Selective expansion and continuous culture of macrophages from adult pig blood

Macrophages were selectively expanded and continuously cultured from adult pig blood. One-half ml of heparinized adult pig blood was inoculated directly into the medium overlaying a feeder layer of STO mouse fibroblasts. After attachment to the feeder cells for 24 h, the culture was washed several times with the medium to remove most of any unattached blood cells and re-fed. Approximately 7×104 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 (2–3×106 cells/T25 flask), and the culture was passaged on to fresh STO feeder layers to begin secondary culture. Over 2–3 months of culture the macrophage replication produced as many as 1.4×109 DiI-Ac-LDL-positive cells. The macrophages grew on top of the feeder cells in two forms: either a semi-attached, round morphology, or a closely adherent, flat ameboid morphology with several extended pseudopods. Electron microscopic examination revealed 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-specific monoclonal antibody (mAb), and were negative for reactivity with pig T- and B-cell-specific mAb. This simple method for isolating and propagating macrophages may indicate the replicative capacity of either adult pig blood monocytes or circulating blood stem cells, and it may be useful in providing macrophages for general research, virological assay, adoptive-immunotherapy models, and somatic gene therapy models.