Maintenance of glucose-sensitive insulin secretion of cryopreserved human islets with University of Wisconsin solution and ascorbic acid-2 glucoside

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 mug/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 mug/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 mug/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

Effect of slow freezing versus vitrification on the recovery of mouse embryonic stem cells

The purpose of this study was to cryopreserve mouse embryonic stem (ES) cells R1 line and determine cell viability, morphology, the number of colonies, and the Alkaline Phosphatase (AP) activity. In addition, the expression of transcription factors such as the stage-specific antigen (SSEA-1) and Octamer-4 (Oct-4) were evaluated before and after cryopreservation. The effects of two methods of cryopreservation, slow freezing and vitrification, were studied. The ES cells were cryopreserved either as single cells or as clumps. The viability of a single cell after slow freezing was 88%, but after vitrification no single cell was recovered. Surviving clumps after slow freezing quickly recovered and exhibited a morphology indistinguishable from noncryopreserved cells. After vitrification, 2 weeks of culture were required for the cells in clumps to proliferate enough for subculturing. Analysis of cloning efficiency and the colonies morphology were based on a mouse colony rating scale and their characteristic. The colonies from the slow-freezing group were compared to colonies from the control group, which were the cells before cryopreservation, and they showed the same cloning efficiency and morphology. The colonies from the vitrified group were compared to the colonies from the control group, and they showed differences in cloning efficiency on the mouse colony rating scale A (<0.05) and C (<0.05) but they did not show differences in their morphology. The biggest clumps from both experimental groups showed a reduction of viability in the center area compared to the fresh ones. The survival rate of the clumps in the slow-freezing and rapid-thawing group was 75% and in the vitrified group 25%. The colonies from the control group and both experimental cryopreservation groups show the same activity of AP, and they were all positive for SSEA-1 and Oct-4. The conventional slow-freezing method of cryopreservation of single cells and clumps is reliable and effective for the cryopreservation of mouse R1 ES cells. Vitrification can be used for cryopreservation of these same cell clumps but with lower recovery using the conditions that we used. © Mary Ann Liebert, Inc