SELECTIVE EXPANSION AND CONTINUOUS-CULTURE OF MACROPHAGES FROM ADULT-PIG BLOOD

Macrophages were selectively expanded and continuously cultured from a dult pig blood. One-half mi of heparinized adult pig blood was inocula ted directly into the medium overlaying a feeder layer of STO mouse fi broblasts. After attachment to the feeder cells for 24 h, the culture was washed several times with the medium to remove most of any unattac hed blood cells and re-fed. Approximately 7x10(4) blood monocytes were initially detected and enumerated by specific binding of DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL). Macrophage outgrowths appeared in the primary culture after 6-7 days. The macrophages grew to relatively high density in 2-3 weeks (2-3 x 10(6) cells/T25 flask), and the culture was passaged on to fresh STO feeder layers to begin s econdary culture. Over 2-3 months of culture the macrophage replicatio n produced as many as 1.4 x 10(9) DiI-Ac-LDL-positive cells, The macro phages grew on top of the feeder cells in two forms: either a semi-att ached, round morphology, or a closely adherent, flat ameboid morpholog y with several extended pseudopods. Electron microscopic examination r evealed the cells to be uniformly of macrophage character and that 4-5 % were giant cells. The macrophages were phagocytic and expressed CD14 on their surfaces. They also reacted positively with pig macrophage-s pecific monoclonal antibody (mAb), and were negative for reactivity wi th pig T- and B-cell-specific mAb. This simple method for isolating an d propagating macrophages may indicate the replicative capacity of eit her adult pig blood monocytes or circulating blood stem cells, and it may be useful in providing macrophages for general research, virologic al assay, adoptive-immunotherapy models, and somatic gene therapy mode ls. (C) 1998 Published by Elsevier Science B.V. All rights reserved.