Alginate Microencapsulation of Islet Cells Using Electrostatic Droplet Generator / 대한이식학회지

BACKGROUND: Microencapsulation of islet cells has been studied for the treatment of type I diabetes to protect islets from immune attack by recipient cells during islet transplantation. In this study, we established optimal preparation conditions for islet microcapsules with good morphology and cell viability by employing an electrostatic droplet generator. METHODS: To obtain good quality islet microcapsules, various parameters such as the inner diameter of the electrostatic droplet generator nozzle, concentrations and infusion rates of alginate, electrostatic strength, and calcium chloride concentrations were tested. The size and shape of the capsules and cell viability were examined by light microscopy, and insulin secretion from the cells was determined by an ELISA analysis. RESULTS: The optimal preparation conditions for microencapsulation were a 0.35 mm inner nozzle diameter, a 1.75% alginate concentration, a 20 mL/hr alginate infusion rate, a 5 kV electrostatic potential, and a 75 mM calcium chloride. Under these conditions, over 90% of the capsules had a proper size (300~350 micrometer) and shape, and cell viability was 91%. Cell viability was maintained at greater than 80% even after the capsules were cultured for 2 weeks. However, glucose-induced insulin secretion of encapsulated islet cells was reduced by 85% compared to that of nonencapsulated cells. CONCLUSIONS: The results showed that an encapsulation technique using an electrostatic droplet generator is useful for making islet microcapsules with good morphology and cell viability. This technique is necessary to study microencapsulation using primary islets, enhancing glucose-induced insulin secretion, and to perform a functional evaluation of encapsulated islets in vivo in the near future.

Down-regulation of MHC class I Expression in Porcine Aortic Endothelial Cells by Introduction of hCMV US Genes / 대한이식학회지

PURPOSE: Direct recognition of porcine MHC proteins by human T cells is an impediment to successful xenotransplantation. Therefore, reducing human T cell response initiated by the interaction between TCR/CD8 cell and MHC class I on pig endothelial cell may be beneficial in successful pig- to-human xenotransplantation. METHODS: We examined MHC expression on porcine endothelial cell line, MYP30 cells in the absence or presence of IFN-g by FACS analysis. We introduced human cytomegalovirus (hCMV) US genes, which are known to be able to reduce MHC class I expression on the cell surface after infection, into MYP30 cells in order to test the feasibility of modifying these cells to reduced MHC class I antigens by the introduction of hCMV US genes such as US2, 3, 6 or 11. RESULTS: MHC class I expressions in MYP30 cells were dramatically induced by IFN-gamma treatment. FACS analysis showed that cells transfected with the hCMV US2, 3, 6 or 11 genes exhibited 30~40% of MHC class I expression compared with mock-transfected cells. We next established stable cell lines expressing US6 gene, which had been found to exert best down-regulation effect on MHC class I expression. Stable cell line expressing US6 gene products exhibited more than 10% reduced expression level of the MHC class I compared with transiently transfected cells. CONCLUSION: Although the further analysis of the cytotoxicities of T and NK cells on the hCMV US gene transfected cells are needed to clarify the feasibility of their application, these results suggest that virus stealth technology can be exploited for xenotransplantation.

MHC Antigen Expressions in Human Embryonic Neural Stem Cells and Adult Breast Epithelial Stem Cells / 대한이식학회지

PURPOSE: Due to their unique capacity to self-renew and for multiple differentiation, stem cells are considered potent candidates for cell replacement therapy in many devastating diseases. However, studies on immune rejection, which is a major problem facing successful stem cell therapy, are rare. Thus, we examined MHC expression of human stem cells and effects of IFN-gamma on the MHC class I expression of the cells in order to determine whether human stem cells might be rejected after transplantation. METHODS: The MHC antigen expressions of human embryonic neural stem cell line (HB1.F3) and human breast epithelial stem cell line (M13SV1) were examined by RT-PCR and FACS. The effects of varying concentrations of IFN-gamma and of varying incubation times with IFN-gamma on the expression of MHC class I antigens in these stem cell lines were also examined by FACS. RESULTS: The results show low expression levels of MHC class I antigens on surfaces of these cells. A dramatic induction of MHC class I expression was observed when the cells were treated with IFN-gamma. Maximal induction of MHC class I antigen expression in HB1.F3 and M13SV1 cells was observed at above the concentrations of 20 ng/mL and 5 ng/mL of IFN-gamma 48 h after treatment, respectively. Elevated MHC class I levels in HB1.F3 and M13SV1 cells were sustained for 48 h and 72 h after withdrawing IFN-gamma, respectively. CONCLUSION: These results suggest that human stem cells express high levels of MHC class I antigens, and thus may be rejected on transplantation unless they are modified. Therefore, in addition to studies on stem cell differentiation, studies on overcoming the immunological barriers to stem cell transplantation are prerequisite for successful clinical application of stem cell therapy.