A Novel Method for the Assessment of Cellular Composition and Beta-Cell Viability in Human Islet Preparations

Abstract

Current methodologies to evaluate islet cell viability are largely based on tests that assess the exclusion of DNA-binding dyes. While these tests identify cells that have lost selective membrane permeability, they do not allow us to recognize apoptotic cells, which do not yet stain with DNA-binding dyes. Furthermore, current methods of analysis do not discriminate between cell subsets in the preparation and, in particular, they do not allow for selectively defining β-cell viability.For these reasons we have developed novel methods for the specific assessment of β-cell content and viability in human islets based on cellular composition analysis through laser scanning cytometry (LSC) coupled with identification of β-cell-specific apoptosis at the mitochondrial level.Our novel analytical methods hold promise to prospectively analyze clinical islet transplantation preparations and predict functional performance, as suggested by the observed correlation with in vivo analysis of islet potency in immunodeficient rodents. Current methodologies to evaluate islet cell viability are largely based on tests that assess the exclusion of DNA-binding dyes. While these tests identify cells that have lost selective membrane permeability, they do not allow us to recognize apoptotic cells, which do not yet stain with DNA-binding dyes. Furthermore, current methods of analysis do not discriminate between cell subsets in the preparation and, in particular, they do not allow for selectively defining β-cell viability. For these reasons we have developed novel methods for the specific assessment of β-cell content and viability in human islets based on cellular composition analysis through laser scanning cytometry (LSC) coupled with identification of β-cell-specific apoptosis at the mitochondrial level. Our novel analytical methods hold promise to prospectively analyze clinical islet transplantation preparations and predict functional performance, as suggested by the observed correlation with in vivo analysis of islet potency in immunodeficient rodents.