Isolation of amelogenin-positive ameloblasts from rat mandibular incisor enamel organs by flow cytometry and fluorescence activated cell sorting

The purpose of this study was to use amelogenin as a marker to examine the feasibility of isolating ameloblasts from enamel organ cell populations by fluorescence activated cell sorting. After treating dissected rat enamel or gans with proteolytic enzymes to loosen cell attachments and labial connect ive tissues, dissociated cell suspensions were fixed, then immunostained wi th rabbit anti-rM179 recombinant amelogenin antibody and FITC-conjugated go at anti-rabbit Ig G antibody. Flow cytometry indicated that about 70% of th e total cell sample and virtually all the larger cells therein were ameloge nin-positive. Fluorescence activated cell sorting yielded a sample of amelo genin-positive cells at 97% purity. Immunofluorescence microscopy indicated that these isolated amelogenin-positive cells varied widely in size and mo rphology, This was attributed to loss of intercellular support for amelobla sts once they were dissociated from each other, and to some fragmentation c aused when the cells were initially physically removed from the teeth. The results demonstrate that viable ameloblast cell fractions, especially repre senting cells at the secretory stage, can be purified from enzymic digests of rat enamel organ by sorting on the basis of cell size alone. From these fractions, subpopulations of ameloblasts may be identified when differentia tion specific cell surface markers become available.