Submitted to: In Vitro Cellular and Developmental Biology - Animals
Publication Type: Peer reviewed journal
Publication Acceptance Date: 8/12/2004
Publication Date: 8/12/2004
Citation: Talbot, N.C., Paape, M.J., Sohn, E., Garrett, W.M. 2004. Macrophage population dynamics within fetal mouse fibroblast cultures derived from c57/bl/6, cd-1, cf-1 mice and il-6 and gm-csf knock out mice. In Vitro Cellular and Developmental Biology - Animals. 40(7):196-210. Interpretive Summary: Macrophages (¿large eater¿) are a type of white blood cell important in fighting disease and clearing the body of unwanted proteins and cellular debris. Because macrophages consume bacteria and viruses they are also a route by which some pathogens, such as HIV, infect the body. In order to study macrophages and their interaction with other immune cell and microbes, it is important to be able to grow the cells in the culture dish, i.e., in vitro. A method is described for the growth of macrophages in vitro from fetal mouse tissues. The method relies on other mouse cells, called fibroblasts, to act as ¿feeder cells¿ to promote the growth of the mouse macrophages. The results showed that macrophages can be grown for several weeks from five different strains of mice. This implies that the method is probably applicable to any strain of mouse. The study also illustrates that what researchers may assume is a population of fibroblast cells growing in their dish is really a mixture of fibroblasts and macrophages. The findings should have broad applicability to the study of macrophage biology since these cells are generally difficult to grow in culture.
Technical Abstract: In vitro models of macrophage growth, differentiation, and function are needed to facilitate the study of their biology as important immune facilitator cells, and as frequent targets of bacterial and viral infection. A simple method for the selective expansion and continuous culture of mouse macrophages from primary explant cultures of mouse embryonic tissue is described. Culture in Dulbecco¿s modified Eagle¿s medium low glucose (1 g/L) formulation (DMEM/L) inhibited fibroblast growth. In contrast, mouse macrophages continued to proliferate in the presence of DMEM/L when in contact with the fibroblasts. Alternating growth in high glucose DMEM with DMEM/L produced a 1.16- to 2.1- fold increase (depending on mouse strain) in the percentage of macrophages within the cell culture with respect to culturing in DMEM with high glucose exclusively. Macrophages yields of over one million cells per T12.5 flask reached maximums by passage 3-4, and, thereafter, declined over the next 5-10 passages. The peak percentage of macrophages within a culture varied depending on the strain of mouse (C57Bl/6, CD-1, and CF-1 and two knock-out C57Bl/6 strains deficient in either IL-6 or G-CSF). G-CSF (-/-)-derived cultures had the lowest peak macrophage content (30%) and CD-1 the highest content (64.9%). The IL-6 (-/-) culture spontaneously transformed to create a cell line (IL6MAC) that was composed of ~75% macrophages. The macrophages were phagocytic, and were positive for CD-14, acetylate-LDL receptors, and F4-80 antigen. Light and electron microscopy showed the cultured macrophage had in vivo-like morphological features, and they could be plated to high purity by differential attachment to petri dishes in serum-free medium.