High-Purity Resident Tissue Macrophage Isolates From Human Synovium and Periprosthetic Tissues Using Immunomagnetic Techniques

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Abstract

Destruction of periarticular and periprosthetic bone by activated macrophages, a process often termed “macrophage mediated osteolysis,” is recognized as a leading mechanism of aseptic arthroplasty failure. To develop effective interventional approaches and increase the longevity of implanted joint prostheses, the pathobiology of activated human-synovium-derived macrophages needs to be better characterized. The first step toward achieving this research objective is the acquisition of pure populations of macrophages from human synovial tissue. A simple, fast, and highly efficient method for isolating a relatively pure population of macrophages from periprosthetic tissue received from either primary or secondary arthroplasty is presented. This technique uses murine monoclonal antibodies (IgG) that recognize a phagocyte-specific marker, CD68, for primary binding, and sheep anti-murine IgG antibodies bound to polystyrene-coated magnetic micro-spheres for secondary binding. While the primary antibody specifically labels CD68-positive phagocytes in the digestion of synovial and periprosthetic tissue, the secondary antibody bound to polystyrene-coated iron oxide beads facilitates the removal of CD68-positive cells from CD68-negative cells by anchoring the former with a magnet. This protocol requires centrifugation only in the washing steps, which reduces the frequency of cell death and altered cell morphology. The patient population includes three primary and eight revision arthroplasties. The tissue macrophage isolation protocol yielded on average 4 X 105 cells/g tissue, of which 91% were viable nonspecific esterase positive macrophages. The experimental results suggest that immunomagnetic beads coupled to anti-CD68 enable the isolation of a purified population of resident tissue macrophages suitable for further biologic characterization.

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