Maintenance of Glucose‐sensitive Insulin Secretion of Cryopreserved Human Islets with University of Wisconsin Solution and Ascorbic Acid‐2 Glucoside

Abstract

Normal human islet cells are an ideal source for pancreas-targeted cell therapies, but the availability of human donor pancreata for islet isolation is severely limited. To effectively utilize such scarce donor organs for cell therapies, it is crucial to develop an excellent isolation, effective cryopreservation, and efficient gene transfer techniques for the transportation of isolated cells. In the present study, we investigate the effect of University of Wisconsin (UW) solution and ascorbic acid-2 glucoside (AA2G) on the cryopreservation of human islets. We also evaluate the gene transfer efficiency of a lentiviral vector expressing the E. coli LacZ gene, Lt-NLS/LacZ, in human islets. Human islets were isolated with a standard digestion method at the University of Alberta. Isolated islets were transported to Japan for 40 h and then subjected to cryopreservation experiments. The following preservation solutions were tested: UW solution with 100 micro g/mL of AA2G, UW solution, 100% fetal bovine serum (FBS), and CMRL supplemented with 10% FBS. Following three months of cryopreservation, the islets were thawed and analyzed for viability, glucose-sensitive insulin secretion, proinsulin gene expression profile, and in vivo engraftment. The islets were also subjected to monolayer formation with 804G-cell-line-derived extracellular matrix (ECM), followed by Lt-NLS/LacZ transduction. The viability, morphology, glucose-sensitive insulin secretion, proinsulin gene expression, and monolayer formation efficiency of the thawed cryopreserved islets are significantly better maintained by the use of UW solution. When AA2G (100 microg/mL) is combined with UW, such parameters are further improved. The adequate engraftment of UW + AA2G-cryopreserved human islets is achieved in the liver of nude mice. Efficient Lt-NLS/LacZ transduction is identified in monolayered islets cryopreserved with UW solution with AA2G. The present work demonstrates that the combination of UW solution with AA2G (100 microg/mL) would be a useful cryopreservation means for human islets. Human islets monolayer-cultured with 804G-derived ECM are efficiently transduced with a lentiviral vector Lt-NLS/LacZ.

Maintenance of stimulus-secretion coupling and single beta-cell function in cryopreserved-thawed human islets of Langerhans

“…Similarly, by selecting for largest diameter, smooth-surfaced single cells after several days in culture, it is likely that we were choosing for study the most viable cells and avoiding the bulk of cells undergoing apoptosis. Recent improvements in cryopreservation technique include: reduction in the concentration of DMSO as cryopreservant [29], substitution of ethylene glycol or trehalose as the cryopreservant [30,31], and addition of antioxidants such as beraprost sodium, butylated hydroxyanisole or ascorbic acid-2 glucoside to the cryopreservant [32][33][34].…”

Improvement of Human Islet Cryopreservation by a p38 MAPK Inhibitor

“…The inclusion of antioxidants in cryopreservation solutions has been shown to improve the islet yield after thawing (9,10). The use of an intracellular-ion composition-based solution, instead of the conventional extracellular solution, has also improved the islet recovery (9,11).…”

Modeling Chick to Assess Diabetes Pathogenesis and Treatment

“…There are many studies on isolation and cryopreservation of mammalian islets [51][52][53][54][55][56], but reports on cryopreservation of chick islets are lacking. We attempted to cryopreserve isolated chick islets following an established protocol used for cryopreservation of mouse islets [57].…”