Isolated osteoclasts and their presumed progenitor cells, the monocyte, in culture

Abstract

Osteoclasts were isolated from the endosteal surface of day 19 embryonic chick tibias by mild trypsinization. Osteoclast enrichment was achieved by passing cell suspensions through Nitex screening of selective sizes, including the eventual selective retention of osteoclasts on 12 μm polycarbonate filters or by sequential sieving through Nitex screens and fractionation on Percoll gradients. The enrichment procedures produced osteoclast populations of 50–75% based on morphological criteria with the latter isolation method providing populations with less matrix debris. The results of light microscopy, transmission and scanning electron microscopic observations indicate that osteoclasts can be maintained in culture for up to 10 days with retention of osteoclast morphology. This morphology includes a specialized ruffled plasma membrane, large numbers of mitochondria, lysosomes, as well as a multinucleated cytoplasm. Furthermore, acid phosphatase and butyrate esterase histochemical measurements support these morphological observations. In addition, chick hatchling circulating monocytes were isolated and purified by Ficoll‐hypaque gradient centrifugation with subsequent adhesion to glass petridishes. With time in culture, these cells form multinucleated cells, but lack the ultrastructural complexity of the isolated osteoclasts. This report describes a unique culture system to study osteoclast function and illustrates the similarities and differences of this system to the monocyte‐to‐giant cell culture system.