Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 2, 1998
Publication Date: N/A
Interpretive Summary: Macrophages are a type of white blood cell of central importance in defending the body against disease causing organisms either by direct ingestion of microbes or by helping to orchestrate an immune response with other types of white blood cells. Marcophages also serve an important function in scavenging the many millions of cells that normally die in the body each day. The in vitro culture of "normal" macrophages for biomedical and veterinary research is currently very difficult or impossible. We have previously shown that pig macrophages could be grown from very early embryonic tissue, termed the epiblast, and from various fetal pig tissues. Their growth was dependent on being cultured with a support cell or "feeder" cell called STO. We now show that from one-half milliliter of adult pig blood (approximately 10 drops) hundreds of millions of macrophages may be grown in culture over a period of 2-3 months in co-culture on STO feeder cells. As determined by a series of macrophage specific assays and by transmission electron microscopy, the macrophage cultures were of high purity as no other blood cells replicated in the culture system. The technique is very simple to perform and requires no specialized equipment. Because macrophages are frequently the target cell of many disease causing organisms, the culture system may aide the study of several economically important infectious diseases of pigs.
Technical Abstract: Normal macrophages were selectively expanded and continuously cultured from adult pig blood monocytes. One-half of a milliliter of heparinized adult pig blood was inocculated directly into the medium overlaying a feeder layer of STO mouse fibroblasts. Blood monocytes, detected and enumerated by specific binding of DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL), attached to the feeder cells after 24 h. The culture was washed several times with medium to remove most of any unattached blood cells and re-fed. Macrophage outgrowths appeared in the primary culture after 6-7 d. The macrophages grew to relatively high density in 2-3 weeks, and the culture was passaged on to fresh STO feeder layers to begin secondary culture. Over 2-3 months of culture the macrophage replication totaled as many as 1 x 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 uniformily 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 adult pig blood monocytes, and may be useful in providing normal macrophages for general research, adoptive-immunotherapy models, and somatic gene therapy models.