairment of Endothelial Cell Viability by Endogenous Islet Mesenchymal Stromal Cells

“…Each individual experiment used islets combined from four to six mice. Human islets were isolated and cultured as described [19]. Five separate batches of human islets (60%-85% purity) were used.…”

“…Mouse and human islets (150) were isolated as described previously [18,19], and RNA was extracted according to manufacturer's guidelines (Qiagen, Crawley, U.K., http://www1. qiagen.com).…”

“…Immunostained islet area was assessed on micrographs using ImageJ software, and the islet EC density was assessed by manual counting of CD34-immunostained ECs. Islet insulin content was quantified by radioimmunoassay measurements of acidified ethanol extracts, as described [18,19].…”